Mutations in NOTCH1 Cause Adams-Oliver Syndrome

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    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: (J.E.N.), (M.S 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


    y of Human Genetics. All rights reserved.

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


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

    Clinical Characteristic


    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



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

    thrombosis of thesagittal sinus


    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 considered both recessive (hemizy-

    gous, homozygous, or compound heterozygous) and

    dominant (heterozygous) models. We searched genome-

    wide for individual variants or an increased gene burden

    of rare disrupting variants (frameshift, splice site, or stop

    gain or loss) in all or a subset of families. No variants or

    genes were found to be significantly enriched. Therefore,

    we relaxed our thresholds to include missense variants

    and restricted our analysis to the genes with known roles

    in AOSARHGAP31, DOCK6, EOGT, and RBPJas well as

    a set of 273 genes reported by Ingenuity Knowledge Base

    to be in direct interaction or relationship with one of the

    four AOS-associated genes. We also assessed copy-number

    variations (CNVs) and structural variants. For CNVs, we

    not only used the CGI annotation but also developed

    an algorithm that normalizes the depth of sequencing

    coverage in 1 kb bins to the median coverage depth of

    comparable in-house genomes and uses a hidden Markov

    model to segment the genome by observed ploidy. We

    identified sequences that differ from the expected diploid

    state and computed frequencies of such events by using

    ISBs in-house database of 5,077 genomes (including

    3,816 CGI-sequenced genomes and 1,261 Illumina-

    sequenced genomes).

    WGS of family 4 was performed on an Illumina HiSeq

    2000. Genomic DNA of the three study participants

    was isolated from peripheral blood (QIAGEN DNAeasy

    kit, QiaSymphony DNA). Paired-end libraries were gener-

    ated, quantified, and quality checked with the protocols

    recommended by Illumina. WGS, alignment to the

    reference genome (NCBI human genome assembly build

    37), and variant calling were performed by Illuminas

    FastTrack WGS service. SNVs and indels with PASS

    annotations and quality scoresR 30 in proband, maternal,

    and paternal genomes were merged with gVCFtools

    v.0.16, annotated with snpEff v.3.3,16 and then queried

    with GEMINI v. We used modified versions of the

    GEMINI tools both to identify variants in known AOS-

    associated genes in the proband and to conduct genome-

    wide analysis for variants on the basis of possible inheri-

    tance patterns. Only variants that were fully called in

    the trio and that had minor allele frequencies < 0.5%

    (for de novo variant queries) or < 1% (for other queries)

    in ESP6500 and 1000 Genomes were considered. Variants

    were filtered for those predicted by snpEff to be of

    medium or high impact or those annotated as possibly

    disease associated for any disease by ClinVar (v.12/30/13)

    or HGMD Professional (v.2013.2, BIOBASE). Variants pre-

    sent with the same inheritance pattern in Inovas internal

    database of 659 healthy families were excluded. Parent-

    offspring relationships were confirmed by GRAB.18278 The American Journal of Human Genetics 95, 275284, SeptembCandidate variants were confirmed with bidirectional

    Sanger sequencing, and where available, additional family

    members were screened by Sanger sequencing.

    We identified candidate variants in NOTCH1 (on the

    basis of the RefSeq transcript NM_017617.3 and NCBI hu-

    man genome assembly build 37) in five families (Figure 2).

    The proband of family 1 (1-II-3) was found to have a

    heterozygous 85 kb deletion of the 50 region of NOTCH1;it includes part of the promoter and the entire first

    exon (chr9: 139,439,620139,524,480; ClinVar accession

    number SCV000172278). We identified this deletion via

    sequence-coverage analysis at 20 bp resolution. This

    finding is supported by an analysis of heterozygous sites

    around the NOTCH1 50 end in family 1, which showed arun of homozygosity in the proband, but not in other fam-

    ily members (Figure S1), and by quantitative real-time PCR

    within the deleted region, which revealed half the amount

    of template in the proband (Figure S2). Because the dele-

    tion is not present in the unaffected parents or two unaf-

    fected siblings, de novo occurrence is apparent. In the

    two affected members from family 2 (2-II-1 and 2-III-1),

    we identified a heterozygous NOTCH1 canonical splice-

    site variant, c.7431G>T, at chr9: 139,414,018 (ClinVarSCV000172277). Sanger sequencing revealed that the

    variant is present in neither the unaffected mother (2-II-

    2) nor the unaffected brother (2-III-2) of the proband.

    The unaffected paternal grandparents of the proband

    were unavailable for genetic testing. In the proband of

    family 3 (3-II-1), we found a heterozygous NOTCH1

    missense variant, c.1285T>C (p.Cys429Arg), at chr9:

    139,412,360 (ClinVar SCV000172279). It has a SIFT score

    of 0.0 (damaging) and is rated as probably damaging

    by PolyPhen-2. Sanger sequencing of the two unaffected

    parents revealed that the variant occurred de novo. Anal-

    ysis of family 4 revealed a heterozygous de novo NOTCH1

    missense variant, c.4487G>A (p.Cys1496Tyr), at chr9:

    139,399,861 (ClinVar SCV000172281) in the proband

    (4-II-1). The variant has a SIFT score of 0 (damaging) and

    a PolyPhen-2 score of 1 (probably damaging). The

    family 5 proband (5-II-1) has a heterozygous NOTCH1

    missense mutation, c.5965G>A (p.Asp1989Asn), at chr9:

    139,393,681 (ClinVar SCV000172280). It has a SIFT score

    of 0.25 (tolerated) and is rated probably damaging by

    PolyPhen-2. No DNA was available from the deceased

    affected father and sister.

    The Notch signaling pathway is highly conserved in

    Bilateria and governs cell-fate decisions by maintaining

    progenitors in an undifferentiated state, initiating dif-

    ferentiation, or maintaining quiescence of differentiated

    cells.19,20 The mammalian family of Notch single-pass

    transmembrane receptors includes NOTCH1, NOTCH2,

    NOTCH3, and NOTCH4. Signaling is initiated by binding

    of one of the Jagged or Delta-like ligandsJAG1, JAG2,

    DLL1, DLL3, or DLL4to the extracellular domain of

    one of the Notch receptors. NOTCH1 has an extracellular

    domain with 36 epidermal-growth-factor (EGF)-like re-

    peats, 21 of which are potentially calcium binding, ander 4, 2014

  • Figure 2. Pedigrees of the AOS-Affected Families and the Respective NOTCH1 Variants(A) Affected males are represented by solid squares, affected females are represented by solid circles, and index individuals are indicatedby arrows. For individuals tested byWGS and/or Sanger sequencing, the genotypes are noted below in boxes; WT indicates the wild-typegenotype, and the five different mutations observed in NOTCH1 (RefSeq accession number NM_017617.3) are numbered as follows: (1)the 85 kb deletion, (2) c.7431G>T (splice site), (3) c.1285T>C (p.Cys429Arg), (4) c.4487G>A (p.Cys1496Tyr), and (5) c.5965G>A(p.Asp1989Asn).(B) For family 1, analysis of sequencing coverage is shown and indicates a 85 kb deletion (chr9: 139,439,621139,524,478) that overlapswith the promoter region and coding sequence of NOTCH1 (chr9: 139,388,896139,440,238) in the proband. For families 25, Sangersequencing chromatograms show the respective variants (red asterisks).(C) The exon-intron structure ofNOTCH1 is shown. The red triangles indicate our reportedmutations inNOTCH1: (1) the 85 kb deletion,(2) c.7431G>T (splice site), (3) c.1285T>C (p.Cys429Arg), (4) c.4487G>A (p.Cys1496Tyr), and (5) c.5965G>A (p.Asp1989Asn). Bluedots indicate previously reported NOTCH1 variants associated with isolated cardiac defects.three Lin-12 NOTCH repeats (LNRs). The Notch intracel-

    lular domain (NICD) contains an RBPJ-associationmodule,

    seven ankyrin repeats, and a C-terminal proline-, glutamic-

    acid-, serine-, and threonine-rich domain. Ligand binding

    leads to two proteolytic cleavage stepsone of which is

    mediated by ADAM-family metalloproteases and the other

    of which is mediated by the g-secretase complexthat

    release NICD to translocate to the nucleus. There, NICD

    activates target-gene transcription in cooperation with

    the DNA-binding protein CSL (named after RBPJ, Su(H),

    and LAG-1) and a coactivator of the Mastermind-like fam-

    ily (MAML1MAML3).19,20 Noncanonical Notch signaling

    also occurs and contributes to the pleiotropic effects of this


    The 85 kb deletion found in the proband of family 1 (1-

    II-3) removes the promoter and the first exon, suggesting

    that it causes disease through haploinsufficiency. We did

    not find any other largeNOTCH1 deletions in our database

    of 5,077 genomes. In a 2010 computational study, the

    probability that NOTCH1 function is susceptible to hap-

    loinsufficiency was predicted to be 0.957.21 The canonical

    splice-site variant (c.7431G>T) in family 2 (2-II-1 and2-III-1) disrupts the exon 5 acceptor splice site, probably

    ablating exon 5 and leading to a 41 amino acid in-frame

    deletion affecting the EGF-like repeats 6 and 7. These two

    EGF repeats have not been implicated with ligand binding

    but might be involved in glycosylation-mediated regula-

    tion of NOTCH1 activity.22 Another possible consequenceThe Americanof this mutation is that a cryptic splice site might be re-

    cruited. Because this would most likely introduce a stop

    codon toward the 50 end of the gene, nonsense-mediateddecay would probably lead to loss of function.23 For the

    three missense variants in families 35, we aimed to esti-

    mate the functional impact on NOTCH1 folding and

    stability by using the macromolecular modeling software

    Rosetta.2426 We modeled these variants on the following

    template structures from the Protein Data Bank (PDB):27

    p.Cys429Arg (PDB ID 2VJ3),28 p.Cys1496Tyr (PDB ID

    3L95),29 and p.Asp1989Asn (PDB ID 3V79).30 For both

    wild-type and altered proteins, we sampled the side-chain

    and backbone conformations of each protein domain at

    least 100 times by using a Monte Carlo procedure to mini-

    mize the estimated total free energy of folding for the pro-

    tein (Figure 3). We calculated the change in the free energy

    upon mutation (DDG) as the estimated free energy of

    folding of the altered protein minus that of the wild-type

    protein. Positive DDG values indicate destabilization of

    the protein upon substitution, and values exceeding

    3.0 kcal/mol indicate significantly reduced protein stabil-ity.31 The two cysteine substitutions, p.Cys429Arg (3-II-1)

    and p.Cys1496Tyr (4-II-1), are predicted to strongly desta-

    bilize the protein domains in which they occur, primarily

    as a result of the disruption of disulfide bridges in both

    cases. The p.Cys429Arg variant (3-II-1) resides in the

    calcium-binding EGF-like repeat 11. EGF-like repeats 11

    and 12 were previously shown to be required for DeltaJournal of Human Genetics 95, 275284, September 4, 2014 279

  • D EF

    A B C

    HG I

    Figure 3. Modeling the Effect of the NOTCH1 Variants p.Cys429Arg, p.Cys1496Tyr, and p.Asp1989Asn on Protein Structureand EnergeticsFor each variant, the left and middle panels show 20 energy-minimized structures from Rosetta modeling (A, D, and G represent thewild-type proteins, and B, E, and H represent the simulated substitutions). In (C), (F), and (I), the predicted change in the free energyof protein folding and stability upon substitution (DDG) is shown for each variant (blue, wild-type; pink, variant protein). Higher valuesindicate decreased domain stability.ligand binding in Drosophila Notch.32 The variant could

    impair calcium binding, which is a prerequisite for ligand

    binding.33 The p.Cys1496Tyr variant (4-II-1) occurs within

    the negative regulatory region (NRR) of the extracellular

    region of NOTCH1 in the second LNR domain. The NRR

    sterically inhibits processing of NOTCH1 in the absence

    of ligand stimulation. Thus, destabilization of this domain

    could increase constitutive Notch signaling and lead to a

    gain of function. Modeling of the third missense substitu-

    tion, p.Asp1989Asn (5-II-1), suggests a more subtle effect280 The American Journal of Human Genetics 95, 275284, Septembon protein stability. The affected aspartic acid is involved

    in a bipartite-charged hydrogen-bonding interaction with

    the backbone nitrogen-hydrogen atoms of p.Asp2020,

    which is located in a neighboring ankyrin repeat. The

    asparagine substitution is predicted to be capable of main-

    taining an uncharged hydrogen bond here. However, the

    different strength and geometry of an uncharged versus

    charged hydrogen bond34 could destabilize or perturb the

    regional domain structure. Although p.Asp1989 itself

    does not appear to directly interact with Notch bindinger 4, 2014

  • partners, the ankyrin repeat domain is involved in the

    binding of NOTCH1 to RBPJ and transcriptional coacti-

    vators of the Mastermind-like family.30 Alignment of

    the three missense-variant positions with the use of

    MUSCLE35 and Jalview36 shows that they are strictly

    conserved in all vertebrates and that the two cysteines

    are conserved in Bilateria (Figure S3).

    Here, we report that NOTCH1 mutations are a frequent

    and possibly the largest single genetic cause of AOS. We

    found five NOTCH1 mutations in 5 of 12 unrelated

    families affected by AOS (42%). None of these variants

    were found in over 10,000 control genomes or exomes.

    For three of the five families, unaffected parents were

    available for analysis, and in all three families, the

    NOTCH1 variants occurred de novo (Figure 2). In one fam-

    ily affected by autosomal-dominant AOS, a canonical

    splice-site variant in NOTCH1 segregated with the disease

    in the father and daughter. The missense variants each

    alter an amino acid that is strictly conserved in vertebrates.

    Further supporting our contention that mutations in

    NOTCH1 cause AOS, previous reports of other individuals

    with AOS showed autosomal-dominant mutations in the

    gene encoding the main NOTCH1-binding partner, RBPJ,

    and recessive mutations in EOGT, which encodes a puta-

    tive NOTCH1 glycosylase.7,8,11 Our findings confirm that

    Notch signaling alterations play a prominent role in the

    development of AOS.

    The majority of our reported AOS-affected individuals

    with NOTCH1 variants show cardiac or vascular defects.

    In addition to observations that Notch1 loss-of-function37

    or gain-of-function38 mutations in mice cause major

    vascular defects, several hereditary cardiovascular disorders

    have unveiled the pivotal role that Notch signaling plays

    in cardiovascular development and homeostasis. Examples

    include bicuspid aortic valve with calcification and/or

    thoracic aortic aneurysm (MIM 109730; caused by

    NOTCH1mutations); defects of the left ventricular outflow

    tract (caused by NOTCH1 mutations); Alagille syndrome

    (ALGS), involving predominantly right-sided congenital

    cardiovascular malformations (ALGS1 [MIM 118450],

    caused by JAG1 mutations; ALGS2 [MIM 610205], caused

    by NOTCH2 mutations); Hajdu-Cheney syndrome, fea-

    turing cardiac defects (MIM 102500; caused by NOTCH2

    mutations); and cerebral autosomal-dominant arteriopa-

    thy with subcortical infarcts and leukoencephalopathy

    (MIM 125310; caused byNOTCH3mutations).3941 Our re-

    sults add to this growing list a wide variety of AOS-related

    vascular defects, including right- and left-sided congenital

    heart defects, pulmonary hypertension, portal hyperten-

    sion, cutis marmorata, venous ectasia, and thrombophilia.

    Still, the AOS phenotype qualitatively differs from

    those previously reported with NOTCH1 variants, and it

    is unclear at present how the mutations we report cause

    AOS. The identified variants might lead to loss of function

    by preventing transcription (the 85 kb deletion), pre-

    venting translation (the splice-site variant c.7431G>T),or destabilizing NOTCH1 (the three missense variantsThe Americanc.1285T>C [p.Cys429Arg], c.4487G>A [p.Cys1496Tyr],

    and c.5965G>A [p.Asp1989Asn]). Given that Notch recep-

    tors bind ligands as tetramers,33 it is also possible that some

    AOS-associated NOTCH1 missense mutations exert domi-

    nant-negative effects by destabilizing normal tetramer

    formation and thus disrupt ligand binding. It is also

    conceivable that some of the NOTCH1 mutations induce

    a gain of function; for example, c.4487G>A (p.Cys1496-

    Tyr), which resides in the negative regulatory region,

    might reduce the restraint on ligand-independent sig-

    naling. There is also a possibility that the 85 kb deletion

    induces utilization of an alternative downstream promoter

    and transcription start site that skips the extracellular

    domain of NOTCH1 and produces a constitutively active

    NICD.42 The possibility that both loss- and gain-of-func-

    tion mutations in NOTCH1 cause AOS could be explained

    if Notch-signaling strength is the critical element in

    determining normal development of the vasculature.43

    An alternate hypothesis for the different phenotypes

    observed with AOS-related NOTCH1 mutations is that

    other genetic, epigenetic, or environmental factors shape

    the NOTCH1 mutant phenotype.

    Several mechanisms have been proposed to explain the

    limb reduction and scalp defects in AOS. Vascular obstruc-

    tion by thromboses during embryonic development

    was suggested as a cause of terminal transverse limb

    defects,44,45 and we report that two of our five probands

    experienced multiple thromboses. Examination of blood

    vessels from an affected individual with hepatoportal

    sclerosis led to the suggestion of endothelial cell dysfunc-

    tion as the root cause of AOS.46 On the basis of the wide-

    spread appearance on autopsy of denuded or reduplicated

    internal elastic laminae in AOS blood vessels (both signs

    of intimal damage), aswell as blood vessel stenosis or dilata-

    tion in association with exuberant or poor, respectively,

    vascular smooth muscle cell coverage, we suggest pericyte

    dysfunction as the basic pathophysiologic mechanism in

    AOS.47 Consistent with this suggestion, a critical role for

    Notch signaling in the vascular smooth muscle cell precur-

    sors of developing mouse limbs was shown by Chang and

    colleagues.48 Using a tetracycline-inducible dominant-

    negativeMastermind transgenicmodel, theydemonstrated

    that inhibition ofNotch signaling during a specificwindow

    in late gestation leads to defective vasculogenesis and hem-

    orrhage at the scalp and tips of all developing limbs in

    mouse embryos. Necrotic, hemorrhagic digits have also

    been observed in a preterm neonate with AOS, in keeping

    with the notion that some of the limb defects in AOSmight

    be due to aNOTCH1-related vasculopathy.49 Underbranch-

    ing of vasculature trees, irregular vascular smooth muscle

    cell coverage, tortuous ectatic vessels, and a general paucity

    of small to medium blood vessels are key features of the

    AOS vasculopathy12,47 and might also explain other symp-

    toms observed with AOS, including CNS microbleeds,

    retinopathy and visual impairment due to failed retinal

    vascularization, portal hypertension, ischemic bowel dis-

    ease, placental insufficiency, and pulmonary hypertension.Journal of Human Genetics 95, 275284, September 4, 2014 281

  • Our data clearly implicate NOTCH1 variants as a cause

    of AOS. There are numerous reported instances of the

    co-occurrence of aplasia cutis congenita with congenital

    cardiovascular anomalies, which seem to form a contin-

    uum within the variability in clinical expression of AOS,

    and NOTCH1 was previously predicted as a candidate

    gene for this spectrum of disorders.50 Some of the most

    serious manifestations of AOS, such as pulmonary hyper-

    tension, can be progressive. Revealing the molecular and

    cellular mechanisms that underlie AOS pathogenesis

    might facilitate the development of rational therapies to

    interrupt or slow this sometimes lethal complication.Supplemental Data

    Supplemental Data include three figures and one table and can be

    found with this article online at


    We are very grateful to the families who took part in this study

    and the Adams-Oliver syndrome Facebook support group. This

    work was funded in part by the Rare Disease Foundation, the BC

    Childrens Hospital Foundation, the University of Luxembourg

    Institute for Systems Biology Program, the Center for Systems

    Biology (2P50GM075647), the Inova Health System, and a gift

    from the Odeen family. A.B.S. was supported by the German

    Academic Exchange Service (Deutscher Akademischer Austausch

    Dienst) and the German Research Foundation (Deutsche For-

    schungsgemeinschaf). J.A. is a Gordon and Betty Moore Founda-

    tion Fellow of the Life Sciences Research Foundation. We thank

    Edward Greenberg and John H. Lee for help with radiologic

    interpretation for the proband from family 4.

    Received: July 2, 2014

    Accepted: July 22, 2014

    Published: August 14, 2014Web Resources

    The URLs for data provided herein are as follows:

    1000 Genomes,


    Family Genomics Group, http://familygenomics.systemsbiology.


    HGMD Professional,



    NHLBI Exome Sequencing Project (ESP) Exome Variant Server,

    Online Mendelian Inheritance in Man (OMIM), http://www.

    RefSeq, Numbers

    The ClinVar accession numbers for the variants reported in this

    paper are SCV000172277, SCV000172278, SCV000172279,

    SCV000172280, and SCV000172281.282 The American Journal of Human Genetics 95, 275284, SeptembReferences

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    Mutations in NOTCH1 Cause Adams-Oliver SyndromeSupplemental DataAcknowledgmentsWeb ResourcesAccession NumbersReferences