1

Ph.D in Genetics, Oncology and Clinical Medicine

Array-CGH and complex developmental disorders: refinements of phenotypes in Xq28 and 6p25 and

new contiguous gene deletion syndrome in 22q13 not involving SHANK3 gene.

Vittoria Disciglio

Supervisor: Prof. Alessandra Renieri

Thesis suitable for the title of “Doctor Europaeus”

Doctoral School in Medical Genetics

Academic year 2011-2012

XXIV cycle

Ph.D dissertation board

Prof. Rosanna Abbate

University of Florence, Florence, Italy

Pro f. Sritharan Kadirkamanathan

University of London, London, UK

Prof. Stylianos Antonarakis

University of Geneva, Geneva, Switzerland

Prof. Alessandra Renieri

University of Siena, Siena, Italy

Ph.D thesis reviewers

Prof. Thomas Liehr

Institute of Human Genetics, University of Jena, Germany

Prof. Béla Melegh

Department of Medical Genetics, University of Pécs, Hungary

TO WHOM IT MAY CONCERN 23 November, 2012 Review of the PhD thesis: ”Array-CGH and complex developmental disorders: refinements of phenotypes in 6p25 and Xq28 and new contiguous gene deletion syndrome in 22q13 not involving SHANK3 gene.” by Vittoria Disciglio

The doctoral thesis written by Vittoria Disciglio comprises 77 pages excluding an appropriately sized list of 107 references. The sections of the thesis are proportionate, with 8 pages of introduction, one page of stating the aims of the study, 4 pages of methods and the most extensive part comprising the results section with 50 pages followed by an elaborate discussion of 4 pages and a brief conclusion of one page.

The introduction is very concise and it provides an extensive overview of the methodological background of the array CGH technique and its clinical utility, the language is precise and easy to follow. The aims of the study first defines a much broader goal of the study than it is focused on in the work of the author, but than narrows the focus of the study to the precise study population of overally 16 patients.

The materials and methods section is well written and gives enough information reflecting the contribution and self-experience of the candidate in the applied methods. In the results section the first two analyses of the candidate are presented as already published articles, in which the candidate participated as a co-author. The reviewed articles provide substantial novel results both in the characterization of females with Xq28 duplication and in delineating the spectrum of the neuroanatomic phenotype associated with alterations of the 6p25 region. The last part of the results section, still a manuscript under preparation, presenting the clinical and molecular data of nine patients with overlapping interstitial deletion in 22q13 chromosome region is very extensive and precise, but perhaps the introduction, methods and discusison sections could have been a bit shortened beacuse they are partly discussed int he appropriate sections of the thesis. The clinincal descriptions are extremely precise and detailed. All the figures and tables are very detailed and correctly reflects the information discussed in the text.

The discussion and conclusion part is clear, the conclusions are adequate but this part of the thesis is much less detailed and precisely formulated and expressed than the introduction and results sections. MAJOR comments and questions to the thesis: 1. On page 11, in section 1.7., line 4: the “genotype-first”, or reverse genomics approach” statement is true, but a careful phenotypic characterization before and after the genotype is still inevitable and is even emphazides through the results of the thesis. 2. In the Materials and Methods section under Patients collection the author states that: „patients with ID and MCA enrolled in this study have been selected among those attending the Medical Genetics Unit of the University Hospital of Siena.”

However, the Aims of the study section and the results clearly show that patients have been enrolled from multiple European Centers. Why is this not mentioned under Patients collection? Also, it is presumed that the statement that all patients have been first investigated by clinical geneticists refers to all enrolled patients from various centers, but there is no mention of it. 3. The conclusions start with the statement: „The application of array-CGH has revolutionized clinical cytogenetic, leading to identification of CNVs:” This formulation is misleading, since the identification of CNVs, as described in the introduction has occurred long before the application of array-CGH. 4. In patient enrollment: what obvious genetic conditions were excluded and what tests were done before enrolling patients with ID and MCA for aCGH testing? 5. There is no mention of ethics approval by the institutional review board. Was the study approved locally or for the multicenter analyses was there a joint ethics approval? 6. In the discussion section, on page 73, paragraph 3, line 8: Why is the Xq28 duplication study designated as second study, since it is presented as a first study throughout the thesis? 7. Why two sets of references are listed at the end of the third study in the results section?

MINOR comments (mostly typing and spelling errors or style): -title: refinements instead of „refiniments” -index: page 2, 1.5.: copy number variation instead of „namber variation” -index: page 2, 3.4.: instead of dipendent in „Multiplex ligation-dipendent Probe Amplification” dependent -page 5, section 1.2.: instead of „Karypotypes technique” perhaps „karyotyping” would be more suitable -page 5, section 1.2.: instead of „Tjio and Levan” the correct spelling is „Tijo and Levan”, this is also misspelled in the References -page 8, section 1.5., line 1: instead of „region” use „regions” -page 9, section 1.6., line 1: instead of „do not led” better „do not lead” -page 9, section 1.6., line 4: instead of „disease …are” better „diseases…are” - page 9, section 1.6., line 9: instead of „CNVs led” better „CNVs lead” -page 10, section 1.7, line 4: instead of „phatogenic” correctly: „pathogenic” - page 10, section 1.7, line 23:the number 15 should be deleted from the following

text: „intellectual disability,15 obesity, schizophrenia, and 1% of sporadic cases of

autism”

- page 10, section 1.7, line 27: instead of „has” plural is correct: „have”

-page 14, section 2, line 2: instead of „phatogenic” correctly: „pathogenic”

-page 16, materials and methods section: titles of 3.1 and 3.2.1. should be rather in

singular (patient collection and sample preparation)

-page 18, section 3.4, line 2 and 5: DiGeorge syndrome is mentioned twice in the

listing

-page 41, Patient 1, line 6: instead of „allux”, „hallux” is correct

-page 72, line 2: instead of „were associated to” correct phrase is „were associated

with”

-page 72, line 6: instead „copy number are common in normal individuals” „copy

number variation is common” would be more appropriate (Copy number variations

are)

-page 75, line 13: the sentence „These genes could be considered in the clinical

phenotype of

patients with larger terminal deletion of 22q13. „ is duplicated

-page 75, last line and page 77, line 20: instead of „a new gene contiguous

syndrome” correctly „new contiguous gene syndrome”

-page 77, line 10: spelling error, instead of „internaltional”, international

-page 77, line 24: instead of „ our results emphatizes” correctly „our results

emphasize”

-page 77, last line: instead of „rielable” correctly reliable

Important results of the candidate 1. The author and her coworkers presented clinical and molecular data on a series of five females with an Xq28 duplication including MECP2 gene expanding the spectrum of the associated phenotype. In contrast to the previously reported series of affected females their series includes a female patient with the typical symptoms of affected boys, therefore expanding the phenotypic spectrum of small Xq28 duplications including MECP2 in females. 2. The author and her group reported two unrelated patients with overlapping terminal 6p25 deletions, who presented with distinctive brain abnormalities, further delineating the spectrum of the neuroanatomic phenotype associated with alteration of this genomic region. 3. Finally, they characterized nine patients with overlapping interstitial deletion in the 22q13 chromosome region not involving the SHANK3 gene, supporting evidence for a new microdeletion syndrome. The topic chosen by the candidate is an uptodate and very exciting field of the postgenomic studies. Apart from the major and minor comments the substantial work and publications of Vittoria Disciglio and her coworkers presented in this doctoral thesis fulfills the requirements of a doctoral thesis and is suitable for achieving the title of “Doctor Philosophiae”. Béla Melegh, M.D., Ph.D., D.Sc. professor

INDEX

1. INTRODUCTION 1

1.1 DNA Copy Number Variant as a source of genetic variability 2

1.2 Classical Cytogenetic Technique for the detection of structural variation 3

1.3 Array-CGH Methodology 4

1.4 Applications of microarray technology in postnatal diagnosis 5

1.5 Mechanisms of formation of Copy Namber Variations 6

1.6 Phenotypic Consequence of Copy Number Variations 7

1.7 Genomic Disorders 8

1.8 Definition of rare genomic disorder through coordinated collaborations 9

2. AIM OF THE STUDY

11

3. MATERIALS AND METHODS 13

3.1 Patients collection 14

3.2 Array based CGH 15

3.2.1 Samples Preparation 15

3.2.2 Human oligonucleotides array 16

3.3 Real Time quantitative PCR 17

3.4 Multiplex ligation-dependent Probe Amplification (MLPA)

18

4. RESULTS 19

4.1 Xq28 duplications including MECP2 in five females: expanding the phenotype to severe mental retardation

20

4.2 Periventricular heterotopia with white matter abnormalities associated with 6p25 deletion

30

4.3 Interstitial 22q13 deletions not involving SHANK3 gene: a new contiguous gene syndrome

36

5. DISCUSSION

73

6. CONCLUSIONS

78

7. BIBLIOGRAPHY

81

Curriculum Vitae 93

Aknowledgements 100

1

1. INTRODUCTION

1.1 DNA Copy Number Variant as a source of genetic variability

Genomes vary from one another in multifarious ways, and the totality of this genetic

variation underpins the heritability of human traits. Human genomic variation is

present in many forms, including single-nucleotide polymorphisms (SNPs), small

insertion-deletion polymorphisms (INDELs), variable numbers of repetitive

sequence and genomic structural variations (Bowcock et al., 1994; Feuk et al., 2006;

Conrad and Hurles 2007; 1000 Genomes Project Consortium 2012). Structural

Variation (SV) is a broad term for genetic variants that alter chromosomal structure.

It includes deletions, duplications, inversions, transpositions, translocations and

complex rearrangements (Hurles et al., 2010). Historically, structural variation was

primarily assayed cytogenetically in diseased genomes. The first report of disease

caused by mega base sized genomic rearrangements was published in the early 1990s

(Lupski et al., 1991). For most of twentieth century geneticists focused on large-

scale genomic rearrangements because these were the only variants that were visible

by early cytogenetic methods. The discovery of the structure of DNA and the

elaboration of genetic code spawned great interest in the role of small-scale variants

such as SNPs, and the development of molecular cloning, DNA sequencing and PCR

during the 1970s and 1980s allowed for researcher to directly ascertain these variants

in an increasingly high-throughput manner. By the turn of the century, as thousands

of automated DNA sequencers were churning out data of Human Genome Project,

conventional wisdom held that the overall structure of a genome was relatively static,

and that most natural variation could be explained by small-scale sequence

differences. This is best exemplified by an oft-repeated statement of this era that the

phenotypic differences between two humans could be explained by the presence of 1

SNP in every 1,000 bp. The first convincing evidence that genome architecture was

more dynamic came from the work of Bailey J. and colleagues who, upon aligning

the draft human genome to itself, discovered that roughly 5% of DNA is contained

within segmental duplications (regions of the genome larger than 1 kb that share 90%

of identity) (Bailey et al. 2001). In 2004, two landmark studies (Iafrate et al., 2004;

Sebat et al., 2004) demonstrated that genomic submicroscopic variations (<500 kb in

size) are widespread in normal human genomes. Intense analysis of this type of

genomic variability followed, revealing the role of DNA copy number variations

(CNVs) as an essential contributor to individual variability and a major driving force

in human evolution (Conrad et al., 2010; Kidd et al., 2008; Korbel et al., 2010).

CNVs are a form of structural variation, defined as genomic segments of 1 kb or

larger in size present at a variable copy number in comparison with a reference

genome. On average, there are > 1000 CNVs in the genome, accounting for ~4

million base pairs of genomic difference (Conrad et al., 2010; Mills et al., 2011).

Although SNPs outnumber CNVs in the genome by three orders of magnitude, their

relative contribution to genomic variation (as measured in nucleotides) is similar.

Thus, in addition to 0.1% of genetic difference at the nucleotide sequence level, we

now recognize another 0.1% of genetic difference at the structural level (Malhotra et

al., 2012).

1.2 Classical Cytogenetic technique for the detection of structural variation

Initially, the only way to visualize our entire genome was by performing

Karyotyping technique that was introduced in the late 1950s (Tijo and Levan 1956).

In the 1960s, cytogenetic analysis became more powerful by the development of

chromosomal banding that permitted to study gross chromosomal abnormalities, both

numerical and structural. Therefore, this technique became a valuable diagnostic

tool. However, a disadvantage of banding is the resolution limitations that make

impossible the detection of small aberrations (< 5 Mb) (Trask BJ 2002; Caspersson et

al., 1968). To overcome this limitation, several new cytogenetic technique were

developed, including Fluorescent in situ hybridization (FISH), introduced in the early

1980s (Van Prooijen-Knegt et al., 1982). FISH is based on the use of chromosome

region-specific, fluorescent-labeled DNA probes. These probes are cloned pieces of

genomic DNA that are able to detect their complementary DNA sequences,

producing a fluorescent signal against background stained chromosomes that can

easily be detected. However, this technique requires prior knowledge of the type and

location of expected aberrations and can only be used for the analysis of a limited

number of chromosomal loci at once. In 1992, Kallioniemi and collegueas introduced

a new chromosome analysis technique called comparative genomic hybridization

(CGH) (Kallioniemi et al., 1992).

In CGH, differentially labelled test and reference DNA were simultaneously

hybridized to chromosome spreads. Conventional CGH is a molecular cytogenetic

technique that detects and maps alterations in copy number of DNA sequences

(Weiss et al., 1999). In this approach, the genomic DNAs of test (patient) and

reference (normal) samples are labeled green and red, respectively. The DNAs are

hybridized to normal human metaphase chromosomes where DNA sequences from

both sources compete for their targets. Subsequently, images of both fluorescent

signals are captured, and ratios of the test and reference hybridization signals are

digitally quantified along the length of each chromosome. The result is that

chromosomal regions equally represented in both samples appear yellow (because

both the red and green fluorochrome are detected) and have a ratio of one. Deleted

regions are red and have a ratio below one, while amplified regions green colored

and have a ratio above one. In this way, a global overview of chromosomal gains and

losses throughout the whole sample genome is obtained (Tachdjian et al., 2000).

1.3 Array-CGH Methodology

Two major types of array targets are currently being utilized. Initially, bacterial

artificial chromosomes (BACs) were the array target of choice (Pinkel et al., 1998).

However, more recently, oligonucleotide arrays have been adopted due to the

increased genome coverage they afford. The design of both array types was made

possible by the availability of the complete map and sequence of the human genome.

The BAC arrays may contain DNA isolated from large insert clones that range in

size from 150–200 Kb, spotted directly onto the array or may employ the spotting of

PCR products amplified from the BAC clones (Ylstra et al., 2006). These arrays are

generally very sensitive and results can be directly validated with FISH using the

BAC DNA as a probe. However, production of BAC DNA is labor intensive and the

resolution is limited to 50–100 Kb, even on a whole genome tiling path array

(Ishkanian et al., 2004). Oligonucleotide arrays offer a flexible format with the

potential for very high resolution and customization. Today, several different

platforms are available for oligonucleotide arrays, some of which were adapted from

genome wide SNP-based oligonucleotide markers and others that were created from

a library of virtual probes that span the genome, and consequently can be constructed

to have extremely high resolution (Shaikh et al., 2007). Both BAC and

oligonucleotide arrays have been used successfully to detect copy number changes in

patients with intellectual deficit (ID), multiple congenital anomalies (MCA) and

autism. A number of different array design approaches have been taken for

diagnostic purposes. A targeted array is one that contains specific regions of the

genome, such as the subtelomeres and those responsible for known

microdeletion/microduplication syndromes, but does not have probes that span the

whole genome (Bejjani et al., 2005; Bejjani et al., 2006; Shaffer et al., 2006). This

type of array was initially used for clinical applications in postnatal specimens but

has also been implemented for prenatal specimens with an abnormal ultrasound

finding or for general screening purposes (Le Caignec et al., 2005; Sahoo et al.,

2006; Kitsiou-Tzeli et al., 2008). A whole genome or tiling path array offers full

genome coverage with a resolution that is dependent on the spacing of the probes.

For clinical testing the resolution generally involves a spacing of 50 Kb to 1 Mb

between adjacent probes on the array often with additional coverage at the

subtelomeric regions (Veltman et al., 2006; Toruner et al., 2007). The enhanced

coverage of whole genome arrays identifies an additional 5% of abnormalities when

compared to a targeted array (Baldwin et al., 2008; Veltman and De Vries 2007). For

research purposes, very high density oligonucleotide whole genome arrays and

region specific custom arrays have been instrumental in defining new syndromes,

detecting target gene deletions and characterizing breakpoint regions (Selzer et al.,

2005; Urban et al., 2006; Wong et al., 2008).

1.4 Applications of microarray technology in postnatal diagnosis

In the last years the implementation of microarray technology in postnatal diagnosis

had led to identification of genomic defects of congenital and developmental

abnormalities including developmental delay (DD), intellectual disability (ID),

autism spectrum disorders (ASD) and/or multiple congenital anomalies (MCA).

An example of clinical utility of array CGH is a study published in 2008. This is one

of a largest published studies that used array CGH to estimate the frequency of

genomic imbalance testing neonates with birth defects. The reported pathological

CNV detection rate for subject samples referred with an indication of “suspected

chromosomal abnormality” was 66,7%. When compared to other studies published

during the previous 15 years this number is about three times greater (Lu XY et al.,

2008). This and other studies had led to consider array CGH a first-line diagnostic

approach for clinically suspected genetic disorders (Shaffer et al., 2011; Kang and

Koo 2012).

1.5 Mechanism of formation of Copy Number Variations

Although CNVs are found throughout the genome, some regions are more prone to

rearrangements than others. Based on their breakpoints and modes of formation

CNVs are divided in recurrent or nonrecurrent. Recurrent CNVs show breakpoints

clustering and junction that are limited to the location of low copy repeats (LCRs).

LCRs or segmental duplication (SDs) are defined as DNA segments that occur more

than twice in the haploid genome, have an extension larger than 1Kb (may extend to

over 300 kb in size) and present more than 95% sequence identity between their

paralogous copies (Edelmann et al., 1999; Bailey et al., 2001). In recurrent

rearrangements LCRs during meiosis, and at lower frequency in mitotically dividing

cells, could mediate a process called non-allelic homologous recombination (NAHR)

that originate CNVs with common size and nearly identical boundaries in carriers.

Several of such recurrent CNVs have been linked to human diseases (Reiter et al.,

1998; Stankiewicz and Lupski 2002). Nonrecurrent CNVs have scattered

breakpoints and the boundaries share limited or no nucleotide homology. This

categories of CNVs are of variable size and may arise via mechanisms like

nonhomologous end joining (NHEJ) and replication-based mechanisms described by

the fork stalling and template switching (FoSTeS) and microhomology-mediated

break-induced replication (MMBIR) models. It is becoming clear that most disease-

causing CNVs are nonrecurrent and generally arise via replication-based mechanisms

(van Binsbergen, 2011; Lee et al., 2007; Hastings et al., 2009). Furthermore, it is

now appreciated that genomic features other than low copy repeats play a role in the

formation of nonrecurrent CNVs. Patients carried nonrecurrent CNVs may share a

critical region whose copy number changes results in common clinical features

(Cooper et al., 2011; Kleefstra et al., 2005; Koolen et al., 2012).

1.6 Phenotypic Consequence of Copy Number Variations

Aggregate data has shown in recent years that all rearrangements do not lead to

disease, and many appear to represent polymorphic variants in the population (see

Database of Genomic Variants, http://projects.tcag.ca/variation/). However CNVs

have been identified as one of the common causes of human disease. Diseases caused

by these structural variants are collectively known as genomic disorders. The most

common mechanism of conveying a phenotype by microdeletion or microduplication

is altering gene expression of a dosage sensitive gene(s) located within the

rearranged region (Vissers et al; 2004; Stankiewicz et al; 2009; Lupski JR. et

al;1993). However, in some case, CNVs lead to manifestation of clinical phenotypes

through other mechanisms. First, the breakpoint of a CNV can disrupt the coding

sequence of a gene (Rujescu et al; 2009), or can generate a novel fusion gene (Lifton

et al; 1992). Second, CNVs can delete or duplicate gene regulatory elements

changing gene function but leaving the protein coding sequence intact. This position

effect has been described mainly in balanced translocation (Velagaleti et al; 2005;

Pop et al; 2004) and microdeletion (Beysen et al; 2005) with breakpoint mapping as

far as 1Mb upstream or downstream from the protein-coding region of the causative

gene (Kleinjan and van Heyningen; 2005). Recently, microduplications of regulatory

elements have been reported also to cause specific phenotypes. A recessive mutation

or functional polymorphism can be unmasked as a consequence of a deletion CNV

(Kurotaki et al; 2005). Moreover CNV may affect microRNAs in the genome, which

in turn may lead to alterations in gene expression.

1.7 Genomic Disorder

Genomic disorders are common diseases with a frequency of ~1 per 100-1,000 new

borns and are often sporadic, due to de novo microdeletion or microduplication

CNVs (Shaffer and Lupski, 2000; Lupski, 2007). Rare, recurrent variants of

pathogenic significance, were originally identified in persons with a characteristic set

of clinically recognizable features, such as the Smith Magenis Syndrome, the Sotos

Syndrome, and the Williams-Beuren syndrome. Some phenotypic traits resulting

from microdeletions or microduplications can show non-Mendelian inheritance, such

as genomic imprinting (e.g. in Prader Willi and in Angelman syndromes) or

mosaicism (e.g. neurofibromatosis type I) (Matsuura et al., 1997; Chai et al., 2003;

Sahoo et al., 2008; Pasmant et al., 2009; Mabb et al., 2011). The worldwide use of

high-resolution platforms on large patient cohort has been instrumental for the

identification of novel microdeletion and microduplication syndromes (Franco et al.

2010; Shaffer et al., 2007; Slavotinek et al., 2008; Portnoï et al., 2009). The recent

discoveries of pathogenic copy-number variants have broadened the phenotypic

range associated with a given variant to include entirely distinct diseases (Deak et al.,

2011). High-throughput analyses of patient populations have implicated the same

copy-number variants in diseases, such as schizophrenia, autism, cardiac disease,

epilepsy, and intellectual disability (Horváth et al., 2011; Wiśniowiecka-Kowalnik et

al., 2012; Southard et al., 2012; Hochstenbach et al., 2011; Girirajan et al., 2012).

For example, a recurrent deletion on chromosome 15q13.3 has been associated with

intellectual disability, schizophrenia, autism, and 1% of idiopathic generalized

epilepsy (Muhle et al., 2011; Crespi et al., 2012; Mulley et al., 2011). Similarly, a

deletion on chromosome 16p11.2 has been associated with intellectual disability,

obesity, schizophrenia, and 1% of sporadic cases of autism (Ciuladaitė et al., 2011;

Jacquemont et al., 2011; Zufferey et al., 2012).

Because of the relative rarity of genomic disorders it has been difficult to draw

reliable conclusions about patterns of concurrent clinical traits, and the cytogenetic

causes of these syndromes have often remained unknown. Even when phenotypic

features are reliably established, the underlying genetic background is often not

homogeneous, since multiple genes and alterations may contribute to the same

pathway and thereby to a similar final phenotype. Thereby, the classical paradigm of

a “phenotype-first” approach, referring to the identification of a disease gene in a

preselected patient-cohort, has been shifted to a “genotype-first”, or reverse

genomics approach, wherein the overlapping CNVs identified in the clinically

heterogeneous patient cohort are analyzed first (Schulze et al., 2004). This approach

is a valuable tool but, in the clinical setting the guidance of expert physiscians in the

medical evaluation of complex patients is important to avoid medical errors of

“overuse” (e.g. testing patients inappropriately as in those case with recognizable

monogenic syndromes). Therefore the medical history and the physical examination

by an expert clinician should always preced the “genotype-first” approach for

patients with complex developmental disorders (Saul, 2009). This “genotype-first”

approach has the most clinical implications since a considerable number of genomic

alterations can be related to the clinical phenotypes of the patients (Poot et al. 2011).

This emphasizes the need for a close collaboration between clinicians and laboratory

professionals.The clinical evaluation of patient after array-CGH results is

recommended in order to provide the best care for the patient, which relies on

habilitation and counseling for his/her family (Romano, 2010). Moreover, more

careful examination of the clinical phenotype of patients may reveal phenotypic

overlap not expected for such heterogeneous disease, hence allowing the

identification of new syndromes (Girirajan and Eichler 2010; Slavotinek 2008).

1.8. Definition of rare genomic disorder through coordinated collaborations

As we already mentioned before, many patients suffering from developmental

disorders harbor submicroscopic deletions or duplications that, by affecting the copy

number of dosage-sensitive genes or disrupting normal gene expression, lead to

disease. As identification of patients sharing a genomic variant and having common

phenotypic features leads to greater certainty in the pathogenic nature of CNVs and

is the prerequisite for defining new syndrome, data sharing and collaboration

between clinical and research centers is crucial. To facilitate the analysis of these

rare events, an interactive web-based database has been developed called

DECIPHER (DatabasE of Chromosomal Imbalance and Phenotype in Humans using

Ensembl Resources; http://www.sanger.ac.uk PostGenomics/decipherl) (Wellcome

Trust Sanger Inst. 2007). It may facilitate widespread appreciation of such

phenotypic variability, which incorporates a suite of tools designed to aid the

interpretation of submicroscopic chromosomal imbalance, inversions, and

translocations. As of November 2012, the database includes 19762 patient reports

from 247 participating centers, as well as descriptions of 65 distinctive syndromes.

DECIPHER catalogs common copy-number changes in normal populations and thus,

by exclusion, enables changes that are novel and potentially pathogenic to be

identified. DECIPHER enhances genetic counseling by retrieving relevant

information from a variety of bioinformatics resources. Known and predicted genes

within an aberration are listed in the DECIPHER patient report, and genes of

recognized clinical importance are highlighted and prioritized. DECIPHER enables

clinical scientists worldwide to maintain records of phenotype and chromosome

rearrangement for their patients and, with informed consent, share this information

with the wider clinical research community through display in the genome browser

Ensembl. By sharing cases worldwide, clusters of rare cases having phenotype and

structural rearrangement in common can be identified, leading to the delineation of

new syndromes and furthering understanding of gene function (Swaminathan et al.,

2012). The possible differences in frequency of particular CNVs in different

populations and the variety of different CNVs observed in a given study may be

significantly limited by the number and ethnic origin of individuals examined. For

this purpose, from a collaboration with some Italian laboratories, an interactive web-

based database, who collect rearrangements, de novo and inherited, not described in

DGVs, has been developed called Human Copy Number Variations Database

(HCNVs) (http://dbcnv.oasi.en.it/gvarianti/index.php).

2. AIM OF THE STUDY

The aim of current study was to investigate rare structural variants in patients with

ID, ASD, MCA and to dissect the phenotype in order to identify genotype-phenotype

correlation and assign a possible pathogenic role to one or some of major genes of

the “minimal critical region”.

In this study we developed partnership with different centers registered in database

DECIPHER and Human Copy Number Variations Database (HCNVs) to enroll two,

five and nine patients with similar rearrangements in chromosomal regions Xq28,

6p25 and 22q13 respectively. Since structural variants under investigation are rare, in

order to reach this aim, we needed collaborative efforts with different centers in

different countries, with the purpose to collect a greater number of subject sharing

similar CNVs. The international collaborative effort, carried out in order to

investigate patients sharing 22q13 interstitial deletions, was extremely relevant since

allowed us to define a new contiguous gene deletion syndrome characterized by

distinctive clinical features that could be recognized by clinical geneticists.

The most important impact of this thesis has been the refinement of the clinical

phenotype of patients affected by rare genomic disorders including Xq28 duplication

syndrome and 6p25 deletion syndrome. Moreover, a new contiguous gene deletion

syndrome on 22q13 was identified. An in depth clinical dissection of these rare

structural variants is fundamental for the clinical geneticists since it provids powerful

diagnostic hints even before the array-CGH analysis is performed. Furthermore,

detailed phenotyping, refinement of clinical diagnostic assignments, and an

increasingly wider understanding of array-CGH data are essential in order to dissect

all influences undergoing a specific genetic condition.

3. MATERIALS & METHODS

3.1 Patient collection

All 16 patients with ID and MCA enrolled in this study have been investigated by

clinical geneticists. Three patients have been selected among those attending the

Medical Genetics Unit of the University Hospital of Siena. The remaining patients

were clinically evaluated and molecularly characterized in different medical centers

of different countries. Clinical and molecular informations on patients were obtained

from the referring physician and molecular cytogenetist respectively. Specifically the

patients enrolled with the purpose to investigate Xq28 microduplication syndrome

were clinically evaluated and molecularly analysed by following clinical and

laboratory centers: Netherlands [Center for Human and Clinical Genetics, Leiden;

Department of Pediatric Neurology, Juliana Children’s Hospital/HAGA teaching

Hospital, The Hague; (Case 1)], United Kingdom [(Wessex Clinical Genetics

Service, Princess Anne Hospital; Wessex Regional Genetics laboratory, Salisbury

District Hospital; (Case 2)], Italy [(Medical Genetics Unit of Siena and Cytogenetics

and Molecular Genetic Unit of Pisa; Child Neuropsychiatry of Siena; (Case 3)],

Spain [(Molecular genetics laboratory, Biochemestry Department, Cruces Hospital,

Barakaldo-Bizkaia; Neuro-pediatrician Section, Txagorritxu Hospital, Vitoria-

Gasteiz; Clinical Genetics-Paediatrics Department, Cruces Hospital, Barakldo-

Bizkaia; Clinical Psycology, Galdakao-Usansolo Hospital, Galdakao; (Case 4)] and

USA [Division of Genetic, Nemours Children’s Clinic, Orlando; (Case 5)].

The patients enrolled in order to investigate 6p25 deletion syndrome were

molecularly evaluated in Medical Genetics Unit of the Univerity Hospital of Siena,

Italy (Patient 1) and in the department of Molecular Medicine, University of Pavia,

Italy (Patient 2). These patients were clinically characterized in Italian and American

clinical centers: Child Neurology Unit, Children’s Hospital A. Meyer, Florence

(Italy); Department of Radiology, University of California, San Francisco; Child

Neurology Unit, Siena; Neurological Clinic C. Mondino, Pavia and IRCCS Stella

Maris, Pisa.

The patients enrolled with the purpose to investigate the interstitial 22q13 deletions

not involving SHANK3 gene were clinically and molecular analysed by different

centers: Italy [Medical Gnetics Unit of University of Siena and Department od

Pediatrics, Ospedale Valdichiana, Montepulciano (Patient 1)]; United Kingdom

[Manchester Academic Health Sciences Centre, Manchester Biomedical Research

Centre, St Mary’s Hospital, Manchester M13 9WL, UK; (Patient 2)]; Sweden

(Department of Molecular Medicine and Surgery , Karolinska Institutet and

HospitalStockholm (Patient 3)]; France [CHU Nantes, Service de genetique medicale

(Patient 4)]; United kingdom [Department of Clinical Genetics, Alder Hey Children's

Hospital, Liverpool, and Liverpool Women's Hospital, Liverpool, (Patient 5)];

France [Service de Génétique Clinique, Hôpital Jeanne de Flandre, Lille, ( Patient

6)]; Italy [Laboratory of Genetics Diagnosis IRCCS Oasi M.SS. Troina; Unit of

Pediatrics and Medical Genetics, IRCCS Oasi M.SS. Troina and Medical Genetics of

university of Catania (Patient 7)]; Italy [IRCCS Casa Sollievo della Sofferenza

Hospital, Mendel Institute, Rome; Department of Developmental Neuroscience,

IRCCS Stella Maris,Calambrone Pisa (Patient 8)]; Italy [S.C. Medical Genetics,

Institute for Maternal and Child Health IRCCS “Burlo Garofolo”, Trieste, Italy

(Patient 9)]. All patients were evaluated by experienced clinical geneticists who

excluded a recognizable syndrome on a clinical ground. Regarding Patient 1

described in 6p25 study, G-bandend karyotype from peripheral lymphocytes and X-

fragile test were first performed. These analysis resulted normal. Patients 7 and

Patients 9 described in 22q13 study were first cytogenetically analysed. Parents or

guardians of the patients gave their informed consent. Local Institutional Review

Board approved this study.

3.2 Array-based CGH

3.2.1 Sample preparation

Genomic DNA of normal controls was obtained from Promega. Genomic

DNA was extracted from peripheral blood using a QIAamp DNA Blood Maxi kit

according to the manufacturer protocol (Qiagen, www.qiagen.com). The

OD260/280 method on a photometer was employed to determine the

appropriate DNA concentration (Sambrook and M.J. Gething; 1989). Patient and

control DNA samples were sonicated to produce a homogeneous smear DNA

extending from approximately 600 bp to 2 Kb. DNA samples were then purified

using the DNA Clean and Concentrator kit (Zymo Research, Orange, CA). Ten

micrograms of genomic DNA both from the patient and from the control were

sonicated. Test and reference DNA samples were subsequently purify using

dedicated columns (DNA Clean and Concentrator, Zymo research, CA92867-4619,

USA) and the appropriate DNA concentrations were determine by a DyNA Quant™

200 Fluorometer (GE Healthcare).

3.2.2 Human oligonucleotides array

Array based CGH analysis was performed using commercially available

oligonucleotide microarrays containing about 43,000 60-mer probes with an

estimated average resolution of about 100-130 Kb (Human Genome CGH

Microarray 44B Kit, Agilent Technologies) and microarrays containing 99,000

60-mer probes with an estimate average resolution of 50-65 Kb (Human Genome

CGH Microarray 105A Kit, Agilent Technologies). Physical positions of the

probes correspond to the UCSC genome browser - NCBI build 36, March 2006.

(http://genome.ucsc.edu). DNA labelling was executed essentially according to

the Agilent protocol (Oligonucleotide Array-Based CGH for Genomic DNA

Analysis 2.0v) using the Bioprime DNA labelling system (Invitrogen). Genomic

DNA (2 µg) was mixed with 20 µl of 2.5X Random primer solution (Invitrogen)

and MilliQ water to a total volume of 41 µl. The mix was denaturated at 95° C

for 7 minutes and then incubated in ice/water for 5 minutes. Each sample was

added with 5 µl of 10X dUTP nucleotide mix (1.2 mM dATP, dGTP, dCTP, 0.6 mM

dTTP in 10 mM Tris pH 8 and 1 mM EDTA), 2.5 µl of Cy5-dUTP (test sample) or

2.5 µl of Cy3-dUTP (reference sample) and with 1.5 µl of Exo-Klenow (40 U/µl,

Invitrogen). Labeled samples were subsequently purified using CyScribe GFX

Purification kit (Amersham Biosciences) according to the manufacturer protocol.

Test and reference DNA were pooled and mixed with 50 µg of Human Cot I DNA

(Invitrogen), 50 µl of Blocking buffer (Agilent Technologies) and 250 µl of

Hybridization buffer (Agilent Technologies). Before hybridization to the array

the mix was denatured at 95°C for 7 minutes and then pre-associated at 37°C

for 30 minutes. Probes were applied to the slide using an Agilent microarray

hybridization station. Hybridization was carried out for 24/40 hrs at 65°C in a

rotating oven (20 rpm). The array was disassembled and washed according to the

manufacturer protocol with wash buffers supplied with the Agilent kit. The

slides were dried and scanned using an Agilent G2565BA DNA microarray

scanner. Image analysis was performed using the CGH Analytics software v.

3.4.40 with default settings. The software automatically determines the

fluorescence intensities of the spots for both fluorochromes performing

background subtraction and data normalization, and compiles the data into a

spreadsheet that links the fluorescent signal of every oligo on the array to the

oligo name, its position on the array and its position in the genome. The linear

order of the oligos is reconstituted in the ratio plots consistent with an

ideogram. The ratio plot is arbitrarily assigned such that gains and losses in

DNA copy number at a particular locus are observed as a deviation of the ratio

plot from a modal value of 1.0.

3.3 Real-time quantitative PCR

Some aCGH data were confirmed by Real-time Quantitative PCR

experiments. To design adequate probes in different regions of the human

genome, we used an TaqMan Gene Expression Assays by design which provides

pre-designed primers-probe set for real-time PCR experiments (Applied

Biosystems, https://products.appliedbiosystems.com) PCR was carried out using

an ABI prism 7000 (Applied Biosystems) in a 96-well optical plate with a final

reaction volume of 50 µl. A total of 100 ng (10 µl) was dispensed in each of the

four sample wells for quadruplicate reactions. Thermal cycling conditions

included a prerun of 2 min at 50°C and 10 min at 95°C. Cycle conditions were 40

cycles at 95°C for 15 sec and 60°C for 1 min according to the TaqMan Universal

PCR Protocol (ABI). The TaqMan Universal PCR Master Mix and Microamp

reaction tubes were supplied by Applied Biosystems. The starting copy number

of the unknown samples was determined using the comparative Ct method as

previously described (Ariani et al., 2004).

3.4 Multiplex Ligation-dependent Probe Amplification (MLPA)

MLPA analysis was performed according to the provider’s protocol with a

specifically designed set of probes for testing critical regions in 1p-deletion

syndrome, Williams syndrome, Smith-Magenis syndrome, Miller-Dieker syndrome,

DiGeorge syndrome, Prader-Willisyndrome, Alagille syndrome, Saethre-Chotzen

syndrome, Sotos syndrome: (SALSA P064B MR1 kit); subtelomere regions (SALSA

P036D subtelomeric primer kit) and Prader-Willi / Angelman syndrome (SALSA

ME028-B2 Kit).

The ligation products were amplified by PCR using the common primer set with the

6-FAM label distributed by the supplier. Briefly, 100 ng of genomic DNA was

diluted with TE buffer to 5 µl, denatured at 98°C for 5 minutes and hybridized with

SALSA Probe-mix at 60°C overnight. Ligase-65 mix was then added and ligation

was performed at 54°C for 15 minutes. The ligase was successively inactivated by

heat, 98°C for 5 minutes. PCR reaction was performed in a 50 µl volume. Primers,

dNTP and polymerase were added and amplification was carried out for 35 cycles

(30 seconds at 95°C, 30 seconds at 60°C and 60 seconds at 72°C). Amplification

products were identified and quantified by capillary electrophoresis on an ABI 310

genetic analyzer, using GENESCAN software (version 3.7) all from Applied

Biosystems (Foster City, CA, USA). The peak areas of the PCR products were

determined by GENOTYPER software (Applied Biosystems). A spreadsheet was

developed in MicrosoftTM Excel in order to process the sample data efficiently. Data

were normalized by dividing each probe’s peak area by the average peak area of the

sample. This normalized peak pattern was divided by the average normalized peak

pattern of all the samples in the same experiment (Koolen et al; 2004).

4. RESULTS

4.1. Xq28 duplications including MECP2 in five females: Expanding

the phenotype to severe mental retardation.

Bijlsma EK, Collins A, Papa FT, Tejada MI, Wheeler P, Peeters EA, Gijsbers AC,

van de Kamp JM, Kriek M, Losekoot M, Broekma AJ, Crolla JA, Pollazzon M,

Mucciolo M, Katzaki E, Disciglio V, Ferreri MI, Marozza A, Mencarelli MA,

Castagnini C, Dosa L, Ariani F, Mari F, Canitano R, Hayek G, Botella MP, Gener B,

Mínguez M, Renieri A, Ruivenkamp CA.

Eur J Med Genet. 2012 Jun.

4.2. Periventricular heterotopia with white matter abnormalities

associated with 6p25 deletion.

Cellini E, Disciglio V, Novara F, Barkovich JA, Mencarelli MA, Hayek

J, Renieri A, Zuffardi O, Guerrini R.

Am J Med Genet A. 2012 Jul

4.3 Interstitial 22q13 Deletions not involving SHAN3 gene: a new

contiguous gene syndrome

Unpublished Results (Manuscript in Preparation)

ABSTRACT

Phelan-McDermid Syndrome (22q13.3 deletion syndrome) is a contiguous gene

disorder resulting from deletion of the distal long arm of chromosome 22. SHANK3,

a gene included within the minimal critical region, is thought to be a candidate gene

for the major neurological features of this syndrome. Here we report the clinical and

molecular data of nine patients with overlapping interstitial deletion in 22q13 not

involving SHANK3. All these interstitial deletions overlap with the largest terminal

deletions, but not with the smallest terminal deletions. The deletion sizes and

breakpoints varied considerable between the different patients, with the largest

deletion spanning 6,9Mb and the smallest deletion spanning 2,7Mb. Eight out of nine

patients have a de novo deletion and in the other one patient the origin of deletion is

unknown. These patients share several clinical features common to Phelan Mc-

Dermid syndrome: developmental delay (11/12), speech delay (11/12), hypotonia

(9/12) and feeding problems (7/12). Moreover, 8 out of 12 patients show

macrocephaly, while normal occipitofrontal circumference, in combination with

overgrowth were observed in one patient. In the minimal deleted region, we

identified two candidate genes, SULT4A1 and PARVB, which could be associated in

our cohort of patients with neurological features and macrocephaly/overgrowth and

hypotonia, respectively

This study suggests that there are additional haploinsufficient genes in the 22q13

region that contribute to cognitive and speech development and are involved in the

phenotype of the at least the larger 22q13 deletions. Moreover, since the deletions in

our patients do not involve SHANK3 gene, we speculate the existence of a new gene

contiguous syndrome proximal to smallest terminal deletions, in the 22q13 region.

INTRODUCTION

Deletions involving the distal long arm of chromosome 22q13 are well

described and associated with the Phelan-McDermid syndrome (PMS (OMIM

#606232), characterized by severe neonatal hypotonia and global developmental

delay, moderate to severe intellectual impairment, normal growth and minor

morphological anomalies. Common facial features include dolicocephaly, flat

midface, wide brows, wide nasal bridge with bulbous nasal tip, deep-set eyes, full

cheeks, puffy eyelids and long eyelashes. Large fleshy hands, dysplastic toenails,

sacral dimple, and large poorly formed ears are frequently observed (Phelan and Mc

Dermid, 2012). More than 50% of patients show autism or autistic-like behavior, and

therefore it can be classified as a syndromic form of autism spectrum disorder (ASD)

(Phelan and Mc Dermid, 2012).

In the minimal critical region, the gene SHANK3 is thought to be the strongest

candidate gene for the major neurological features of this syndrome (Luciani et al.,

2003; Wilson et al., 2003; Bonaglia et al., 2011).

Interstitial deletions of 22q13 chromosomal region, not involving SHANK3, have

been rarely reported totaling no more than four patients (Romain et al., 1990; Wilson

et al., 2008). Only three cases are finely defined at molecular level through array-

CGH and clinically described by Wilson et al., in 2008. Their phenotype shows

many similarities to the terminal 22q13 deletion syndrome including hypotonia,

speech impairment, developmental delay and morphological anomalies (Wilson et

al., 2008). We present the clinical and molecular data of an additional 9 patients

with overlapping interstitial deletions in 22q13 not involving the SHANK3 gene

diagnosed by array-CGH. After identification of the 22q13 deletion in Patient 1, a

search for further patients revealed eight individuals with overlapping deletions

through the DECIPHER database (http://decipher.sanger.ac.uk) and the Database of

Human CNVs (http://gvarianti.homelinux.net/gvarianti/index.php). In all 9 patients

the deleted region overlaps with deletions found in the three cases previously

reported with a common deleted region spanning about 950 Kb. This cohort of

patients, sharing common clinical features, helped to delineate the emerging clinical

phenotype of 22q13 interstitial deletions not involving the SHANK3 gene.

METHODS

In total, nine patients with an 22q13 interstitial deletion were included in this

study. DNA samples of the patients were analyzed in clinical centers of different

countries: Italy, United Kingdom, France and Sweden. Clinical information on

patients were obtained from the referring physician. The clinical features of our

patients are presented below and the main clinical details are listed in Table I. The

age of all patients, reported below in each clinical description, is referred to the time

of genetic evaluation. The pictures of patients are shown in Figure 1. Parents or

guardians of patients gave their informed consent.

ARRAY-CGH ANALYSIS

Table II provides details of the microarray-platforms used, other cytogenetic and

molecular genetics methods applied in evaluation of the families, deletion size and

heritability. This table summarizes the results of the array-CGH analyses in Patient

1-9 and provides the genomic coordinates of the deletions according to the Feb. 2009

human genome sequence assembly [hg19]. Eight out of nine patients have a de novo

deletion while in patient 7 the origin of deletion is unknown. The size of deletions

was variable, ranging from 2,7 Mb to 6,98 Mb. The size of minimally deleted region

was ~950 Kb and encompassed 12 genes. In patient 7 standard chromosome

karyotyping was first performed revealing the presence of pericentric inversion of

chromosome 5 (inv5p15.1-q11.2) inherited from his father. Molecular

characterization through array-CGH analysis did not identify aberrations involving

chromosome 5. Cytogenetic analysis was first conducted on patient 9 that revealed a

female karyotype with the presence of small supernumerary chromosome ring

(sSCR) originated from chromosome 19 in 100% of cells examined. To confirm the

extent of the small supernumerary chromosomal marker 19, SNP array analysis was

performed revealing the presence of the following aberrations: arr

19p13.11q13.12(16,790,859-36,722,832)x3; arr 22q13.1(37,799,188-38,621,456)x3;

arr 22 (43704364-49832693)x1. SNP array analysis conducted on DNA of parents

showed that these aberrations occurred de novo. Figure 1 shows the extent of

interstitial 22q13 deletion in each subject of this report and compares this to the

published individuals with an overlapping 22q13 deletion (Wilson et al. 2008).

Additional information regarding these patients are provided in the supplementary

online material.

CLINICAL REPORTS

Patient 1 (Siena, MCA 146)

Male, age 4 months.

The patient was one of twins born at 37 weeks of gestation by caesarean section.

Auxological parameters at birth were: weight 2.56 kg (25-50°cnt), length 47 cm (25-

50°cnt) and head circumference (OFC) 34 cm (75°cnt). He showed poor sucking.

The physical examination, performed at the first week of life, disclosed several

peculiar facial features including triangular face, posteriorly rotated and apparently

low set ears, high nasal bridge, prominent columella, bilateral broad thumb and

hallux, clenched hands and flexed upper limbs. At the follow-up the patient was 4

months and 10 days; the weight was 7.800 gr (90°cnt), the length was 63 cm (50-

75°cnt) and OFC was 45.5 cm (>97°cnt). He was still hypotonic and he did not show

head control. He had prominent frontal bossing with depressed ocular region,

prominent columella, pointed chin and short neck. He had tapered fingers and

elongated halluces and he was unable to extend his thumbs. The echocardiogram

showed a patent foramen ovale. Brain MRI showed a thin corpus callosum and little

layers in frontal brain region.

Patient 2

Female, age 11 years

The pregnancy was complicated by bleeding between 8-10 weeks and a pregnancy-

related rash at 37 weeks. She was delivered by lower segment caesarean section at 41

weeks gestation for fetal distress with a birth weight of 2.920 gr and required

resuscitation. Feeding problems were present at birth and she required nasogastric

feeding from 16 weeks. She had transient low thyroxine with normal TSH, now

resolved. The echocardiogram showed a large patent ductus arteriosus requiring

closure, small Ventricular Septal Defect and patent foramen ovale. She smiled at 6

months. When first seen in the genetic service at 11 months her OFC was 42.5cm

(<0.4th centile), she vocalized only with consonants, would sit unaided for a few

minutes but was not reaching out much. She showed no interest in exploring her

environment and parents were concerned about possible autism. By 39 months she

was bottom-shuffling, using a rollator with support and had a couple of words. By 8y

3months she had walked independently on only one occasion and has never repeated

this. Autistic features were more evident. At the age of 11 she had severe learning

disability and autism. She has developed a progressive in-turning of her feet and is

not now able to walk. She communicates in single words. Physical examination

showed a girl with weight and height on 2nd centile and OFC below 0.4th centile, fine

facial features with small nose and anteverted nostrils, tapered fingers and small slim

hands. She had an asymmetric buttock crease. She was farsighted with a squint.

Initial brain MRI showed delayed myelination but repeat scan at 10 years was normal

except for a small pituitary gland. Spinal MRI was normal.

Patient 3

Male, age 5 years.

The boy was the 5th of 6 siblings and the parents were not related. There was no

heredity for developmental disorders. The pregnancy was uneventful. Amniocentesis

was performed but revealed no chromosomal abnormalities. He was born at 38

weeks of gestation by vacuum extraction because of signs of asphyxia. Auxological

parameters at birth were: weight 3.170 gr (50-75°cnt) and length 51 cm (75-90°cnt).

OFC was not available. During infancy he had severe feeding problems. Ultrasound

of the pyloric muscle showed an enlargement but no stenosis. He had a mild

hypospadia and slight asymmetry of the face with a larger ear on the right side.

Kidney-ultrasound revealed agenesis of the right kidney, enlargement of the left one

and a further evaluation disclosed reflux of the 5th grade. He underwent surgical

correction for urethral reflux at 3 years. His motor development was delayed and

muscular tone decreased. The speech-development was delayed (first words at 18

months). At age 2.5 years he developed a squint. Brain-CT scan, performed when he

was 3 years and 9 months, revealed slight broadening of the liquor space in the left

Sylvian fissure but no major pathological findings. At the age of 4 years his growth

was within the normal range.

Patient 4

Female, age 6 years and 2 months.

The girl was born at term from uneventful pregnancy. Auxological parameters at

birth were: weight 2.400 gr (<10°cnt) and length 48 cm (25°cnt), OFC was not

available. She showed developmental delay (walking alone 15 months) and language

delay. She had school difficulties with attention deficit. She had strabismus and an

ocular-constructive apraxia. Auxological parameters at the time of examination were:

height 115 cm (50°cnt), weight 24 kg (95°cnt) and OFC 55.5 cm (>97°cnt). She had

dysmorphic facial features with a high forehead and a widow peak. She also had a

supernumerary nipple.

Patient 5

Male, age 4 years.

He is the first child of unrelated parents. He was born by elective caesarean section

because of breech presentation. Echocardiography showed a large Ventricular Septal

Defect. He underwent cardiac surgery at 5 months of age. Developmental delay was

noted at 6 months. He was floppy and inactive with lack of response. As a result of

the hypotonia, he developed plagiocephaly and torticollis. With regard to his lack of

responsiveness, the ophthalmologic examination showed no structural eye problems.

By 15 months of age he was more visually alert but was extremely floppy and he

also had a poor speech-development. He had feeding difficulties. Plagiocephaly and

torticolls were reported. He weighed 9.91 kg (just below 25th centile) with length of

77.5 cm (25th centile) and head circumference 47.3 cm (between 9th and 25th centile).

He had no facial dysmorphism. He had slightly small toe and fingernails and a slight

triangular shape to his little toes. The brain-MRI scan was normal. He had a left

orchidopexy for cryptorchidism. His hands were soft and chubby and the feet

presented with deep palmar creases and tapering digits. He had overlapping toes with

very small toenails. In view of the severe persistent plagiocephaly and torticollis he

was referred to the craniofacial unit who found that he had atlanto-axial subluxation

and commenced traction therapy. He also had a mid-thoracic syrinx at T3-T6 and a

very mild thoracic scoliosis. He showed developmental delay (he began walking at 3

years 9 months). Presently he speaks a few words and has some autistic traits.

Patient 6 Female, age 2 years and 9 months.

Information about the pregnancy was not available. She was born by cesarean

section. She was hypotonic. Auxological parameters at birth were not available. At

the time of examination length was 83 cm (90°cnt) and OFC was 52.5 cm (>97°cnt).

She had developmental and speech delay. Brain MRI showed periventricular

leucomalacia. She also had finger hyperlaxity and strabismus. She had dysmorphic

features.

Patient 7

Male, age 1 year and 3 months.

He was the second child of unrelated parents. The family history was negative for

neurological disease. He was born at term after an uneventful pregnancy with a birth-

weight of 3050 gr (>25th cnt), length and OFC were not available. He was hypotonic

and he had feeding-problems. He presented a delayed psychomotor development and

he has not started to walk unsupported. He presented psychomotor retardation. He

presented 3 seizures episodes. The phenotype is characterized by persistent anterior

fontanelle, chubby cheeks, prominent frontal bossing, downslanting palpebral

fissures, depressed nasal root with downward bulbous nasal tip, high palate and

fleshy hands and feet. Neurological examination revealed generalized muscular

hypotonia and absent head and trunk control with poor standing control.

Patient 8

Male, age 5 years and 6 months.

His parents were non consanguineous. Information about family history of the

mother was not available. The mother presented facial features analogue to those of

her son. At 5 years he presented a mild cognitive impairment. The pregnancy was

achieved after in-vitro fertilization-embryo transfer (IVF-ET). During pregnancy a

vertebral cleft and micrognathia were noted by ultrasonography. Amniocentesis

examination was performed and fetal karyotype was normal. He was born at 38

weeks after an uneventful vaginal delivery with normal birth weight (3650 g;75-

90°cnt), length (51 cm; 75-90°cnt) and head circumference (OFC, 33.5 cm; 50°cnt).

In the third day of life an intestinal abnormality (intestinal volvulus and malrotation

with sub-obstruction) was diagnosed and gastrointestinal surgery was performed.

Computerized tomography (TC) of spinal column showed spinal abnormalities such

as butterfly-shaped vertebrae and scoliosis. The baby had hypotonia and facial

dysmorphisms including epicanthal folds, dolichocephaly, large ears, frontal

eminences, bulbous nose and pointed chin. He had delayed psychomotor

development: sitting position was acquired after 15 months, walking independently

at 5 years. At present he is able to use only a few common words. In the first year of

life he underwent orchidopexy surgery for bilateral cryptorchidism and

dermatological intervention for abnormal toenail growth. At 3 years of age he

presented pronounced joint hyperlaxity, axial hypotonia and severe mental

impairment. The already observed facial dysmorphisms were more evident and

relative large and fleshy hands were also present. Postural and tonal abnormalities

were noted, including the absence of anticipation movement when falling, sitting

with trunk in kyphosis and walking on the knees. Hypersalivation and absence of

language were also observed. At 6 years of age auxological parameters are: height

114 cm (50°cnt), weight 19 kg (25-50°cnt) and OFC 48 cm (<3°cnt).

Patient 9

Female, 1 year and 6 months.

The parents were not related and the family history was negative for others genetic

diseases. The propositus was born at 39 weeks by a vaginal delivery. During

pregnancy, fetal echography revealed a left cerebral ventriculomegaly. The postnatal

MRI confirmed the ventriculomegaly and showed also mild dilatation of both frontal

horns and thin corpus callosum. Auxological parameters at birth were: weight 3320

gr (50°cnt), length 49.5 cm (25°cnt) and head circumference 35 cm (50°cnt). The

Apgar score was 7 at I’ and 8 at V’. She showed poor sucking. She had recurrent

conjunctivitis caused by immaturity of lacrimal ducts. The ophthalmological

evaluation was normal. Her general examination showed: weight of 9.650 gr (75°-

90°), length of 67 cm (3°-10°), head circumference of 47 cm (90°), hypotonia, head

deviation, bilateral presence of preauricular peduncles bilaterally, abnormal lobes,

bilateral epicanthus, inverted nipples, small fingernails, handheld deep folds, single

transverse palmar crease in right hand, brevity of first metatarsal, laryngomalacia and

Mongolian spots on the back.

DISCUSSION

We enrolled in the present study nine patients with novel overlapping interstitial

microdeletions in 22q13 chromosomal region and we evaluated their clinical data.

Detailed molecular analysis of the deletions by whole genome array analysis

demonstrated that in all nine cases proximal and distal breakpoints and deletion sizes

(between 2,7 Mb and 6,98 Mb) differed (see Table II and Fig. 2). In patients 7 and 9,

additional chromosomal rearrangements were identified. Specifically, in patient 7 a

pericentric inversion of chromosome 5 inherited from his father was identified and in

patient 9 a copy number gain on 22q13 (820 kb) and a ring chromosome 19 were

found. All 22q13 interstitial deletions identified overlap with the largest terminal

ones, but do not include the smallest terminal deletions involving SHANK3 gene.

Several studies support the hypothesis that the SHANK3 gene is a strong candidate

for the neurobehavioral symptoms in Phelan-McDermid syndrome, since SHANK3

maps within region of common deletion observed in patients (Luciani et al., 2003;

Wilson et al., 2003; Bonaglia et al., 2011; Delahaye et al., 2009, Gong et al., 2012).

In patients with the 22q13 deletion syndrome phenotype, a balanced translocation

disrupting SHANK3 or deletions restricted to SHANK3 were identified (Bonaglia et al

2001; Anderlid et al., 2002; Bonaglia et al, 2006). To date more than 600 patients

with 22q13.3 deletion syndrome have been reported, ranging in size from ~100 Kb to

more than 9 Mb, resulting from terminal or interstitial deletions, ring chromosomes,

and unbalanced or balanced translocations (Bonaglia et al. 2011; Phelan and

McDermid 2012). Previous genotype-phenotype studies have shown that many

clinical features of 22q13 deletion syndrome are associated with deletion size. In a

study of 50 patients, Wilson et al. (2003) found a positive correlation between

increased deletion size and developmental delay, hypotonia, head circumference, ear

infections, pointed chin, dental anomalies and several measures of independent

behavior and daily living abilities. In a case series of 33 patients a genotype-

phenotype study showed that the severity of hypotonia increased with the size of the

deletion but that the degree of severity of speech delay did not correlate with deletion

size (Luciani et al. 2003). Sarasua et al. (2011) found an association of increased

deletion size with neonatal hypotonia, head circumference and facial features.

Only three cases with 22q13 interstitial deletion not encompassing SHANK3 gene, all

molecularly characterized with array-CGH, have been reported (Wilson et al., 2008).

Another case has been described as an interstitial deletion of 22q13 in 1990. This

patient showed a phenotype similar to 22q13 deletion syndrome. Nevertheless the

involvement of SHANK3 was unknown because the deletion was not characterized at

molecular level, but only cytogenetically (Roman DR et al. 1990). The phenotype of

patients described in the present study closely match with that of the patients

reported by Wilson et al. 2008 with developmental delay, delay of speech, hypotonia,

overgrowth and macrocephaly. A comparison of physical findings of our patients

with those reported by Wilson et al. 2008 is summarized in Table I. In figure 1 the

images from 6 out of 9 patients presented in this report at the time of genetic

evaluation are shown and compared to those of 2 patients reported by Wilson et al

2008. The most common facial features are high frontal hairline, broad forehead,

wide nasal bridge and thin upper lip. The phenotype of all patients show many

similarities to Phelan-McDermid syndrome. Like all patients with terminal 22q13

deletion, the 12 patients without SHANK3 deletion have developmental delay

(11/12), speech delay (11/12), hypotonia (9/12) and feeding problems (7/12). The

mother of patient B (patient BX) described by Wilson et al. 2008 exhibited a very

mild phenotype. Supporting our findings of severe clinical phenotype similar to the

Phelan-McDermid syndrome are reports of genotype-phenotype study regarding

associations between features of PMS and deletion sizes. These data suggest that the

etiology for the neurological features must be due to loss of genes located more

proximal to SHANK3. These genes are also deleted in many patients with 22q13

terminal deletions, contributing to their phenotype. The deleted regions in patients 1-

12 overlap and share the genomic segment with many genes in common with Phelan

McDermid syndrome. In this study we define the minimal critical region associated

to phenotype (Fig. 2). A simple model for genotype-phenotype correlation may

consider each single gene mapping in the critical region. UCSC genome database

shows that the minimally deleted region spans about 950 Kb from SULT4A1 to

ARHGAP8 genes. Among these, SULT4A1 is of particular interest. The protein

encoded by this gene is a brain specific cytosolic sulfotransferase that catalyzes the

transfer of a sulfonate group from 3’-phosphoadenosine 5’-phosphosulfate (PAPs) to

an acceptor group of the substrate (Minichin et al., 2007; Allali-Hassani et al., 2007).

In both humans and rodents, SULT4A1 is mainly, but not exclusively, expressed in

selected regions of the brain. Immunohistochemical studies have localized SULT4A1

to areas of the cerebral cortex, cerebellum, and brainstem. Within the cerebral cortex,

the strongest staining was seen in pyramidal neurons of the motor cortex as well as

structures within the cingulate and insular cortex. Its high expression in specific

regions of the brain suggests a role in the central nervous system (Liyou et al. 2003).

Different studies have linked polymorphisms upstream, of the SULT4A1 coding

sequence with schizophrenia susceptibility (Brennan and Condra 2005; Condra et al.,

2007; Meltzer et al., 2008). This protein, and other members of the sulfotransferase

superfamily, are involved in the sulfation and inactivation of neurotransmitters,

including dopamine (DA) and norepineprhine, steroids (including neurosteroids),

drugs and xenobiotics (Yu et al.,1985; Falany et al., 2000; Sakakibara et al., 2002;

Liyou et al., 2003). Since this gene is particularly abundant in the cortex and it has

been implicated in schizophrenia we suggest that the deletion of this gene may be

associated with neurological symptoms in these patients.

In our cohort of patients, two clinical features recurred with high frequency:

macrocephaly, present in 8 out of 12 patients (67%) and hypotonia, present in 9 out

of 12 patients (75%). All patients with macrocephaly presented height at 90°

percentile, while patient 7 has normal OFC in combination with overgrowth (height

and weight at 90° percentile). The study of each single gene mapping in the critical

region led us to propose that haploinsufficiency of the gene PARVB could be

responsible for macrocephaly and/or overgrowth and hypotonia. PARVB gene

encodes a member of the parvin family proteins. The parvins are a family of actin-

binding proteins that play a critical role in transducing signals from integrins to the

actin cytoskeleton and intracellular signaling proteins (Olski et al. 2001).

Analysis of human tissue shows that ParvB is ubiquitously expressed but its

expression levels are highest in the heart and skeletal muscle. ParvB does not possess

intrinsic catalytic activity and exerts its effect through interaction with other proteins

such as ILK, α-actinin and α-PIX (Suplveda and Wu, 2006). Important functions

have been identified for ParvB in integrin-mediated cell adhesion, regulation of cell

spreading and cell motility. Data from several studies showed that Parvin- β has an

inhibitor effect on intracellular ILK function (Zhang Y. et al. 2004). Literature data

provides evidence that ParvB has a pivotal role in cell growth and invasion in tumor

cells. It has been demonstrated that loss of ParvB expression leads to the

upregulation of ILK activity (Mongroo et al. 2004) and that ILK activation triggers a

cascade of Akt/PKB phosphorilation in tumor cells (Majumder et al. 2004). Kimura

et al. (2010) have shown that ParvB preferentially binds to Akt1 rather ILK under

living cell conditions and that ParvB might acts as a modulator to prevent the

transmission of excess signals, such as those derived from growth factors, in the

ILK-Akt/PKB signaling cascade. Mutations in genes that encode proteins belonging

to the receptor tyrosine kinase (RKT)-PI3K-AKT pathway have been associated to

overgrowth disorders (Lindhurst M. et al. 2012; Riviere J-B. et al. 2012). Germline

loss of function mutation in PTEN, a negative regulator of PI3K signaling, causes

overgrowth, and a somatic second hit in PTEN causes the type 2 segmental Cowden

syndrome (Happle R. et al. 2010). Somatic mutation in the AKT1 gene that

constitutively activates PI3K-AKT signaling has been associated with Proteus

syndrome, a progressively deforming regional overgrowth syndrome that affects

bone, adipose and other mesenchimal tissue (Lindhurst M. et al. 2011). Recently

mutations in three core components of the phosphatidynositol 3-kinase (PI3K)-AKT

pathway encoded by AKT3, PIK3R2 and PIK3CA genes have been identified in

patients with megalencephaly-capillary malformation (MCAP) and megalencephaly-

polymicrogyria-polydactyly-hydrocephalus (MPHH) syndromes. All these

syndromes are overgrowth disorders associated with markedly enlarged brain

(Lindhurst M. et al. 2012; Riviere J-B. et al. 2012). Since ParvB is one of the

identified cellular inhibitors of ILK signaling, including PTEN, we suggest that

deletion of this gene might be responsible for the activation of PI3K-AKT signaling

causing macrocephaly and overgrowth.

ParvB is a dysferlin binding protein that colocalizes with dysferlin at the

sarcolemma of normal human skeletal muscle. The dysferlin gene is defective in

Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B).

Matsuda C. et al (2005) showed that a deficiency of dysferlin caused a secondary

reduction of ParvB at the sarcolemma of LGMD2 and MM muscles (Matsuda C. et

al., 2005). LGMD2B is characterized by progressive wasting and weakness of the

muscles of the proximal lower limb girdle, whereas MM mostly affects the distal

muscle groups of the limb girdle. Both disorders are considered to result from a loss

of dysferlin (DYSF) protein from the plasma membrane of muscle fibers, leading to

abnormalities in vesicle traffic and membrane repair this process is collectively

referred to as “dysferlinopathy” (Han R et al. 2007, Glover L et al., 2007). Since the

majority of the present cohort of patients showed hypotonia (9/12) and carried

deletion of the ParvB gene it is possible to suggest that hapolinsufficiency of this

gene may be responsible of this symptoms affecting colocalization of dysferlin at the

sarcolemma of skeletal muscle.

Autism Spectrum Disorder (ASD) is the neurodevelopmental disorder that is

associated with the 22q13 deletion syndrome with a frequency higher than 50%

(Phelan McDermid et al., 2012). Haploinsufficiency of the SHANK3 gene is

considered to be responsible for the presence of autistic behavior as well as other

neurological symptoms in patients with 22q13 deletion syndrome. This hypothesis is

also supported by recent genetic studies that have indicated that several SHANK3

mutations cause neuronal developmental disorders, including ASD and psychiatric

disorders.

In our cohort of patients ASD was reported in two patients over age 3 years.

In four out of the remaining seven patients we were not able to establish the presence

of autistic-like behavior because of the young age (< 3 years). The presence of

autism in patients described by Wilson et al. 2008 was not reported. Since the

possibility that those patients could elicit autistic-like behavior phenotype in the

future cannot be excluded, we are not able to suggest if other genes different from

SHANK3 could contribute to manifestation of ASD in patients with interstitial

deletions proximal to SHANK3 gene.

In order to verify the role of PARVB and SULT4A1 genes we compared a set

of clinical features (developmental delay, speech delay, hypotonia, overgrowth,

macrocephaly, autism) manifested in our cohort of patients with those manifested in

individuals affected by Phelan McDermid syndrome previously reported in the

literature. We classified these patients in four categories on the base of genomic

rearrangements (Fig. 3):

Group A: Patients with interstitial deletion not involving SHANK3 gene

Group B: Patients with terminal deletion that include the minimal deleted

region

Group C: Patients with terminal deletion that not include the minimal deleted

region.

Group D: Patients with intragenic deletion and chromosomal aberration

disrupting the SHANK3 gene.

The relative frequency of the clinical signs (developmental delay, speech delay,

hypotonia, overgrowth, macrocephaly, autism) in groups of patients A,B, C and D

are rappresentedin table III.

Χ-square statistical analysis revealed that macrocephaly was present with higher

frequency in groups A and group B in comparison with groups C and D (p=0,001).

These results support the hypostesis that PARVB gene coulb be invovlved in

etiology of the manifestation of macrocephaly in our chort of patients.

In summary, in this report we describe a cohort of patients with 22q13

deletions not involving SHANK3 gene and propose the involvement of two new

candidate genes, SULT4A1 and PARVB, in the etiology of clinical manifestations.

These genes could be considered in the clinical phenotype of patients with larger

terminal deletion of 22q13. Characterization of additional patients with smaller

interstitial deletions could be helpful to confirm this hypothesis and to better define a

genotype-phenotype correlation. In conclusion we speculate the existence of a new

gene contiguous syndrome proximal to the smallest terminal deletions in the 22q13

region.

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URLS

DECIPHER database (http://decipher.sanger.ac.uk)

Database of Human CNVs (http://gvarianti.homelinux.net/gvarianti/index.php)

Thomas Liehr’s website (http://www.fish.uniklinikum-jena.de/sSMC/sSMC+by+chromosome/sSMC+19.html#Symptoms)

OMIM (http://www.ncbi.nlm.nih.gov/omim/)

UCSC Genome Browser (http://genome.ucsc.edu/cgi-

bin/hgGateway?hgsid=309187673)

AKNOWLEDGMENTS

Antonio Massarelli1, Valentina Canocchi1, Britt Marie Anderlid2, Kay Metcalfe3,

Cédric Le Caignec4, Albert David4, Alan Fryer5, Odile Boute6, Andrieux Joris7,

Donatella Greco12, Vanna Pecile8, Roberta Battini9, Antonio Novelli10, Marco

Fichera11,13, Corrado Romano12

1 Dipartimento di Pediatria, Ospedale Valdichiana, Montepulciano, Italy

2 Department of Molecular Medicine and Surgery, CMM, Karolinska Institutet and

Hospital Stockholm, Sweden

3 Manchester Academic Health Sciences Centre, Manchester Biomedical Research

Centre, St Mary’s Hospital, Manchester M13 9WL, UK

4 CHU Nantes, Service de génétique médicale, France

5 Department of Clinical Genetics, Alder Hey Children's Hospital, Liverpool, and

Liverpool Women's Hospital, Liverpool, UK.

6 Service de Génétique Clinique, Hôpital Jeanne de Flandre, Lille, France

7 Institut de Génétique Médicale, Hôpital Jeanne de Flandre, Lille, France

8 S.C. Medical Genetics, Institute for Maternal and Child Health IRCCS “Burlo

Garofolo”, Trieste, Italy

9 Department of Developmental Neuroscience, IRCCS Stella Maris,Calambrone

Pisa, Italy

10 IRCCS Casa Sollievo della Sofferenza Hospital, Mendel Institute, Rome, Italy

11 Laboratory of Genetics Diagnosis, IRCCS Oasi M. SS., Troina, Italy

12 Unit of Pediatrics and Medical Genetics, IRCCS Associazione Oasi M. SS.,

Troina, Italy

13 Medical Genetics, University of Catania, Italy

Fig. 1 (A) Photographs of six of nine patients described in this paper at the age of 4

months (#1), 10 years and 4 months (#2), 5 years (#3), 1 year and 3 months (#7), 5

years and 6 months (#8), 1 year and 6 months (#9) and patient described by Wilson

et al. (Wilson et al. 2008) at the age of 4 years and 2 months (#A) and 8 years (#B).

Frontal view showing high front hairline, broad forehead, epicanyhal folds,

boulbouse nose and pointed chin. (B) Picture of small and slim hand of patient 2 (#2)

showing tapered fingers. Pictures of chubby hand and feet of patient 7 (#7). Picture

of ear of patient 7 (#7) showing large lobe.

#1 #2 #3

#7 #8 #9 #B

A

B

#2 #7 #7 #7

#A

Fig.2 Localization of overlapping deletion detected in Patients 1-10 and deleted genes on chromosome 22q13.2q13.33. The minimal deletion overlap is marked by a yellow box. Array data were uploaded into UCSC Genome Browser, GRCh 37, hg 19 (http:/www.genome browser.ucsc.edu)

Patient 1Patient 2Patient 3Patient 4Patient 5Patient 6Patient 7Patient 8Patient 9Patient APatient B/BX

Fig. 3 Image that illustrate the genomic rearrangements involving chromosomal region 22q13 classified in four group:

(A) Interstitial deletions that not involve SHANK3 gene and that include the minimal deleted region.

(B) Terminal deletions that include the minimal deleted region

(C) Terminal deletions that not include the minimal deleted region

(D) Intragenic deletions and chromosomal aberrations disrupting SHANK3 gene

The minimal deleted region is marked by yellow segments.

Table I Summary of clinical manifestations of patients with 22q13 deletion (for patients 1-9 and patients A, B and BX described by Wilson et al., 2008)

Phenotypic features Patient 1

Patient 2

Patient 3

Patient 4

Patient 5

Patient 6

Patient 7

Patient 8

Patient 9

Patient A

(Wilson et

al. 2008)

Patient B

(Wilson et

al. 2008)

Patient BX

(Wilson et

al. 208)

Developmental Delay + + + + + + + + + + + -

Speech delay NA + + + + + + + + + + +

Hypotonia + - + - + + + + + + + NK

Overgrowth + - - + - NK + - + + + NK

Macrocephaly + - - + + + - - + + + +

Neurological scan

abnormalities

+ + + - - + ND ND + + + ND

Seizures - - - - - - + - - - + -

Kidney defects - - + - - - - - + - - -

Abnormal toenails - - - - - - - - - - - -

Autism NA + - - + NA NA - NA NK NK NK

Feeding problems + + + - + - + + - - + -

(NA, Not Assessed; ND, Not Done; NK, not Known)

Table II . Details of the Microarray-Platforms Used, Other Cytogenetic and Molecular Genetics Methods Applied in Evaluation of the Families, Deletion Size, and Heritability. Patient ID

Decipher / ID Database of Human CNVs

Type of Array Flanking locus proximal to the deletion (non-deleted)

First proximal deleted locus

Last distal deleted locus

Flanking locus distal to the deletion (non-deleted)

Minimal Deletion size (Mb)

Additional not polymorphic Aberrations

Confirmed by Method of investigation (relatives investigated)

Inheritance

1 261452a Agilent Human Genome CGH Microarray 44k

42170084 42204882 48139205 48224528 5.93 Agilent Human Genome CGH Microarray 44k

Agilent Human Genome CGH Microarray 44k

De Novo

2

251299a Genome-Wide Human SNP Array 6.0 (Affimetrix)

41822793 41834214 45630709 45643694 3.80 FISH: probe RP3-526I14 BAC probe

FISH: probe RP3-526I14 BAC probe

De Novo

3

248768a Agilent Human Genome CGH Microarray 244k

43344370 43355693 49933743 49939664 6.578 FISH: probes RP1-102D24; RP3-439F8; CTA-299D3; CTA-722E9

FISH: probes RP1-102D24; RP3-439F8; CTA-299D3; CTA-722E9

De Novo

4

257430a Agilent Human Genome CGH Microarray ISCA 180K

42141788 42160270 45160369 45193119 3 FISH: RP11-236I15

FISH: RP11-236I15

De Novo

5

250716a Cytochip_V3.0 Unknown 42176139 47543953 Uknown 5.368 Unknown Unknown De Novo

6 250049a Agilent Human Genome CGH Microarray 44k

42663297 42776398 48312768 5.536 FISH: probe N85A3 (BAC probe)

FISH: probe N85A3 (BAC probe)

De Novo

7 14955b Agilent Human Genome CGH Microarray 60kc

44154742 44223471 46928267 46988921 2.705 MLPA Agilent Human Genome CGH Microarray 60k (father). Mother not available

Unknown

8 263478a / 11530b

Agilent Human Genome CGH Microarray 44k

43670702 43709818 50692268 50706908 6.982 FISH: probes RP1-50E2; RP11-94B12

FISH: probes RP1-50E2; RP11-94B12

De Novo

9 16758b Human-OmniExpress SNP array analysis (Illumina)c

43701918 43704364 49832693 49841479 6.128 arr19p13.11q13.12 (16,790,859-36,722,832)x3; arr22q13.1 (37,799,188-38,621,456)x3

NO Human-OmniExpress SNP array analysis (Illumina)

De Novo

Methods and results of array-CGH investigations a Patients that are registered in the Decipher Database, http://decipher.sanger.ac.uk b Patients that are registered in the Database of Human CNVs, http://gvarianti.homelinux.net/gvarianti/index.php (User: Guest; Pasword: Guest) c First diagnosis was by conventional karyotyping

Tab. III Relative frequency of clinical features correlated with four gro

ical features correlated with four groups of genomic rearrangements illustrated in fig. 3

ups of genomic rearrangements illustrated in fig. 3

SUPPLEMENTARY MATERIAL.

Additional findings in patient 5

The karyotype ananlysis was performed on patient 7 revealing a pericentric inversion

of chromosome 5. The proband karyotype was interpreted as

46,XYinv(5)(p15.1q11.2) and the same inversion was present in the proband’s

father. In the literature is described one family in whom a pericentric inversion of

chromosome 5 [inv(5)(p15.1q11.2)], with the same apparently breakpoints in both

arm, segregates with benign familial neonatal convulsion (BFNC) (Concolino et al.,

2002). Patient 7 manifested 3 seizures episodes during early infancy, but his father

didn’t elicit this symptoms during the infancy. Given the absence of segregation of

seizures episodes in the proband’s family and the impossibility to compare

breakpoints of pericentric inversion of chromosome 5, identified in our proband and

in the family described by Concolino et al. (2002), it is difficult to demonstrate if this

symptom is caused by this structural rearrangement.

Additional findings in patient 9

In patient 9, in addition to the copy number loss on 22q13.2→q13.33, a copy number

gain on 22q13.1 (820Kb) and a ring chromosome 19 (47,XX +r[19]) were identified.

Array-CGH analysis conducted on DNA of relative parents revealed that these

aberration occurred de novo The duplication involving chromosome region 22q13.1

spans 820Kb and encompasses 26 genes, 18 of them are reported in OMIM database

(Table III). In the literature are not present data about patients that carried

duplication in this region.

Chromosome 19 supernumerary rings (sSCR19s) are very rare and most of those

described so far have not been fully molecularly characterized. Almost all cases

reported of sSMC19 can be found on the Thomas Liehr’s website

(http://www.fish.uniklinikum-

jena.de/sSMC/sSMC+by+chromosome/sSMC+19.html#Symptoms). Of these

patients 15, collected in the table IV, were molecularly defined through array-CGH

and in 3 of these the clinical information were not reported because diagnosed during

the pregnancy (Patients 19-U-14; 19-U-17; 19-U-18). Seven out of twelve remaining

patients were suitable for comparison with our patient with regard to their genetic

content, but not with regard to the structure of the sSMC. In fact four concern min

and not ring chromosome. We excluded from this study five patients because carried

additional rearrangements (patient 19-O-p13.11/4-1) or complex chromosomal

rearrangements (patients 19-Npt13.2/1-1; 19-Wp11/3-1; 19-U-15); moreover in one

patient (19-CW-5) the breakpoints of duplication were not specifically described.

Six patients (19-O-p13.11/3-1; 19-Wp12/2-1; 19-Wp12/4-1; 19-Wp12/5-3; 19-

Wp11/1-2; 19-Wp12/5-1) carried duplications derived from material within the

19p13.11q13.1 that are completely included within the duplication region seen in our

patient, while 1 patient (19-Wp12/6-1) have a duplication that is larger and that

overlap with the region seen duplicated in our proband. The clinical features that

these patients showed include developmental delay (5/6); Asperger syndrome (1/6),

tall stature (1/6) and brain malformation(1/6). A review of literature data about

duplications and deletions involving chromosomal regions 19p13.11→19p12 and

19q12→19q13.12 revealed that only few cases are described with partial trisomy 19p

and 19q. Duplications involving both arms of chromosome are more larger than

duplications identified in our proband. The phenotype associated with partial trisomy

19p is characterized by dysmorphic features, severe mental retardation, abnormalities

of brain morphology and anomalies in the fingers (Novelli et al., 2005; Vraneković et

al., 2008). The phenotype associated with partial trisomy of 19q is variable among

cases and includes facial dysmorphism, growth and mental retardation,

macrocephaly, hearth malformation and anomalies of the genitourinary tracts (Quack

et al., 1992; Qorri et al., 2002; Zung et al., 2007; Palomares Bralo et al., 2008;

Davidsson et al., 2009; Hall et al., 2010; Wilson et al., 2012).

Given the phenotypic variability in patient carried supernumerary ring chromosome

and the data in the literature about larger deletion and duplication involving

chromosome 19p and 19q we suggest that our patient’s phenotype is unrelated to the

presence of the supernumerary ring chromosome 19 but dependent on the 22q13

interstitial deletion. Nevertheless we don’t exclude the possibility that the phenotype

of our patient is the result of concurrent presence of 22q13 interstitial deletion and

duplications involving chromosome regions 22q13.1 and 19p13.11→q13.12.

Table III. Omim associated genes included in chromosomal region 22q13(37,799,188-38,621,456)

OMIM # Gene Function Associated Disease (OMIM #)

Inheritance/ Molecular Basis

602577 MFNG

This gene is a member of the fringe gene family which also includes radical and lunatic fringe genes. They all encode evolutionarily conserved secreted proteins that act in the Notch receptor pathway to demarcate boundaries during embryonic development. While their genomic structure is distinct from other glycosyltransferases, fringe proteins have a fucose-specific beta-1,3-N-acetylglucosaminyltransferase activity that leads to elongation of O-linked fucose residues on Notch, which alters Notch signaling

607209 CARD10 The caspase recruitment domain (CARD) is a protein module that consists of 6 or 7 antiparallel alpha helices. It participates in apoptosis signaling through highly specific protein-protein homophilic interactions. Like several other CARD proteins, CARD10 belongs to the membrane-associated guanylate kinase (MAGUK) family and activates NF-kappa-B (NFKB) through BCL10

606084 CDC42EP1 CDC42 is a member of the Rho GTPase family that regulates multiple cellular activities, including actin polymerization. The protein encoded by this gene is a CDC42 binding protein that mediates actin cytoskeleton reorganization at the plasma membrane. This protein is secreted and is primarily found in bone marrow.

150571 LGALS2 The protein encoded by this gene is a soluble beta-galactoside binding lectin. The encoded protein is found as a homodimer and can bind to lymphotoxin-alpha. A single nucleotide polymorphism in an intron of this gene can alter the transcriptional level of the protein, with a resultant increased risk of myocardial infarction.

Susceptibility to Myocardial infarction (608446)

606004 GGA1 This gene encodes a member of the Golgi-localized, gamma adaptin ear-containing, ARF-binding (GGA) protein family. Members of this family are ubiquitous coat proteins that regulate the trafficking of proteins between the trans-Golgi network and the lysosome. These proteins share an amino-terminal VHS domain which mediates sorting of the mannose 6-phosphate receptors at the trans-Golgi network. They also contain a carboxy-terminal region with homology to the ear domain of gamma-adaptins. Multiple alternatively spliced transcript variants encoding different isoforms have been found for this gene.

609246 PDXP Pyridoxal 5-prime-phosphate (PLP) is the active form of vitamin B6 that acts as a coenzyme in maintaining biochemical homeostasis. The preferred degradation route from PLP to 4-pyridoxic acid involves the dephosphorylation of PLP by PDXP

150570 LGALS1 The galectins are a family of beta-galactoside-binding proteins implicated in

modulating cell-cell and cell-matrix interactions. This gene product may act as an autocrine negative growth factor that regulates cell proliferation.

609761 TRIOBP This gene encodes a protein with an N-terminal pleckstrin homology domain and a C-terminal coiled-coil region. The protein interacts with trio, which is involved with neural tissue development and controlling actin cytoskeleton organization, cell motility and cell growth. The protein also associates with F-actin and stabilizes F-actin structures. ively spliced transcript variants that would encode different isoforms have been found for this gene, however some transcripts may be subject to nonsense-mediated decay (NMD).

Deafness, autosomal recessive 28 (#609823)

Autosomal Recessive

142708 H1F0 H1F0 is a replacement H1 histone whose transcription is independent of DNA replication. Unlike the transcripts for replication-dependent histones, transcripts for replacement histones, including that for H1F0, are polyadenylated.

607422 GCAT GCAT (EC 2.3.1.29) is the second mitochondrial enzyme in a 2-step reaction that converts L-threonine to glycine.

603692 GALR3 The neuropeptide galanin modulates a variety of physiologic processes including cognition/memory, sensory/pain processing, hormone secretion, and feeding behavior. The human galanin receptors are G protein-coupled receptors that functionally couple to their intracellular effector through distinct signaling pathways.

13383 ANKRD54 Homo sapiens ankyrin repeat domain 54

604414 POLR2F This gene encodes the sixth largest subunit of RNA polymerase II, the polymerase responsible for synthesizing messenger RNA in eukaryotes, that is also shared by the other two DNA-directed RNA polymerases. In yeast, this polymerase subunit, in combination with at least two other subunits, forms a structure that stabilizes the transcribing polymerase on the DNA template.

602229 SOX10 This gene encodes a member of the SOX (SRY-related HMG-box) family of transcription factors involved in the regulation of embryonic development and in the determination of the cell fate. The encoded protein may act as a transcriptional activator after forming a protein complex with other proteins. This protein acts as a nucleocytoplasmic shuttle protein and is important for neural crest and peripheral nervous system development.

PCWH syndrome (#609136)

Autosominal Dominant / Loss of function

Waardenburg syndrome, type 2E, with or without neurologic involvement (#611584)

Autosominal Dominant / Loss of function

Waardenburg syndrome, type 4C (#613266)

Autosominal Dominant / Loss of function

605926 PICK1 The protein encoded by this gene contains a PDZ domain, through which it interacts with protein kinase C, alpha (PRKCA). This protein may function as an adaptor that binds to and organizes the subcellular localization of a variety of membrane proteins. It has been shown to interact with multiple glutamate receptor subtypes, monoamine plasma membrane transporters, as well as non-voltage gated sodium channels, and may target PRKCA to these membrane proteins and thus regulate their distribution and function. This protein has also been found to act as an anchoring protein that specifically targets PRKCA to mitochondria in a ligand-specific manner.

610409 SLC16A8 SLC16A8 is a member of a family of proton-coupled monocarboxylate transporters that mediate lactate transport across cell membranes

603604 PLA2G6 The protein encoded by this gene is an A2 phospholipase, a class of enzyme that catalyzes the release of fatty acids from phospholipids. The encoded protein may play a role in phospholipid remodelling, arachidonic acid release, leukotriene and prostaglandin synthesis, fas-mediated apoptosis, and transmembrane ion flux in glucose-stimulated B-cells.

Infantile neuroaxonal dystrophy 1 (#256600)

Autosomal recessive

Karak syndrome (#610217)

Autosomal recessive

Neurodegeneration with brain iron accumulation 2B (#610217)

Autosomal recessive

Parkinson disease 14 (#612953)

Autosomal recessive

604877 MAFF The protein encoded by this gene is a basic leucine zipper (bZIP) transcription factor that lacks a transactivation domain. It is known to bind the US-2 DNA element in the promoter of the oxytocin receptor (OTR) gene and most likely heterodimerizes with other leucine zipper-containing proteins to enhance expression of the OTR gene during term pregnancy. The encoded protein can also form homodimers, and since it lacks a transactivation domain, the homodimer may act as a repressor of transcription. This gene may also be involved in the cellular stress response. Multiple transcript variants encoding two different isoforms have been found for this gene.

Table IV. Patients with Small Supernumerary Marker Chromosomes 19 (http://www.fish.uniklinikum-jena.de/sSMC/sSMC+by+chromosome/sSMC+19.html#Symptoms) Case no. Liehr’s Database

age at diagnosis

De Novo / Inherited

GTG-Banding result grade of mosaicism

Final results of the sSMC Test methods Clinical Symptoms Reference

19-U-14 prenatal n.a. 47,+mar[60%]/ 46[40%]

min(19)(:p12→q13.1:) aCGH: 24.08-40.50

wcp19; cep19, subcenM; aCGH

n.a.

19-U-17 prenatal

n.a. 47,+mar[100%] min(19)(:p12→q12:) aCGH: 33.10-33.59

cenM; subcenM; array-CGH

amniocentesis due to advanced maternal age; no US abnormalities. No further info available

19-U-18 prenatal de novo 47,XY,+mar[56]/ 46,XY[47]

mar(19)(p1? 2→q13.11) aCGH: 32.54-37.60

aCGH normal sonography, TOP

Zagorac A (Abstract)

19-O-p13.11/4-1 43y n.a. 47,XX,der(7)inv(7) (p22q36)inv(7) (q31.2q36),+mar [15]/ 46,XX,der(7)inv(7) (p22q36)inv(7) (q31.2q36)[67]

r(19)(:p13.1→q11:) locus spec. probes, array-CGH

normal female, detected due to clin. abnormal daughter

Argiropoulos B

19-Npt13.2/1-1 11m n.a. 47,+mar[30%]/ 46[70%] acc. to FISH

?inv dup(19) (pter→q13.2: :q13.2→pter)

aCGH; RP11- 19I2

developmental delay, short stature

Bruno DL

19-Wp11/3-1 postnatal de novo 47,XX,+mar[30] der(19) (:p11→q11::q12→q13.2: :q13.2→q13.31:) aCGH: 30.74-36.12 and 42.89-43.92

aCGH autsim, DD, dysmorphic face, obesity, friendly personality

Reddy KS

19-U-15 postnatal n.a. 47,XX,+mar[?%] der(19)t(18;19) array-CGH psychomotor disorder Kulikowski L (Poster)

19-CW-5 postnatal n.a. 47XX,+mar[?%] min(19) 44.9 MB size

array-CGH ophthalmologic disease Kulikowski L

19-O-p13.11/3-1 28y De novo 47, XX + mar[60%]/ 46,XX[40%]

Min(19)(:p13.11→q11:)[3]/ r(19)(::p13.11→q11::) [9] / r(19)(::p13.11→q11::p13.11→q11::) [2] FISH data: RP11-22G10 (22.98MB) on sSMC aCGH:17.50-33.59

Midi; MCB; subcenM; array-CGH

Normal Female

19-Wp12/2-1 3 m n.a. 47,XY,+mar[60- 90%]/ 46,XY[40-10%]

min(19)(:p12→q12:) size in p 2.59MB (= position 25.91 MB) , in q 2.3 MB (= position 32.4MB)

MCB; subcenM midi, array- CGH (Agilent 244k-chip)

Born in week 31 spontaneously, weight 2060g (93centile), length 42,5cm (58 centile), OFC 31.5 cm (92 centile) , APGAR 7/8/8 hyperbilirubinamia, subluxation of hip joint, Pes calcaneovalgus congenitus, periodic breathing, stridor cong, feeding problems. at 3 months: extreme restlessness, nearly opstotone posture; OFC now at 10. centile; at 5m: hyperexitability, developmental delay

Caliebe A (Poster)

19-Wp12/4-1 15y

de novo 47,XX,+r[100%] r(19)(::p12→q12::) aCGH: 23.12-33.29MB

M-FISH, diff. FISH-probes, aCGH

As newborn child seemed normal. Later in childhood, developmental delay, particularly of language skills. At 15y height >95th centile, high forehead, down-slanting palpebral fissures, wide diastema between upper incisors, high palate, short fraenulum of the upper lip, prominent lips and scoliosis, long and tapering fingers, fifth finger campodactyly, hallux valgus, fifth toe clinodactyly, pes planus. Overall neuropsychological picture: borderline intelligence with specific cognitive deficits in the processing of verbal information as well as learning disorders. Brain magnetic resonance imaging: enlarged cisterna magna with slight hypoplasia of the basal part of the cerebellar hemispheres as well as of the inferior vermis; the posterior fossa was normal.

Melis D

19-Wp12/5-3 4y 10m n.a. 47,XY,+mar[44- 60%]/46,XY[54- 40%]

mar(19)(:p12→q13.11:) positions: 19.80 to 38.21

aCGH normal pregnancy apart from oligoamnion in last month. weight length and APGAR normal at birth; Asperger syndrome = autism; mild hypotonia; milestones in development delayed

Faucz FR

19-Wp11/1-2 2y de novo 47,+mar[100%] min(19)(:p11→q13.11:) * size ~0.4MB

n.a.; subcenM with 3 BACs; array CGH

Metopic craniosynostosis; ventricular septal defect (VSD); strabismus; significant developmental delay; not rolling or sitting at 7 months of age; “not talking” at 27 months of age.

Baldwin EL

19-Wp12/5-1 13y

de novo 47,XY,+mar[23]/ 46,XY[7]

min(19) (:p12→q13.11~13.12:) size in p: RP11-22G10 in subcenM present on sSMC (22.98 MB - no signal in p on array), in q according to array: sSMC goes to 36.3 MB

cep; wcp; subcenM; array-CGH

Normal pregnancy and birth. At birth weight 4080g, length 52cm, APGAR 9/10; OFC 37cm; sitting with 6 months, walking with 13 months, speaking with 3 years. Developmental delay noticed from ~5 years. Anxious and impulsive behavior,

19-Wp12/6-1

7y n.a. 47,XX,+r[3]/46,XX [7]

r(19)(::p12→q13.2::) aCGH: 23.73 to 43.47

different FISH -probes; aCGH

global developmental delay

Yu S

71

SUPPLEMENTARY MATERIAL BIBLIOGRAPHY Argiropoulos B, Carter M, Brierley K, Hare H, Bouchard A, Al-Hertani W, Ryan SR, Reid J, Basik M, McGowan-Jordan J, Graham GE. Discordant phenotypes in a mother and daughter with mosaic supernumerary ring chromosome 19 explained by a de novo 7q36.2 deletion and 7p22.1 duplication. Am J Med Genet A. 2011; 155:885-891. Baldwin EL , May LF, Justice AN, Martin CL, Ledbetter DH. Mechanisms and consequences of small supernumerary marker chromosomes: from Barbara McClintock to modern genetic-counseling issues. Am J Hum Genet. 2008 Feb;82(2):398-410. Bruno DL , White SM, Ganesamoorthy D, Burgess T, Butler K, Corrie S, Francis D, Hills L, Prabhakara K, Ngo C, Norris F, Oertel R, Pertile MD, Stark Z, Amor DJ, Slater HR. Pathogenic aberrations revealed exclusively by single nucleotide polymorphism (SNP) genotyping data in 5000 samples tested by molecular karyotyping. J Med Genet. 2011 Dec;48(12):831-839. Caliebe A, Husemeyer N, Martin-Subero J-I, Gesk S, Stefanova I, Gillessen-Kaesbach G, Bruhn K, Stephani U, Kautza M, Krüger G, Weimer J, Tönnies H, Siebert R. Oligonucleotide array-CGH in postnatal cytogenetics - Kiel experiences. MedGen 2008, Vol 20, p 92 (Abstractnr. P037 - information from poster).

Concolino D, Iembo MA, Rossi E, Giglio S, Coppola G, Miraglia Del Giudice E, Strisciuglio P. Familial pericentric inversion of chromosome 5 in a family with benign neonatal convulsions. J Med Genet. 2002 Mar;39(3):214-6.

Davidsson J, Jahnke K, Forsgren M, Collin A, Soller M. dup(19)(q12q13.2): array-based genotype-phenotype correlation of a new possibly obesity-related syndrome. Obesity (Silver Spring). 2010 Mar;18(3):580-7.

Faucz FR, Souza J, Filho AB, Sotomaior VS, Frantz E, Antoniuk S, Rosenfeld JA, Raskin S. Mosaic partial trisomy 19p12-q13.11 due to a small supernumerary marker chromosome: A locus associated with Asperger syndrome? Am J Med Genet A. 2011 Sep;155(9):2308-10. Hall CE , Cunningham JJ, Hislop RG, Berg JN. A boy with supernumerary mosaic trisomy 19q, involving 19q13.11-19q13.2, with macrocephaly, obesity and mild facial dysmorphism. Clin Dysmorphol. 2010 Oct;19(4):218-21. Kulikowski L , Jehee FMS, Pelegrino R, Rosolen DCB, Antonangelo L, Buratini MN, Smith MAC, Melaragno MI. Small Supernumerary Marker Chromosomes characterization elucidated by array techniques.ASHG 2009; abstract only online, information from poster.

Melis D, Genesio R, Del Giudice E, Taurisano R, Mormile A, D'Elia F, Conti A, Imperati F, Andria G, Nitsch L. Selective cognitive impairment and tall stature due to chromosome 19 supernumerary ring. Clin Dysmorphol. 2012 Jan;21(1):27-32. Novelli A, Ceccarini C, Bernardini L, Zuccarello D, Digilio MC, Mingarelli R, Dallapiccola B. Pure trisomy 19p syndrome in an infant with an extra ring chromosome. Cytogenet Genome Res. 2005;111(2):182-5. Palomares Bralo M, Delicado A, Lapunzina P, Velázquez Fragua R, Villa O, Angeles Mori M, Luisa de Torres M, Fernández L, Pérez Jurado LA, López Pajares I. Direct tandem duplication in chromosome 19q characterized by array CGH. Eur J Med Genet. 2008 May-Jun;51(3):257-63. Quack B, Van Roy N, Verschraegen-Spae MR, Klein F. Interstitial deletion and ring chromosome derived from 19q. Proximal 19q trisomy phenotype. Ann Genet. 1992;35(4):241-4. Qorri M , Oei P, Dockery H, McGaughran J. A rare case of a de novo dup(19q) associated with a mild phenotype. J Med Genet. 2002 Oct;39(10):E61. Reddy KS, Aradhya S, Meck J, Tiller G, Abboy S, Bass H. A systematic analysis of small supernumerary marker chromosomes using array CGH exposes unexpected complexity. Genet Med. 2012 Aug 30. Vraneković J, Brajenović-Mili ć B, Modrusan-Mozetić Z, Babić I, Kapović M. Severe psychomotor retardation in a boy with a small supernumerary marker chromosome 19p. Cytogenet Genome Res. 2008;121(3-4):298-301. Wilson BT, Newby R, Watts K, Hellens SW, Zwolinski SA, Splitt MP. A case of mosaic trisomy 19q12-q13.2 with high BMI, macrocephaly, and speech delay: does USF2 determine size in the 19q phenotypes? Clin Dysmorphol. 2012 Jan;21(1):33-6. Yu S, Fiedler SD, Brawner SJ, Joyce JM, Zhou XG, Liu HY. Characterizing small supernumerary marker chromosomes with combination of multiple techniques. Cytogenet Genome Res. 2011 Nov 23. Zagorac A, Ogrizek Pelkic K, Kokalj Vokac N. Array-CGH identification of de novo mosaic supernumerary marker chromosome 19 in prenatal diagnosis. Europ J Hum Genet 20 (Suppl. 1), p 149, Abstractnr. P05.33). Zung A, Rienstein S, Rosensaft J, Aviram-Goldring A, Zadik Z. Proximal 19q trisomy: a new syndrome of morbid obesity and mental retardation. Horm Res. 2007;67(3):105-10.

5. DISCUSSION

Microarray based comparative genomic hybridization (array-CGH) has

revolutionized clinical cytogenetics, as it provides a relative quickly methods to scan

the genome for gain and losses of chromosomal material with significantly higher

resolution. The wide application of array-CGH has remarkably improved the

detection of copy number variations and complex chromosome rearrangements

(Edelmann and Hirschhorn, 2009). These genomic changes were associated with a

range of neurodevelopmental disorders including ID, ASD, DD, schizophrenia,

bipolar disorder, epilepsy and attention deficit disorder (ADHD) (McCarthy et al.,

2009; Girirajan and Eichler 2010; Rosenfeld et al., 2010; Cooper et al., 2011;

Sanders et al., 2011). Because copy number variations are common in normal

individuals, determining the functional and clinical significance of rare copy number

variants in patients remains challenging. Large copy number variant dataset have

been generated through the adoption of whole-genome chromosome microarray

analysis in individuals with unexplained developmental disabilities. There are now

many published reports of the significant role of rare, de novo CNVs with major

phenotypic effects in various disease populations. In some cases, a specific CNV is

necessary and sufficient to result in a suite of characteristic features. These

“syndromic CNVs” most often result in patients with moderate to severe ID and are

by-and-large sporadic in origin. Contrasting with these “syndromic CNVs” are CNVs

which are more variable in their outcome and more likely to be inherited. Large-scale

control series are of particular value in assessing clinical relevance. These type of

studies can be used to objectively determine frequency of particular CNVs and

compare them between case and control populations (Cooper et al., 2011; Kaminsky

et al., 2011). An example of genome-wide study was conducted on cases carried

16p11.2 deletions and duplications. Both reciprocal rearrangemets have been

observed in multiple conditions with significant enrichment compared to healthy

controls (1%). The deletion is associated with more severe phenotypes, including

cases with dysmorphic features, and is strongly enriched in ID and autism with a

strong association to obesity. The reciprocal duplication is not associated with any

common dysmorphic features and is seen in a wider range of conditions, including

clinically underweight cases mirroring the obesity phenotype of the deletion (Walters

et al., 2010; Jacquemont et al., 2011; Sanders et al., 2011).

However, although such statistical analysis is possible for recurrent CNVs, where the

frequency is high this strategy is more difficult for rare and nonrecurrent CNVs

causing highly penetrant rare genetic disease. Therefore, other approach need to be

explored to address this class of CNVs. Collaborative international efforts to collect

results systematically from genomic microarray together with clinical information by

using database such as Database of Chromosomal Imbalance and Phenotype in

Human using Ensemble resource (DECIPHER) are needed to define the clinical

consequence of copy number variations. These database facilitate the careful

phenotypic and microarray data collection and improve clinical interpretation of

genomic array-CGH results from individuals with rare genetic disorders, leading to

better definition of phenotype as well as the characterization of new syndromes.

Thanks to collaborative efforts by multiple centers we collected patients carrying the

same structural variant. We described clinical and molecular data on a series of five

females with an Xq28 duplication including MECP2 gene expanding the spectrum of

the associated phenotype. We reported two unrelated patients with overlapping

terminal 6p25 deletions, who presented with distinctive brain abnormalities, further

delineating the spectrum of the neuroanatomic phenotype associated with alteration

of this genomic region. Finally, we described clinical and molecular data of nine

patients with overlapping interstitial deletion in 22q13 chromosome region not

involving SHANK3 gene, supporting the evidence for a new microdeletion syndrome.

Duplications leading to functional disomy of chromosome Xq28 are associated with

a distinct clinical phenotype in males, characterized by severe mental retardation,

infantile hypotonia, progressive neurological impairment, recurrent infections,

bladder dysfunction, and absent speech. Female patients with Xq28 duplications

including MECP2 are rare. Whereas the phenotype effect of a MECP2 duplication is

well documented in males, it is a comparatively rare cause of mental retardation in

females (Sanlaville et al., 2009; Ramocki et al., 2010; Sanlaville et al., 2005). In our

second study, we present clinical and molecular data on a series of five females with

an Xq28 duplication including the MECP2 gene. Moreover we compare these series

of patients with the previously reported case of small duplications in females.

Previously, it has been stated that female with a MECP2 duplication lack the typical

symptoms of affected boys, such as seizures, poor speech development, and severe

recurrent infections. In contrast to the previously reported series of affected females

our series includes a female patient with the typical symptoms of affected boys,

therefore expanding the phenotypic spectrum of small Xq28 duplications including

MECP2 in females.

Terminal deletion involving the short arm of chromosome 6 is a contiguous gene

deletion syndrome characterized by various anomalies including ocular anterior

segment dysgenesis reminiscent of Axenfeld-Rieger syndrome (ARS), hypertelorism,

down-slanting palpebral features, flat nasal bridge, congenital heart defects, Dandy-

Wolker malformation, hearing loss and developmental delay (Law et al., 1998;

Nishimura et al., 1998; Gould et al., 2004; LeCaignec et al., 2005; Martinez-Glez et

al., 2007; DeScipio et al., 2008; Martinet et al., 2008). The haploinsufficiency of

FOXC1 is a leading candidate for the clinical features of the syndrome (DeScipio

2007). In the second study, we describe two unrelated patients with 6p25 terminal

deletions that were molecularly characterized using array-CGH. Brain MRI scans in

both patients revealed multiple cystic areas within the white matter represented

mainly by prominent perivascular (VR) spaces. Instead many of the white matter

areas were composed by abnormalities of unclear origin probably represented by

hypomielination, demyelination or reactive astrogliosis due to injury. Additional

brain abnormalities were found in the two patients. Among previously reported

patients prominent perivascular spaces were mentioned in some cases (Gould et al.,

1997; Mears et al., 1998; Mirza et al., 2004; Lin et al., 2005; Chanda et al., 2008).

Dysmielination of the cerebral white matter has been recently reported with

microdeletion at 6p25, but for this report, no MRI images are available for

comparison (Kapoor et al., 2011). Since brain MRI scans have been performed only

in some patients carrying the 6p25 deletion , the contribution of anatomical changes

to phenotype has not been adequately defined yet. Our clinical reports contribute to

delineate the spectrum of developmental brain anomalies associated with terminal

6p25 deletions, which appears to be wide and also include periventricular heterotopia

and multifocal alterations of white matter.

Deletions involving the terminal distal long arm of chromosome 22q are associated

with Phelan-McDermid syndrome. Data on genotype-phenotype correlation propose

that SHANK3, a gene included within the minimal critical region, is responsible for

the major neurological features of this syndrome (Phelan and McDermid 2012). Here

we report the clinical and molecular data of nine patients with overlapping interstitial

deletions in 22q13 not involving SHANK3 and compare our cohort of patients with

other three previously reported cases. All deletions differ in breakpoints and vary in

size between 2.7 and 6.98 Mb, resulting in a minimal deletion overlap of ~950 Kb

including at least 12 genes. These patients share several clinical features common to

Phelan Mc-Dermid syndrome: developmental delay (11/12), speech delay (11/12),

hypotonia (9/12) and feeding problems (7/12). Moreover, 8 out of 12 patients show

macrocephaly, while normal occipitofrontal circumference, in combination with

overgrowth were observed in one patient. In the minimal deleted region, we

identified two candidate genes, SULT4A1 and PARVB, which could be associated in

our cohort of patients with neurological features and macrocephaly/overgrowth and

hypotonia, respectively. These genes could be considered in the clinical phenotype of

patients with larger terminal deletion of 22q13. Characterization of additional

patients with smaller interstitial deletions could be helpful to confirm this hypothesis

and to better define a genotype-phenotype correlation. In conclusion, we speculate

the existence of a new contiguous gene syndrome proximal to the smallest terminal

deletions in the 22q13 region.

6.CONCLUSIONS

The biggest challenge arising from the clinical application of genomic analysis

remains one of being able to accurately distinguish between causal (pathogenic) and

non causal (often termed “benign”) variation. Highly penetrant structural variants are

rare in population and are the cause of rare genomic disorder. In this study we

elucidated the clinical consequence of rare copy number variants involving

chromosomal regions Xq28, 6p25and 22q13. Through collaborative efforts with

other centers registered in DECIPHER database and Human Copy Number

Variations Database (HCNVs), we collected patients sharing the rearrangements

overlapping with that found in our series of patients.

Thanks to these international collaborations we collected clinical and molecular data

of patients harboring aberration in chromosomal regions with previously established

clinical significance.

This approach enabled us to describe clinical and molecular data on a series of five

females with an Xq28 duplication including MECP2 expanding the spectrum of the

associated phenotype. In contrast to the previously reported series of affected

females this study includes a female patients with the typical clinical manifestation

of affected boys, therefore expanding the phenotypic spectrum of small Xq28

duplications including MECP2 in females.

Moreover through collaboration with other centers we described two unrelated

patients with overlapping terminal 6p25 deletions, who presented with distinctive

brain abnormalities, further delineating the spectrum of the neuroanatomic phenotype

associated with alteration of this genomic region.

Finally by participating in collaborative investigations we collected clinical and

molecular data of nine patients with nonrecurrent CNV in 22q13 chromosome. This

study allowed us to define the minimal critical region and identify two candidate

genes that could be responsible of the clinical phenotype for this series of patients.

Moreover, since the deletions in our patients do not involve SHANK3 gene, we

supported the hypothesis of the existence of a new gene contiguous syndrome

proximal to smallest terminal deletions, in the 22q13 region.

In conclusion, the present study highlights the importance of the “genotype-first”

approach of array-CGH analysis in characterization of previously unrecognized rare

genomic disorders.

In addition, our results emphasize the utility and the importance to establish the

collaborative efforts in order to collect more cases sharing the same rearrangements

in order to evaluate the common clinical traits.

This approach could be useful to provide a prime powerful and valuable clinical tool

for the clinician to recognize emerging syndromes even before array-CGH analysis.

Through the identification of rare genomic imbalances responsible for the clinical

presentation in patients, clinicians can provide an accurate diagnosis, predict the

potential risk in the future and alter the clinical management in the patients and/or

their families.

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8. Curriculum Vitae

PERSONAL INFORMATION

First name/Surname Vittoria Disciglio

Place and date of birth Mola di Bari (Ba)-(Italy), 17th September 1981

Nationality Italian

E-mail disciglio@unisi.it

Phone Number +39 3937420269 / +39 3482665899

PRESENT POSITION

November 2008-November 2012: Scholarship for a 4 years PhD program in Oncology and Genetics. Biotechnology Department, University of Siena EDUCATION August 2009: Professional Qualification 07/2008: Master’s Degree in Biological Sciences (Health address), University of Bari (Italy) with marks 110/110 Thesis: “Cytogenetic and molecular aspects of Roberts syndrome ”. Tutor: Prof. Ginevra Guanti 10/2006: Bachelor’s Degree in Biological Sciences (Health address), University of Bari (Italy) with marks 102/110. Thesis: “Experession of SDH1p in S. Cerevisiae studied by “Epitope tagging”: effect of FLX1 gene deletion”. Tutor: Prof. Maria Barile

DESCRIPTION OF THE RESEARCH ACTIVITY 2008-today Medical Genetics Unit, Biotechnology Department, University of Siena (Prof. Alessandra Renieri). Research activity has been focused on the following topics: retinoblastoma, intellectual disability, multiple congenital anomalies, autism and Rett syndrome. Research activity is substantiated by 7 original publications (total impact factor = 46,959). Furthermore, research activity has been recognized in several congresses by oral presentations and posters. November 2008-February 2010: I contributed to clarify the molecular basis of retinoblastoma. In particular my research was aimed to establish the relationship between the type of RB1gene mutations and the phenotype of Retinoblastoma affected patients. I actively contributed to perform mutational screening of RB1 gene using DHPLC, sequencing and MLPA. Furthermore I actively contributed to perform Pyrosequencing assay in a case-control study of 111 Italian hereditary retinoblastoma patients in order to verify the role of two functional polymorphisms (p53 p.Arg72Pro and MDM2 309 SNP) in retinoblastoma predisposition (3-J Hum Genet. 2011 Sep). Moreover I have contributed to investigate a set of oncosuppressor genes methylation status and copy number changes using Methylation Specific Multiplex Ligation Probe Assay in 12 retinoblastoma tissue (5 bilateral and 7 unilateral) (5-Pathol Oncol Res. 2012 Jan 26). March 2010-Today From March 2010, my research project has been focused on the identification of the genetic causes in patients with intellectual disability, autism and multiple congenital anomalies. I contributed to perform array CGH analysis (Aglient platform) in order to identify copy number variations (CNVs) and correlate them with the specific phenotype. I learned to use the main bioinformatic databases (UCSC Genome Browser, Ensemble, GeneCards, Database of Genomic Variant, etc.). As molecular cytogenetist I contributed to insert data entry of patients in Decipher database (http://decipher.sanger.ac.uk/) and in Italian Database of Human CNVs (http://gvarianti.homelinux.net/gvarianti/index.php). I also actively contributed to analyse array-CGH results in two discordant pairs of Rett sisters and four additional discordant pairs of unrelated Rett girls matched by mutation type in order to identify phenotypic modifier genes/regions (1-J Hum Genet. 2011 Jul). Moreover I performed array-CGH analysis on DNA extracted from clones of induced pluripotent stem cells (iPSCs) derived from reprogrammed fibroblasts of two Rett patient carrying different CDKL5 mutations (2-Eur J Hum Genet. 2011 Dec). I participated in a collaborative study about gene dosage at the chromosome 16p11.2 locus supervised by Medical Genetic Unit of Lausanne (Switzerland). In particular I identified copy number variations (six deletions and one duplication) at 16p11.2 in our cohort of 632 patient with intellectual disability, multiple congenital anomalies

and autism. Moreover I contributed to collect and send clinical and molecular data of our patients with 16p11.2 deletion/duplication (4-Nature. 2011 Aug 31). Moreover I contribute to identify patients sharing a genomic rearrangements in order to define new syndrome and extend the spectrum of associate phenotype (6- Eur J Med Genet. 2012; 7- Am J Med Genet A. 2012). During this period I performed array-CGH in 18 cases of prenatal diagnosis in which were characterized fetal chromosome rearrangements, fetal chromosome marker or increased nuchal translucency (>4 mm) with normal karyotype. Specifically this technique was cunducted on DNA extracted from amniotic fluid and small villi fragments. I performed whole genome amplification (WGA) in cases in which DNA amount was insufficient. June 2012-August 2012: I have attended for three months the Division of Molecular Pediatrics of Centre Hospitalier Universitaire Vaudois (CHUV) Lausanne (Switzerland), directed by Professor Andrea Superti Furga. During this period I contribute to set up the library preparation protocol of Ion Torrent technology. Moreover I have performed mutational analysis of MAFB, IDH1, IDH2, and CMG2 genes using sequencing. I characterized the genomic alterations in one patient affected by TAR syndrome using array-CGH and sequencing of RBM8A gene. DIAGNOSTIC ACTIVITY Starting from 2008 I have been involved in molecular diagnosis at the Medical Genetics Unit (University Hospital of Siena) for the following diseases: Retinoblastoma: Mutation screening of RB1 gene using DHPLC, sequencing and MLPA in patients affected by sporadic and familial retinoblastoma. Intellectual Disability: array CGH analysis and MLPA. Prader-Willi/Angelman syndrome: MS-MLPA analysis in patients with Prader-Willi/Angelman phenotype. PUBLICATIONS

1) Artuso R, Papa FT, Grillo E, Mucciolo M, Yasui DH, Dunaway KW, Disciglio V, Mencarelli MA, Pollazzon M, Zappella M, Hayek G, Mari F, Renieri A, Lasalle JM, Ariani F. Investigation of modifier genes within copy number variations in Rett syndrome. J Hum Genet. 2011 Jul; 6(7):508-15. 2) Amenduni M, De Filippis R, Cheung AY, Disciglio V, Epistolato MC, Ariani F, Mari F, Mencarelli MA, Hayek Y, Renieri A, Ellis J, Meloni I. iPS cells to model CDKL5-related disorders. Eur J Hum Genet. 2011 Dec;19(12):1246-55.

3) Epistolato MC, Disciglio V, Livide G, Berchialla P, Mencarelli MA, Marozza A, Amenduni M, Hadjistilianou T, De Francesco S, Acquaviva A, Toti P, Cetta F, Ariani F, De Marchi M, Renieri A, Giachino D. p53 Arg72Pro and MDM2 309 SNPs in hereditary retinoblastoma. J Hum Genet. 2011 Sep;56(9):685-6 4) Jacquemont S, Reymond A, Zufferey F, Harewood L, Walters RG, Kutalik Z, Martinet D, Shen Y, Valsesia A, Beckmann ND, Thorleifsson G, Belfiore M, Bouquillon S, Campion D, de Leeuw N, de Vries BB, Esko T, Fernandez BA, Fernández-Aranda F, Fernández-Real JM, Gratacòs M, Guilmatre A, Hoyer J, Jarvelin MR, Kooy RF, Kurg A, Le Caignec C, Männik K, Platt OS, Sanlaville D, Van Haelst MM, Villatoro Gomez S, Walha F, Wu BL, Yu Y, Aboura A, Addor MC, Alembik Y, Antonarakis SE, Arveiler B, Barth M, Bednarek N, Béna F, Bergmann S, Beri M, Bernardini L, Blaumeiser B, Bonneau D, Bottani A, Boute O, Brunner HG, Cailley D, Callier P, Chiesa J, Chrast J, Coin L, Coutton C, Cuisset JM, Cuvellier JC, David A, de Freminville B, Delobel B, Delrue MA, Demeer B, Descamps D, Didelot G, Dieterich K, Disciglio V, Doco-Fenzy M, Drunat S, Duban-Bedu B, Dubourg C, El-Sayed Moustafa JS, Elliott P, Faas BH, Faivre L, Faudet A, Fellmann F, Ferrarini A, Fisher R, Flori E, Forer L, Gaillard D, Gerard M, Gieger C, Gimelli S, Gimelli G, Grabe HJ, Guichet A, Guillin O, Hartikainen AL, Heron D, Hippolyte L, Holder M, Homuth G, Isidor B, Jaillard S, Jaros Z, Jiménez-Murcia S, Helas GJ, Jonveaux P, Kaksonen S, Keren B, Kloss-Brandstätter A, Knoers NV, Koolen DA, Kroisel PM, Kronenberg F, Labalme A, Landais E, Lapi E, Layet V, Legallic S, Leheup B, Leube B, Lewis S, Lucas J, MacDermot KD, Magnusson P, Marshall C, Mathieu-Dramard M, McCarthy MI, Meitinger T, Mencarelli MA, Merla G, Moerman A, Mooser V, Morice-Picard F, Mucciolo M, Nauck M, Ndiaye NC, Nordgren A, Pasquier L, Petit F, Pfundt R, Plessis G, Rajcan-Separovic E, Ramelli GP, Rauch A, Ravazzolo R, Reis A, Renieri A, Richart C, Ried JS, Rieubland C, Roberts W, Roetzer KM, Rooryck C, Rossi M, Saemundsen E, Satre V, Schurmann C, Sigurdsson E, Stavropoulos DJ, Stefansson H, Tengström C, Thorsteinsdóttir U, Tinahones FJ, Touraine R, Vallée L, van Binsbergen E, Van der Aa N, Vincent-Delorme C, Visvikis-Siest S, Vollenweider P, Völzke H, Vulto-van Silfhout AT, Waeber G, Wallgren-Pettersson C, Witwicki RM, Zwolinksi S, Andrieux J, Estivill X, Gusella JF, Gustafsson O, Metspalu A, Scherer SW, Stefansson K, Blakemore AI, Beckmann JS, Froguel P. Mirror extreme BMI phenotypes associated with gene dosage at the chromosome 16p11.2 locus. Nature. 2011 Aug 31;478(7367):97-102. 5) Livide G, Epistolato MC, Amenduni M, Disciglio V, Marozza A, Mencarelli MA, Toti P, Lazzi S, Hadjistilianou T, De Francesco S, D'Ambrosio A, Renieri A, Ariani F. Epigenetic and Copy Number Variation Analysis in Retinoblastoma by MS-MLPA. Pathol Oncol Res. 2012 Jan 26.

6) Bijlsma EK, Collins A, Papa FT, Tejada MI, Wheeler P, Peeters EA, Gijsbers AC, van de Kamp JM, Kriek M, Losekoot M, Broekma AJ, Crolla JA, Pollazzon M, Mucciolo M, Katzaki E, Disciglio V, Ferreri MI, Marozza A, Mencarelli MA, Castagnini C, Dosa L, Ariani F, Mari F, Canitano R, Hayek G, Botella MP, Gener B, Mínguez M, Renieri A, Ruivenkamp CA. Xq28 duplications including MECP2 in

five females: Expanding the phenotype to severe mental retardation. Eur J Med Genet. 2012 Jun;55(6-7):404-13. Epub 2012 Mar 29.

7) Cellini E, Disciglio V, Novara F, Barkovich JA, Mencarelli MA, Hayek J, Renieri A, Zuffardi O, Guerrini R. Periventricular heterotopia with white matter abnormalities associated with 6p25 deletion. Am J Med Genet A. 2012 Jul;158A(7):1793-7

PARTECIPATION AT CONGRESSES PERSONALLY PRESENTED PLATFORM COMUNICATION:

Disciglio V., Mucciolo M., Mencarelli M.A., Castagnini C., Pollazzon M., Marozza A., Mari F., Renieri A. “Fenotipi opposti di BMI si associano al dosaggio genico della regione 16p11.2” XIV Congresso Nazionale della Società Italiana di Genetica Umana (SIGU), November 13-16, 2011-Milano Disciglio V., Mencarelli M.A., Mucciolo M., Ndoni N., Frullanti E., Marozza A., Di Marco C., Lo Rizzo C., Baldassari M., Massarelli A., Canocchi V., Anderlid B.M., Metcalfe K., Le Caignec C., David A., Fryer A., Boute O., Pecile V., Battini R., Novelli A., Fichera M., Romano C., Mari F., Renieri A. INTERSTITIAL 22q13 DELETIONS NOT INVOLVING SHANK3 GENE: A NEW CONTIGUOUS GENE SYNDROME? XV Congresso Nazionale della Società Italiana di Genetica Umana (SIGU), November 21-24, 2012-Sorrento

ABSTRACTS ACCEPTED AS PLATFORM COMMUNICATIONS:

Papa FT, Katzaki E, Disciglio V, Mucciolo M, Mencarelli MA, Uliana V, Pollazzon M, Marozza A, Bruccheri MG, Mari F, Renieri A. “Array-CGH analysis of two hundred patients with parents investigation and analysis of surrounding genes.” 5th International Meeting on Cryptic Chromosomal Rearrangements and Genes in Mental Retardation and Autism, April 17-18, 2009 - Troina (IT). Katzaki E, Papa FT, Disciglio V, Mucciolo M, Mencarelli MA, Uliana V, Pollazzon M, Marozza A, Bruccheri MG, Mari F, Renieri A. “Array-CGH analysis in a cohort of 200 patients: the importance of parents investigation and analysis of surrounding genes.” 5th International Decipher Symposium, Wellcome Trust Genome Campus, May 20-22, 2009 – Hinxton (UK). Disciglio V., Marozza A., Mucciolo M., Mencarelli M.A., Fichera M., Romano C., Anderlid B.M., Clayton-Smith J., Metcalfe K., David A., Le Caignec C., Tümer Z., Fryer A., Andrieux J., Novelli A., Pecile V., Mari F., Renieri A. “Evidence for a new gene contiguous syndrome at 22q13, not involving SHANK3 gene”. 7th International Meeting on CNVs and Genes in Intellectual Disability and Autism, Troina (Italy) April 13 and 14, 2012.

ABSTRACTS ACCEPTED AS POSTERS:

Papa FT, Katzaki E, Mucciolo M, Mencarelli MA, Uliana V, Pollazzon M, Marozza A, Bruccheri MG, Disciglio V, Ariani F, Meloni. “Array-CGH analysis to identify novel microdeletion/duplication syndromes and to extend the clinical phenotype associated with susceptibility regions.” 59th ASHG Annual Meeting, October 20-24, 2009 - Honolulu, Hawaii (USA)

Amenduni M, Papa FT. , Clark RD, Bruttini M, Disciglio V, Mencarelli MA, Epistolato MC, Toti P, Marozza A, Mari F, Hadjistilianou T, De Francesco S., Acquaviva A, Katzaki E, Mucciolo M, Ariani F, Renieri A. “Dissecting the 13q14 microdeletion syndrome to define the critical region for mental retardation.” 59th ASHG Annual Meeting, October 20-24, 2009 - Honolulu, Hawaii (USA) Mencarelli MA, Papa FT, Katzaki E, Mucciolo M, Disciglio V, Uliana V, Pollazzon M, Marozza A, Castagnini C, Dosa L, Bruccheri MG, Mari F, Renieri A. “Sindromi emergenti da microdelezioni.” XII Congresso Nazionale SIGU, November 8-11, 2009 – Torino (IT)

Papa FT, Mencarelli MA, Mucciolo M, Katzaki E, Disciglio V, Bruccheri MG, Hayek J, Weber RG, Mari F, Renieri A. “Sindrome emegente da microdelezione 14q32.31-qter: descizione di due nuovi casi e revisione della letteratura.” XII Congresso Nazionale SIGU, November 8-11, 2009 – Torino (IT) Disciglio V., Berchialla P., Giachino D., Amenduni M., Livide G., Bruttini M.,. Mencarelli M.A, Marozza A.,. Mari F, Ariani F., Renieri A., De Marchi M.. “Ruolo dei polimorfismi dei geni MDM2 (SNP309T>G) e TP53 (R72P) nella modulazione della variabilità fenotipica del retinoblastoma.” XIII Congresso Nazionale: Società Italiana di Genetica Umana, Ottobre 2010 Firenze Disciglio V, Berchialla P, Giachino D , Amenduni M, Livide G, Mencarelli MA, Marozza A, Mari F, Ariani F, Renieri A, De Marchi M. “MDM2 and p53 are modifier gene of retinoblastoma.” European Human Genetics Conference 2010 Gothenburg, Sweden - June 12 - 15, 2010

Mucciolo M., Disciglio V., Papa F.T., Hayek J., Dosa L., Castagnini C., Marozza A., Pollazzon M., Mencarelli M.A., Canitano R., Renieri A., Mari F.. “Microriarrangiamenti in 95 pazienti con diagnosi di spettro autistico.” XIII Congresso Nazionale: Società Italiana di Genetica Umana, Ottobre 2010 Firenze Mucciolo M, Disciglio V, Mencarelli M.A., Pollazzon M., Marozza A., Castagnini C., Dosa L. , Lo Rizzo C.,Di Marco C., Mari F., Renieri A. "Clinical signs, disease categories and CNVs.” 7th International DECIPHER Symposium, Hinxton (UK), May 23-25, 2011

Mucciolo M, Disciglio V, Mencarelli MA, Marozza A, Castagnini C, Dosa L, Lo Rizzo C, Di Marco C , Mari A, Renieri A. “Clinical signs, disease categories and CNVs.” XIV Congresso Nazionale SIGU, November 13-16, 2011 – Milano (MI). Boddi G., Tuccio A., Luccherini M., Salvi G., Disciglio V., Dosa L., Renieri A., Cioni M., Balestri P. “Deficit cognitivo e dimorfismi in un bambino affetto da sindrome di Dent: possibile ruolo patogenetico della duplicazione della regione 14q11.2” 68° Congresso Nazionale SIP (Società Italiana di Pediatria), 9-11 Maggio 2012 – Roma. Disciglio V., Fichera M., Ciccone R., Mucciolo M., Ndoni E., Fernandez Jaen A., Galesi O., Mari F., Zuffardi O., Romano C., Renieri A. ‘Mirror effects for Autism Spectrum Disorder due to gene dosage at 10q11.22 affecting GPRIN2 gene, a regulator of neurite outgrowth and PPYR1 gene involved in energy homeostasis.’ European Conference of Human Genetics 2012, June 23-26, 2012, Nürnberg, Germany ADDITIONAL SKILLS LANGUAGE Italian: excellent (native speaker) English: good (spoken and written) IT Microsoft Office, Adobe Photoshop, Corel-Draw.

Aknowledgments At the end of this wonderful and exciting experience called PhD, there are many people that I would like to thank… My special thank goes to Prof. Alessandra Renieri for giving me the possibility to realize this dream. These formative years have been for me an opportunity to grow up professionally, but also to grow up personally...Thanks for your guidance and support over the duration of this study… I would like to give a warm thank to my second co-supervisor Francesca M. for her excellent guidance, both technical and academic which built my passion for medical genetics… Many thanks to Francesca A. who always gave me precious help and suggestions especially during the first years of my study… A special thanks to all the seniors of this magnificent group that have always been the headlights in the dark moments… A special thank to Professor A. Superti-Furga, S. Unger and L. Bonafe for giving me the opportunity to live a research exciting experience in the laboratory of Lausanne… I would like to thanks Belinda for her constant teaching and for her assistance during these months. I would like to thanks several people that I met in laboratory of Lausanne for their friendship: Karol, Esra, Laureanne, Philippe and Sonia… A special thanks to my fisrt teachers that introduced me in the unexplored and magical land of array-CGH: Eleni and Filomena…I enjoyed working with you…I will always keep you in my hearth… A cheerful thank to my friend of array-CGH room…Mafalda…thanks for all these moments spent togheter…I will miss our scientific conversations on Friday afternoon… A special thanks to all the girls of the laboratory and also to few courageous Y chromosomes that I met since when I crossed the blu door…I will always keep all of you in my heart…thanks because you shared with me smiles, joys, work, satisfactions or dark moments…thanks because live with all of you has been a beautiful experience… Finally a special thank goes to Maria Elena…merely thank for your sincere friendship… Thanks to my family that always supported and encourage me…because of you I learned to follow the voice of the hearth… A lovely thank to my companion Antonio, always present to guide me in this beautiful and long journey called life…