Mutations in NOTCH1 Cause Adams-Oliver Syndrome

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  • REPORT

    Mutations in NOTCH1 Cause Adams-Oliver Syndrome

    Anna-Barbara Stittrich,1,9 Anna Lehman,2,9 Dale L. Bodian,3,9 Justin Ashworth,1 Zheyuan Zong,2

    Hong Li,1 Patricia Lam,2 Alina Khromykh,3 Ramaswamy K. Iyer,3 Joseph G. Vockley,3 Rajiv Baveja,4

    Ermelinda Santos Silva,5 Joanne Dixon,6 Eyby L. Leon,7 Benjamin D. Solomon,3,8 Gustavo Glusman,1

    John E. Niederhuber,3,10,* Jared C. Roach,1,10 and Millan S. Patel2,10,*

    Notch signaling determines and reinforces cell fate in bilaterally symmetric multicellular eukaryotes. Despite the involvement of Notch

    in many key developmental systems, human mutations in Notch signaling components have mainly been described in disorders with

    vascular and bone effects. Here, we report five heterozygous NOTCH1 variants in unrelated individuals with Adams-Oliver syndrome

    (AOS), a rare disease with major features of aplasia cutis of the scalp and terminal transverse limb defects. Using whole-genome

    sequencing in a cohort of 11 families lacking mutations in the four genes with known roles in AOS pathology (ARHGAP31, RBPJ,

    DOCK6, and EOGT), we found a heterozygous de novo 85 kb deletion spanning the NOTCH1 50 region and three coding variants(c.1285T>C [p.Cys429Arg], c.4487G>A [p.Cys1496Tyr], and c.5965G>A [p.Asp1989Asn]), two of which are de novo, in four unrelated

    probands. In a fifth family, we identified a heterozygous canonical splice-site variant (c.7431 G>T) in an affected father and daughter.These variants were not present in 5,077 in-house control genomes or in public databases. In keeping with the prominent develop-

    mental role described for Notch1 in mouse vasculature, we observed cardiac and multiple vascular defects in four of the five families.

    We propose that the limb and scalp defects might also be due to a vasculopathy in NOTCH1-related AOS. Our results suggest that

    mutations in NOTCH1 are the most common cause of AOS and add to a growing list of human diseases that have a vascular and/or

    bony component and are caused by alterations in the Notch signaling pathway.Adams-Oliver syndrome (AOS [MIM 100300]) is a rare

    developmental disorder with an incidence of approxi-

    mately 1 in 225,000 individuals and is defined by the

    combination of aplasia cutis congenita of the scalp vertex

    and terminal transverse limb defects (e.g., amputations,

    syndactyly, brachydactyly, or oligodactyly).1,2 In addition,

    vascular anomalies, such as cutis marmorata telangiecta-

    tica congenita, pulmonary hypertension, portal hyper-

    tension, and retinal hypovascularization, are recurrently

    observed.3 Congenital heart defects have been estimated

    to be present in 20% of individuals with AOS; reported

    malformations include ventricular septal defects, anoma-

    lies of the great arteries and their valves, and tetralogy

    of Fallot.46 Both familial and sporadic AOS occurrences

    have been described, and genetic heterogeneity is evident

    given that AOS has been shown to be caused by muta-

    tions in four different genes: heterozygous mutations

    in Rho GTPase activating protein 31 (ARHGAP31 [MIM

    610911]) or recombination signal binding protein for

    immunoglobulin kappa J region (RBPJ [MIM 147183]) or

    biallelic mutations in dedicator of cytokinesis 6 (DOCK6

    [MIM 614194]) or EGF-domain-specific O-linked N-

    acetylglucosamine transferase (EOGT [MIM 614789]).710

    Collectively, mutations in these four genes have not

    been shown to account for more than 10% of individuals

    with AOS.1Institute for Systems Biology, Seattle, WA 98109, USA; 2Department of Medi

    Columbia, Vancouver, BC V6H 3N1, Canada; 3Inova Translational Medicin

    Neonatal Associates, Inova Health System, Falls Church, VA 22042, USA;

    4050-111, Portugal; 6Genetic Services, Wellington Hospital, Capital & Coast D

    and Metabolism, Childrens National Medical Center, Washington, DC 20010

    System, Falls Church, VA 22042, USA9These authors contributed equally to this work10These authors contributed equally to this work

    *Correspondence: john.niederhuber@inova.org (J.E.N.), mpatel@cw.bc.ca (M.S

    http://dx.doi.org/10.1016/j.ajhg.2014.07.011. 2014 by The American Societ

    The AmericanWe identified heterozygous variants in NOTCH1 (MIM

    190198) as an additional cause of AOS in multiple families.

    Phenotypic analysis of affected individuals with NOTCH1

    mutations indicates a high rate of vascular anomalies

    (Table 1); in contrast, neither RBPJ nor ARHGAP31 muta-

    tions have yet been shown to associate with abnormal

    vascularization (Table S1, available online). Both of the

    known recessive genes, however, have shown such an

    association: homozygous EOGT mutations have been

    found in AOS individuals with septal defects, patent duc-

    tus arteriosus, and brain infarcts, and homozygous or com-

    pound-heterozygous DOCK6 mutations have been found

    in AOS individuals with congenital heart defects andmark-

    edly abnormal blood vessels (data not shown).7,9,11

    We used whole-genome sequencing (WGS) to analyze

    14 AOS-affected individuals from 12 unrelated families,

    11 of which are of European descent and one of which is

    of mixed European-Asian descent. In addition to analyzing

    the entire genome, we also screened for variants in genes

    with a known role in AOS and found one individual with

    compound-heterozygous mutations in DOCK6 (data not

    shown). Here, we report on five families in which we found

    mutations in NOTCH1 (Figure 2). Four of these families

    were recruited with informed consent through a study

    protocol (H08-02077) that was approved by the institu-

    tional review board (IRB) at the University of Britishcal Genetics and Child and Family Research Institute, University of British

    e Institute, Inova Health System, Falls Church, VA 22042, USA; 4Fairfax5Pediatric Gastroenterology Service, Centro Hospitalar do Porto, Porto

    istrict Health Board, Wellington 6242, New Zealand; 7Division of Genetics

    , USA; 8Department of Pediatrics, Inova Childrens Hospital, Inova Health

    .P.)

    y of Human Genetics. All rights reserved.

    Journal of Human Genetics 95, 275284, September 4, 2014 275

    mailto:john.niederhuber@inova.orgmailto:mpatel@cw.bc.cahttp://dx.doi.org/10.1016/j.ajhg.2014.07.011http://crossmark.crossref.org/dialog/?doi=10.1016/j.ajhg.2014.07.011&domain=pdf

  • Table 1. Clinical Characteristics of the AOS-Affected Individuals

    Clinical Characteristic

    Individual

    1-II-3 2-II-1 2-III-2 3-II-1 4-II-1 5-II-1

    Aplasia cutis of the scalp forme fruste

    Terminal transverse limb defects

    Cutis marmorata

    Intracranial vascular lesions

    Pulmonary hypertension

    Cardiac malformation narrow pulmonaryarteries

    ? ? pulmonary valve stenosis distal narrowingof the aortic arch

    ?

    Otherfeatures

    NA NA NA thrombosis of the portal veinand splenorenal shunt, spasticdiplegia, intellectual disability

    thrombosis of thesagittal sinus

    NA

    The following abbreviations are used: ?, not assessed; and NA, not applicable.Columbia. A fifth family was recruited at the Inova

    Translational Medicine Institute through an IRB-approved

    protocol with informed consent (20121680). The clinical

    phenotypes of affected individuals are summarized in

    Table 1.

    The proband of family 1 (1-II-3) has aplasia cutis conge-

    nita affecting the occiput and marked cutis marmorata.

    At birth, white vesicles (or areas of focal calcinosis cutis)

    were present at the tips of the fingers, which were other-

    wise well formed. The toenails were hypoplastic and

    dystrophic bilaterally (Figure 1), some vesicles were pre-

    sent, and subtle, semicircumferential constriction of the

    skin and soft tissue was present at the bases of several

    toes. An echocardiogram was normal apart from mild

    narrowing of the branch pulmonary arteries. Cardiac

    assessment was repeated in the first year, and there

    appeared to be no hemodynamic consequence to this

    finding. Renal ultrasound and brain MRI were normal.

    The vesicles on the hands and feet would occasionally

    rupture to release a thick chalky substance; this substance

    was radio-opaque, suggesting that it was of bony origin.

    Cutis marmorata persisted into early childhood, and at

    the last follow-up at age 6 years, height was on the tenth

    percentile, the branch pulmonary arteries were normal,

    the main pulmonary artery was dilated but stable for years,

    the vesicles or calcinosis cutis were persistent but nonerup-

    tive on the feet, and moderate micrognathia with a

    retruded tongue caused intermittent airway obstruction

    during sleep. The family history is negative for aplasia

    cutis and terminal transverse limb defects.

    In family 2, a father and daughter are affected by AOS.

    The daughter (2-III-1), age 20 months at enrollment, was

    born with severe aplasia cutis of the scalp (Figure 1), which

    was complicated by recurrent hemorrhage during a

    lengthy healing process. She has hypoplastic toes on the

    left foot and nail hypoplasia of the second and third

    toes. Neurodevelopmental milestones have been age

    appropriate. Her father (2-II-1) was also born with a

    cutaneous and bony defect affecting two-thirds of his276 The American Journal of Human Genetics 95, 275284, Septembcranium, brachydactyly of the right hand, and terminal

    transverse limb defects of both feet, including soft-tissue

    syndactyly of hypoplastic toes (Figure 1). Bony in-growth

    never fully bridged the cranial defect. There is no

    other family history of aplasia cutis or limb defects. Two

    maternal first cousins of the father reportedly had cardiac

    septal defects but were not available for enrollment.

    The clinical phenotype of 3-II-1 from family 3 was

    described in detail by Silva et al.12 In summary, this now

    14-year-old boy was affected by aplasia of the scalp, under-

    lying cranial defect, cutis marmorata, brachysyndactyly

    of the toes, hypoplastic fingernails, inguinal and umbilical

    hernias, mild pulmonary stenosis, and hypoplasia of

    the intrahepatic portal venous tree. He developed portal

    venous thrombosis in infancy, which led to symptomatic

    portal hypertension; a mesenteroportal shunt was placed,

    but this was also complicated by thrombosis. On the sec-

    ond day following this surgery, he suffered an ischemic

    stroke. Since then, no further thrombotic events have

    occurred. Brain imaging demonstrated evidence of water-

    shed infarctions, and he has mild intellectual disability

    and spastic diplegia. The family history is negative for evi-

    dence of AOS.

    The family 4 proband (4-II-1) was born at term with

    severe aplasia cutis affecting most of the scalp superior to

    the ears, as well as the posterior neck. She had bilateral

    prominent, tortuous scalp vessels, truncal cutis marmor-

    ata, and bilateral toe hypoplasia with absent toenails. Neu-

    roimaging (brainMRI and intracranial magnetic resonance

    angiogram and venogram) on day of life (DOL) 1 showed

    small focal areas of bilateral parietal and left frontal

    white-matter acute infarction; the superior sagittal sinus

    was patent but had a focal abnormality felt to be consistent

    with a partial superior sagittal sinus thrombosis with

    recanalization. Repeat neuroimaging 1 week later revealed

    evolving biparietal and left frontal lobe infarcts, near-

    complete sagittal sinus thrombosis, and biparietal cortical

    venous thromboses (Figure 1). Serial neuroimaging

    showed stabilization and improvement of the thromboseser 4, 2014

  • Figure 1. Clinical Vignettes for AOS Indi-viduals from Families 1, 2, and 4(AC) Family 1 proband (1-II-3): scarredaplasia cutis lesion affecting the scalp atage 5 years (A), calcific deposits in thesubcutaneous tissue of the distal firsttoe (arrow, B), and terminal transversedefect of the toes and cutis marmorata ininfancy (C).(DF) Family 2 proband (2-III-1): distalhypoplasia of the digits of the left hand(D) and toes of the feet (E) and aplasia cutisof the scalp (F).(G and H) Family 2 father of the proband(2-II-1): aplasia cutis of the scalp (G) andterminal transverse defects of both feet (H).(IK) Family 4 proband (4-II-1): MRI of thebrain on day of life (DOL) 9 shows areasof infarcts (solid arrows) and an area ofpartial thrombus (outlined arrow) fromaxial sections on diffusion weighted MRI(I and J) and axial T2-weighted MRI (K).over the next several months. She did not undergo anti-

    coagulation therapy, and there was no clinical or labora-

    tory-based evidence for thrombophilia. Echocardiograms

    showed pulmonary hypertension, estimated to be almost

    half of systemic pressure, on DOL 1, and mild mitral valve

    annulus hypoplasia (annulus diameter of 8.7 mm) with no

    stenosis on DOL 4. A trileaflet aortic valve with mild distal

    aortic arch narrowing felt to be of no hemodynamic conse-

    quence and multiperforate patent foramen ovale with

    insignificant shunting were observed on DOL 9, and the

    pulmonary hypertension resolved by DOL 10. No other

    congenital heart defects were detected. Renal ultrasound

    was normal except for mildly echogenic kidneys felt to

    be secondary to dehydration. The family pedigree, having

    both European and Asian ancestry, is negative for any

    similar anomalies.

    The clinical phenotype of the proband of family 5

    (5-II-1) at age 24 years has been previously described byThe American Journal of Human GenVandersteen and Dixon.13 She has a

    family history consistent with auto-

    somal-dominant AOS. Her deceased

    sister had severe aplasia cutis, cutis

    marmorata, nail aplasia of the toes,

    brachydactyly, tricuspid valve incom-

    petence, absence of the inferior

    medullary velum of the cerebellum,

    and hypoplasia of the dentate nuclei.

    She died of pulmonary hypertension

    at 3 years of age. Their deceased father

    had aplasia cutis, cutis marmorata,

    myopathy, and epilepsy. The proband

    was noted at birth to have short

    digits and toes and a macular heman-

    gioma around the circumference

    of her anterior fontanelle. Other

    features, including generalized cutismarmorata telangiectatica congenita and periventricular

    and gray-white-matter-junction hyperintense lesions on

    brain MRI, became apparent over time. Her intelligence

    is normal.

    For WGS, two different platforms and analysis pipelines

    were used. For families 1, 2, 3, and 5, genomic DNA was

    extracted from peripheral blood or saliva (DNA Genotek

    kit, Orasure Technologies). Paired-end library preparation,

    WGS, alignment to the reference genome (NCBI human

    genome assembly build 37), and variant calling were per-

    formed by Complete Genomics Inc. (CGI). Further variant

    annotation and analysis were performed with Ingenuity

    Variant Analysis software (QIAGEN) and the Family Geno-

    mics Toolkit, including Kaviar, a software program that

    estimates variant frequencies by taking into account not

    only the 1000 Genomes data set14 and the NHLBI Exome

    Sequencing Project Exome Variant Server (ESP6500) but

    also publically available personal genomes and exomesetics 95, 275284, September 4, 2014 277

  • and in-house genomes from the Institute for Systems

    Biology (ISB) and combines all available data to compute

    integrated variant frequencies.15 To identify candidate

    variants, we filtered for single-nucleotide variants (SNVs)

    and small indels that had CGI sequencing quality scores

    R 35 and had no reported variants in any data encom-

    passed by Kaviar. We...

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