Cytogenetic Studies in Children with Developmental Delay

The Egyptian Journal of Hospital Medicine (2008) Vol., 30: 104– 114

Cytogenetic Studies in Children with Developmental Delay

*Hassan S.A. El-Dawi, *El-Sayed G. Khedr, *Tarek A. Atia, **Hassan Ali,

and *Mostafa E. El-Sawy Departments of Histology* and Pediatrics**, Faculty of Medicine,

Al-Azhar University, Cairo

Abstract

Introduction: Developmental delay (DD) could be syndromic or non-syndromic, and

collectively it affects 10% of all children. There are numerous causes of DD that could be

genetical, hormonal and/or neurological. The frequency of defected chromosomal anomalies in

patients with DD is variable and estimates between 9% and 36%. However, the accurate

diagnosis needs further tests based on the information gather from parents and the findings on

physical examination. Objective: We aim to evaluate the pattern of chromosomal abnormalities in children with

non-syndromic DD, in order to detect the treatable cases, and offering an appropriate genetic

counseling.

Methodology: 50 children suffering from DD with or without mental retardation(MR) and/or congenital anomalies were subjected to the present study. Additionally, another 50

normally developed children were considered as control group. Peripheral blood samples were

collected, cultured, harvested, metaphase spread and then chromosomes were stained for G-banding using Trypsin-Giemsa technique. Chromosomes were analyzed, metaphase spreads

were captured, and karyotyping has been done.

Result: Seven cases (14%) out of the 50 affected children carried structural chromosomal rearrangements. Six (85.7%) out of the seven structural chromosomal abnormalities were

detected in autosomal chromosomes and one (14.3%) in sex chromosome. Surprisingly, we

have found a case (2%) carrying pericentric inversion of chromosome 3 within the normal

control group. Conclusions: Chromosomal studies are valuable in detecting such cases with DD.

Prenatal genetic diagnosis is of clinical importance to prevent and offer genetic counseling.

Additionally, small proportion of apparently normal population could carry some types of structural chromosomal anomalies.

Key words: developmental delay, mental retardation, congenital anomalies, chromosomal

anomalies.

Introduction

Development refers to how a child becomes able to do more complex things as

he gets older. Human development runs in

three parallel lines: physical, cognitive, and

behavioral, and so, any defect of one of these parameters could affect the normal

development. Developmental delay is

characterized by cognitive impairment or mental retardation (MR); growth retarda-

tion (intra-uterine or extra-uterine); and/or

behavior abnormalities. Developmental

delay (DD) shows slower rate of

development, in which a child exhibits a functional level below the norm for his/her

age (Leonard et al., 2002). Significant delay

in two or more of the developmental

domains; gross/fine motor, speech/lang-uage, cognition,

social/personal, and

activities of daily living; is defined as

Global Developmental Delay (GDD). The term GDD is usually reserved

for young

children (i.e., typically less than 5 years of

age), whereas the term mental retardation

(MR) is usually applied to older children

104


105

when IQ (intelligence quotient) testing is

more valid and reliable (Lichten, et al., 2007).

DD can be categorized as syndromic,

or nonsyndromic; and could or could not be

associated with dysmorphic features. However, DD/MR is considered a big

problem for family and community, where

affected child needs not only financial support, but also special educational and

medical services throughout life (Shevell,

2000). The etiology of DD/MR could be

genetics, metabolic, neurologic or others,

where genetic disorders represent the major

cause. Genetic disorders could be chromo-somal, monogenic or multifactorial; howe-

ver chromosomal abnormalities have been

documented as a single most common cause. The frequency of chromosome ano-

malies detected by karyotyping in patients

with DD/MR was variable and estimated between 9% and 36% (de Vries et al.

2001). Where as, 40% of patients with

sever MR, and up to 10% of patients with

mild MR are documented to have chromosomal abnor-malities. The

frequency could be higher than what we

expect when more accurate techniques are used in diagnosis such as high resolution

banding, FISH, M-FISH, and others

(Granzow et al., 2000).

The human genome is composed of ~25,000 -35,000 genes carried on

chromosomes, approximately 50% of

which are of paternal and 50% are of maternal origin. It is estimated that about

65% of human genes contribute to the

development of the nervous system. Therefore; gene copy number alteration due

to gene(s) and/or chromosome(s) deletion

or duplication, or an abnormal pattern of

allelic inheritance could affect neuronal development (Capone, 2001). On the other

hand, multifactorial disorders are variable

and cause the majority of birth defects e.g. diabetes, spina bifida, anencephaly, cleft lip

and cleft palate, clubfoot and congenital

heart defects (Leonard and Wen, 2002). However, chromosomal disorders

happened sporadically, in most cases the

parent's karyotype is normal, but during cell

division (gametogenesis) an error in segregation or recombination may occur.

Moreover, inherited unbalanced chromo-

somal rearrangements are responsible for a

large proportion of familial disorders (Gardiner and Sutherland, 2004).

Most cases of DD do not show

clinical signs suggesting a particular

chromosome abnormality, while in others there is a strong suspicion of underlying

abnormality. On the other hand, small

proportional of apparently normal individuals carry structural chromosomal

anomalies, which sometimes runs in family

throughout several generations (familial benign chromosome abnormality). Such

cases are rare, and are only detected by

chance, so the accurate population rate is

unknown (Bourne et al., 2000; and Boyle and Cooper, 2001).

Screening of children with DD/MR

for genetic disorders (chromosomal or genes) is of great value especially to those

of unknown etiology. It will also help in

offering an appropriate counseling for those parents owing to minimize the number of

affected children.

Patients And Methods

50 children (27 girls and 23 boys)

suffering from DD with or without MR

and/or congenital anomalies were subjected to this study. Also, 50 apparently normally

developed children were used as control

group. Children were selected at the outpa-tient clinic of the Pediatric Department,

Neurological Unit, Al-Azhar University

Hospital, after their parent's consent. The

selection criteria were based on positive family history, perinatal history, dysmo-

rphic features, and neurological manifes-

tations. Moreover, we have excluded all other causes associated with known

syndromes and other neurological and/or

metabolic disorders.

Our experiment was designed as follow: -

(1) Children evaluation: Children were selected and diagnosed separately

through history, physical and clinical

examinations. The history included the family pedigree; family history, as will

as prenatal and natal history. The

examination includes; measurement of growth parameters (by using the

Hassan S.A. El-Dawi et al

106

percentile chart) and behavioral

observations. (2) Blood samples collection and setting up

blood cultures :addion of 0.4 ml of

heparinized blood to 8 ml of RPMI

1640 medium supplemented with L-glutamine, fetal calf serum, penici-

llin/streptomycin, and phytohaemagg-

lutinin. Cultured samples incubated at 37ºC for 72h, harvested cultured

samples, making slides, and then

staining chromosomes for G-banding using Trypsin-Giemsa technique

according to Fan,( 2002).

(3) Chromosome analysis at 400-500 band

per haploid set, captured metaphase spreads, and finally karyotyping was

prepared for the spreading metaphases.

At least, 10 metaphases were scored

for each case; two cells were captured and

karyotyped per case. All chromosomal abnormalities were recorded according to

the International System for Human

Cytogenetics Nomenclatures (ISHCN).

Result

We found that 7 cases (14%) out of

the 50 affected children carried chrom-osomal rearrangements; table (1)

summarizes all data about patients, their

karyotyping and their clinical presentations.

Six (85.7%) out of the seven structural chromosomal abnormalities were detected

in autosomal chromosomes and one

(14.3%) was detected in sex chromosomes. Twenty nine of the diseased children have

some congenital anomalies (especially in

face, hand, and feet), where five of them (24.1%) have structural chromosomal

abnormalities. Moreover, two cases (9.5%)

in the remaining twenty one diseased cases

were without dysmorphic features but carrying chromosomal anomalies.

We found ,in the control group a case

(2%) with pericentric inverted chromosome 3 in an apparently normal boy aged 6 years,

Unfortunately his parent, karyotyping was

not available.

Table I: List of the chromosomal rearrangements associated with clinical presentation and

phenotype in the current study.

Phenotype Karyotype Sex Age

(year)

Cases

MR with short stature

46, XX, del(x)(q12)

Female

13

Case 1

MR with multiple congenital anomalies and

dysmorphic features including cleft lip, and palate,

small hands and feet, short stature & congenital heart

disease.

46, XX, del(1)(q23;q25)

Female

2

Case 2

Mild MR and delay in motor function; she was not

able to walk until the age of 2.5 years.

46, XX, t(15;22)(q26;p12)

Female

5

Case 3

Severe DD with hypotonia at birth associated with

some dysmorphic features (low-set ear, microcephally, hypertelorism, and epicanthal folds).

46,XY, t(5;18)(p15;q21)

Male

2

Case 4

Growth retardation and mild delay of motor function

with slight dysmorphic features (midface hypoplasia, trigonocephaly, upward-slanting palpebral fissures,

and a long philtrum)

46,XY, t(1;9)(q43;p22)

Male

4

Case 5

Severe DD, hypotonia, epilepsy, dysmorphic features

including round face, and small head.

46,XY, ins(22)(q13)

Male

3

Case 6

MR with mild delay of motor function, dysmorphic

features including upslanding palpebral fissures, high

arched palate, small widely-spaced teeth, and

bilateral clinodactly.

46,XX, dup(16)(q11;q12)

Female

3

Case 7

Apparently Normal boy 46, XY, inv(3)(p14;q11) Male 6 Control


107

Case 1: 46, XX, del(x)(q12)

Case 2: 46, XX, del(1)(q23;q25)


108

Case 3: 46, XX, t(15;22)(q26;p12)

Case 4: 46,XY, t(5;18)(p15;q21)


109

Case 5: 46,XY, t(1;9)(q43;p22)

Case 6: 46,XY, ins(22)(q13)


110

Case 7: 46,XX, dup(16)(q11;q12)

Control Case: 46, XY, inv(3)(p14;q11)

Discussion

Evaluation of cases suffering

developmental delay and mental retardation (MCA/MR) with multiple congenital

anomalies is always a challenge to clinic-

ians, as routine chromosomal analysis is the starting point to investigate such cases.

Subsequent investigations might consider

achieving an accurate diagnosis. Usually, visible loss or gain of chromosome material

will lead to abnormal development,

resulting in a malformed phenotype, but in

a lesser instant they could show normal morphology. The morphologic defect is

greatly variable, and depends on the size of

chromosome lesion and gene content involved (McKinlay et al, 2004).

MR and congenital anomalies are

characteristics of many chromosome

anomalies. It had been suggested that the

size of deleted or duplicated segment may

have a direct effect on severity of the morphologic defect (McKinlay et al, 2004).

Our study showed that the percentage

of autosomal anomalies (~ 80%) was much higher than those of the sex chromosomes

(20%). This could be due to the fact that

sex chromosome defect has a much lesser deleterious effect on the phenotype than

autosomal anomalies do (Brown et al,

2004). In contrast to this chromosomal

study in neonates showed that autosomal chromosome anomalies are usually as

common as sex chromosome anomalies

(Gardner and Sutherland, 2004). Also, the numerical anomalies of sex chromosomes

are more common than the structural

anomalies (Schinzel, 2001). On the other

hand, several studies based on phenotypic


111

anomalies (Sungsoo et al., 1999; Clara et

al., 2005) are in agreement with the present results.

The present study reported chromo-

somal abnormalities in seven cases (14%)

out of 50 diseased children. In deed, our result could be similar, higher or lower than

those of other investigators. Berry (1995)

has reported a frequency of 15.8% out of 114 cases, Verma et al. (1980) reported a

frequency of 27% out of 357 cases, while

Singh (1977) has reported a frequency of 28.8% out of 451 patients. However; much

lower frequencies (1% to 6%) have been

reported in other studies (Kenue, 1995; and

Hook et al., 1977). The variable frequencies shown could contribute to the

size of the population sample, patient

selected criteria, and /or to the techniques used in investigation.

Our study showed that the frequency

of chromosomal anomalies was 9.5% out of 21 mentally retarded children without

dysmorphic features, but it was much

higher (24.1% out of 29) in cases associated

with dysmorphic features. Also the current investigation and others (Tetsuji et al.,

2003; Donnenfeld, et al., 2003) indicated

that chromosomal anomalies are frequently associated with multiple malformations and

MR. We found three patients had reciprocal

translocation, where two of them had

dysmorphic features and MR. Such translocations have been reported to be

harmless, but they are commonly seen in

mentally retarded individuals. Donnenfeld, et al., (2003) reported that abnormal

phenotype is usually uncommon with

balanced chromosomal translocations. But, some apparent cytogenetically balanced

translocations could be molecularly

unbalanced i.e. the break points could

interrupt gene/s resulting in haploin-sufficiency for the gene product in this

region, which in turn result in unexpected

phenotypic anomalies (Cohen et al., 2001; and Robert et al., 2006).

Moreover, we have found a case [case

3: 46, XX, t(15;22)(q26;p12)] with translo-cation between chromosomes 15 and 22,

which need further investigation like FISH

using whole chromosome paint (WCP)

probes to hybridize both chromos-omes to confirm this type of translocation. Also,

another case showed insertion of DNA

material to chromosome 22 [case 6:

46,XY,ins(22)(q13)] which need further investigation like M-FISH, or panel of

FISH probes to hybridize all chromosomes

in order to identify the origin of the extra-

DNA material.. Finally, several case reports have been

published since about 1980 of microsco-

pically visible euchromatic (G-Band negative) chromosome deletions, duplic-

ations, or inversion in individuals with an

apparently normal phenotype. Such cases are rare. Unfortunately, without an abnor-

mal phenotype, individuals are not usually

referred for karyotyping, and so cases are

only detected by chance. In the present investigation we detected a case carrying

chromosomal inversion but with apparently

normal development and phenotype. Chromosomal inversion could be balanced,

and usually is associated with normal

phenotype, but carriers of such anomaly are at risk of producing abnormal gametes that

may lead to unbalanced offspring.

Lindberg, et aI. (1992) has reported that

balanced familial pericentric inversion in chromosome 3 (p 14; q 11) with no adverse

effects and they have concluded that this

chromosome aberration could be an example of a harmless chromosome poly-

morphism. Awareness of such situations

without an abnormal phenotype is impo-

rtant for prenatal diagnosis/counselling; and reinforces the importance of parental

cytogenetic analysis to interpret an

abnormal karyotype, and estimate risks, to exclude the possibility of a familial benign

chromosome abnormality.

Conclusion

Chromosome studies are relatively expensive

thus; the consult of pediatricians,

neurologists, and dysmorphologists could

lead to exclude cases with single gene defects, and syndromes with known

non-

genetic etiology. This in turn will minimize

the need for standard karyotyping .Our

study concluded that chromosomal studies

are a valuable diagnostic technique to

evaluate cases with DD/MR. Investigating

parents for chromosomal abnormalities is important as the risk of inheritance is

usually high in such cases. It helps to

provide proper genetic counseling .


112

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دراسات كروموسومية بين االطفال ذوى النمو المتاخر

طارق عبد هللا, السيد جالل السيد خضر, حسن صبرى علي الضوى

حسن علي حسن و مصطفي السعيد الصاوى, قس اىسزىجيب طت االطفبه ثنييخ اىطت جبعخ االصش ثبىقبشح

اى اىزبخش اىظحة ثجد رخيف عقي خيو ف مو اى اىسيك قذ يجذا

قذ الرظش ا عالبد امييينيخ عي , جنشا ف اىجي زيجخ ىخيو ف اىجيبد اىساثيخ

مثيش االطفبه اىشض ثبىخيو اىنشس يز امزشبف زا اىخيو ثبىعذيذ

خزجبساد اىعييخ يجذ اىعذيذ االخزجبساد ىعو فحض جبع ىزح اىحبالد ثو االرحييو اىنشسبد اخزجبس اىساثخ اىخييخ اىجضيئيخ ثبسزخذا جس اىحبض

اى ىنشس عي مزاىل اخزجبس اىشبد االظبه ز االخزجبساد قذ رسبعذ

الد اىقبثي ىيعالج مزىل اعطبء اىظيحخ ىالثبء ىزقييو فشص ف اىزشخيض اىجنش ىيحب

مب اىذف زا اىجحث يظت عي رقيي اىنشسبد ف ؤالء . االطبثخ فيب ثعذ

االطفبه رحذيذ رج اىنشس اىساث ع طشيق فحض اىنشسبد االثي

ف اى فحض اشخبص طجيعيي ايضب اىنشسبد عبئالد اخش ظبث ثبالضب

مجعخ ضبثطخ قذ ر اخز اىافقخ ز اىعبئالد عي اجشاء ز اىفحطبد ر

اخز خسي االطفبه ر اى اىزبخش اثبئ عذد افشاد اىعبئيخ اخضبع مب ر اخز خسي االطفبه اىطجيعيي مجعخ ضبثطخ . ىزحييو مشس

ر اخز عيخ د مو شخض صسعب ف سظ خبص ث ر . خضبع ىفس اىزحبىيو ا

ث . جع اىخاليب ف شحيخ االقسب اىخي اىزسظ ضعب عي ششائح صجبجيخ

اخضعذ اىعيبد ىيفحض اىنشس ثبسزخذا اىزقيبد اىعبديخ ثعذ رششيظ

قذ ر عو اخزجبساد . طجغ ثظجغخ اىجيسباىنشسبد ثاسطخ اضي اىزشيجسي ث

ثفحض حبالد اىظبثي . ربميذيخ ىجعض اىعيبد اىز رحز عي خيو مشس عقذ

رجي جد رسعخ عششي طفال ثي اىخسي اىظبثي ثبىزخيف اىعقي يعب

ى رظش عيي ا رشبد خيقيخ ظبشح ثخالف اىاحذ اىعششي طفال اىجبقي اىزيثفحض اىحبالد اىزسعخ اىعششي رجي ا خس حبالد فقظ ث عية . رشبد ظبشح

رشميجيخ اضحخ ف اىنشسبد مب اظش فحض اىحبالد اىاحذ اىعششي االخش

رجي ايضب جة عيت رشميج . جد عية رشميجيخ ف اىنشسبد ف حبىزي فقظ

رط اىذساسخ ثفحض . ذ فقظ ف االطفبه اىخسي االطحبءربب ف حبى اح

اىنشسبد ف االطفبه ر اى اىزبخش اىغيش زالص ال رىل يفيذ ف اىزشخيض

اىجنش ىيحبالد اىقبثيخ ىيعالج ايضب يفيذ اعطبء اىظيحخ ىالثبء ب يؤد اى رقييو فشص

.االطبثخ ف االجيبه اىقبدخ

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Cytogenetic Studies in Children with Developmental Delay