Human MutationRESEARCH ARTICLE

Molecular Analysis Expands the Spectrum of PhenotypesAssociated with GLI3 Mutations

Jennifer J. Johnston,1� Julie C. Sapp,1 Joyce T. Turner,1 David Amor,2 Salim Aftimos,3 Kyrieckos A. Aleck,4

Maureen Bocian,5 Joann N. Bodurtha,6 Gerald F. Cox,7 Cynthia J. Curry,8 Ruth Day,9 Dian Donnai,10 Michael Field,11

Ikuma Fujiwara,12 Michael Gabbett,13 Moran Gal,14 John M. Graham Jr,15 Peter Hedera,16 Raoul C.M. Hennekam,17

Joseph H. Hersh,18 Robert J. Hopkin,19 Hulya Kayserili,20 Alexa M.J. Kidd,21 Virginia Kimonis,5 Angela E. Lin,22

Sally Ann Lynch,23 Melissa Maisenbacher,24 Sahar Mansour,25 Julie McGaughran,13 Lakshmi Mehta,26 Helen Murphy,10

Margarita Raygada,27 Nathaniel H. Robin,28 Alan F. Rope,29 Kenneth N. Rosenbaum,30 G. Bradley Schaefer,31 Amy Shealy,32

Wendy Smith,33 Maria Soller,34 Annmarie Sommer,35 Heather J. Stalker,24 Bernhard Steiner,36 Mark J. Stephan,37

David Tilstra,38 Susan Tomkins,39 Pamela Trapane,40 Anne Chun-Hui Tsai,41 Margot I. Van Allen,42 Pradeep C. Vasudevan,43

Bernhard Zabel,44 Janice Zunich,45 Graeme C.M. Black,10 and Leslie G. Biesecker1

1Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland; 2Murdoch

Children’s Research Institute, Royal Children’s Hospital, Parkville, Victoria, Australia; 3Northern Regional Genetic Service, Auckland City Hospital,

Auckland, New Zealand; 4St. Joseph’s Hospital and Medical Center, Phoenix, Arizona; 5Division of Genetics and Metabolism, Department

of Pediatrics, University of California, Irvine Medical Center, Orange, California; 6Department of Human and Molecular Genetics, Pediatrics,

Obstetrics–Gynecology, Epidemiology and Community Health, Virginia Commonwealth University, Richmond, Virginia ; 7Division of Genetics, Children’s

Hospital Boston and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts; Clinical Research, Genzyme Corporation,

Cambridge, Massachusetts; 8Genetic Medicine Central California/University of California, San Francisco, California; 9Cheshire and Merseyside

Clinical Genetics Service, Liverpool, United Kingdom; 10Genetic Medicine, The University of Manchester, Manchester Academic Heath Science

Centre, Central Manchester University Hospitals NHS Foundation Trust, Manchester, United Kingdom; 11Clinical Genetics Department, Nepean

Hospital, Penrith, New South Wales, Australia; 12Department of Pediatrics, Tohoku University School of Medicine, Tohoku University Hospital, Sendai,

Miyagi, Japan; 13School of Medicine, The University of Queensland and Genetic Health Queensland, Royal Brisbane & Women’s Hospital, Brisbane,

Australia; 14Medical Genetic Institute, Shaare Zedek Medical Center, Jerusalem, Israel; 15Medical Genetics Institute, Cedars Sinai Medical Center,

Department of Pediatrics, David Geffen School of Medicine at UCLA, Los Angeles, California; 16Department of Neurology, Vanderbilt University,

Nashville, Tennessee; 17Department of Pediatrics, Academic Medical Center, University of Amsterdam, Meibergdreef, AZ Amsterdam, The

Netherlands; 18Department of Pediatrics, University of Louisville School of Medicine, Louisville, Kentucky; 19Division of Human Genetics, Cincinnati

Children’s Hospital Medical Center, Cincinnati, Ohio; 20Medical Genetics Department, Istanbul Medical Faculty, Istanbul University, Capa 3439

Istanbul, Turkey; 21Canterbury Health Laboratories, Christchurch, New Zealand; 22Massachusetts General Hospital, Boston, Massachusetts;23National Centre for Medical Genetics, Our Lady’s Children’s Hospital, Crumlin, Dublin 12, Republic of Ireland; 24Division of Genetics and Metabolism,

Department of Pediatrics, University of Florida, Gainesville, Florida; 25SW Thames Regional Genetics Service, St. George’s, University of London,

London, United Kingdom; 26Division of Medical Genetics, Department of Genetics & Genomic Sciences, Mount Sinai School of Medicine, New York;27Section on Clinical and Developmental Genomics, Program on Reproductive and Adult Endocrinology, Eunice Kennedy Shriver National Institute of

Child Health and Human Development, National Institutes of Health, Bethesda, Maryland; 28Department of Genetics and Pediatrics, University

of Alabama at Birmingham, Birmingham, Alabama; 29University of Utah School of Medicine, Division of Medical Genetics, Salt Like City, Utah;30Department of Medical Genetics, Children’s National Medical Center, Washington, DC; 31Department of Genetics and Pediatrics, University of

Arkansas for Medical Sciences, Section of Genetics and Metabolism, Department of Pediatrics, Arkansas Children’s Hospital, Little Rock, Arkansas;32Genomic Medicine Institute, Cleveland Clinic, Cleveland, Ohio; 33The Barbara Bush Children’s Hospital Maine Medical Center, Portland, Maine;34University and Regional Laboratories Region Skane, Division Clinical Genetics, Lund University Hospital, Lund, Sweden; 35Department of Pediatrics,

The Ohio State University College of Medicine, Columbus, Ohio and Nationwide Children’s Hospital, Columbus, Ohio; 36Institute of Medical Genetics,

University of Zurich, Schwerzenbach, Switzerland; 37Department of Pediatrics, Madigan Army Medical Center, Tacoma, Washington; 38Department

of Pediatrics, CentraCare Clinic, St. Cloud, Minnesota; 39Clinical Genetics, University Hospitals Bristol, Bristol, United Kingdom; 40Department

of Pediatrics, University of Iowa Hospitals & Clinics, Iowa City, Iowa; 41Department of Pediatrics, University of Colorado Health Sciences Center,

Denver, Colorado; 42Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada; 43Medical Genetics, University of

Leicester, University Hospitals of Leicester NHS Trust, Leicester Royal Infirmary, Leicester, United Kingdom; 44Department of Pediatrics, University

Hospital Freiburg, Freiburg, Germany; 45Genetics Center, Indiana University School of Medicine–Northwest, Gary, Indiana

Communicated by Ravi SavarirayanReceived 26 March 2010; accepted revised manuscript 18 July 2010.

Published online 29 July 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/humu.21328

OFFICIAL JOURNAL

www.hgvs.org

& 2010 WILEY-LISS, INC.

�Correspondence to: Jennifer J. Johnston, Genetic Disease Research Branch,

National Human Genome Research Institute, National Institutes of Health, Building

49, Room 4C64, Bethesda, MD 20892-4472. E-mail: jjohnsto@mail.nih.gov

ABSTRACT: A range of phenotypes including Greigcephalopolysyndactyly and Pallister-Hall syndromes(GCPS, PHS) are caused by pathogenic mutation of theGLI3 gene. To characterize the clinical variability of GLI3mutations, we present a subset of a cohort of 174probands referred for GLI3 analysis. Eighty-one probandswith typical GCPS or PHS were previously reported, andwe report the remaining 93 probands here. This includes19 probands (12 mutations) who fulfilled clinical criteriafor GCPS or PHS, 48 probands (16 mutations) withfeatures of GCPS or PHS but who did not meet theclinical criteria (sub-GCPS and sub-PHS), 21 probands(6 mutations) with features of PHS or GCPS and oral-facial-digital syndrome, and 5 probands (1 mutation)with nonsyndromic polydactyly. These data supportpreviously identified genotype–phenotype correlationsand demonstrate a more variable degree of severity thanpreviously recognized. The finding of GLI3 mutations inpatients with features of oral–facial–digital syndromesupports the observation that GLI3 interacts with cilia.We conclude that the phenotypic spectrum of GLI3mutations is broader than that encompassed by theclinical diagnostic criteria, but the genotype–phenotypecorrelation persists. Individuals with features of eitherGCPS or PHS should be screened for mutations in GLI3even if they do not fulfill clinical criteria.Hum Mutat 31:1142–1154, 2010. & 2010 Wiley-Liss,Inc.

KEY WORDS: GLI3; Greig syndrome; Pallister-Hall syn-drome; oral–facial–digital syndrome

Introduction

Mutations in the zinc-finger transcription factor encoding geneGLI3 (MIM] 165240) on chromosome 7p14.1 cause Greig cephalo-polysyndactyly syndrome (GCPS; MIM] 175700) [Vortkamp et al.,1991], Pallister-Hall syndrome (PHS, MIM] 146510) [Kang et al.,1997] and, less frequently, other phenotypes such as acrocallosalsyndrome (MIM] 200990) [Elson et al., 2002] and nonsyndromicpolydactyly (MIM] 174700) [Radhakrishna et al., 1999] (MIM]174200) [Radhakrishna et al., 1997]. The GCPS and PHSphenotypes are clinically distinct, and there is a robust genotype–phenotype correlation for truncating mutations in GLI3 for thesetwo phenotypes [Johnston et al., 2005]. Truncating mutations in themiddle third of the gene generally cause PHS, whereas largedeletions or truncating mutations elsewhere in the gene (aminoterminal-encoding or carboxy terminal-encoding thirds of the gene)cause GCPS. There are important biologic correlates for thisgenotype–phenotype correlation. The mutations that predicttruncations in the amino-terminal third of the gene are predictedto be null mutations, caused by loss of the zinc-finger DNA bindingdomain. In contrast, the truncations in the middle third of theprotein are predicted to generate a constitutive repressor proteinthat skews the balance of activator and repressor forms of GLI3,which is a key downstream modulator of SHH signaling. Themutations that predict truncations in the carboxyterminal third ofthe gene are predicted to cause the loss of a transactivation domainof GLI3 [Shin et al., 1999]. To date, genotype–phenotype studieshave been predominantly based on mutations found in patients withtypical forms of GCPS and PHS, and it therefore remains unclearwhether there are variant phenotypes that are caused by mutations

in GLI3 and if so, whether the same correlations hold for these otherphenotypes. To address these questions, we have continued toanalyze a large cohort of 174 probands with a wide spectrum ofphenotypic manifestations that include features of GCPS or PHS. Ofthese 174 probands, we present data on 93 patients not previouslyreported representing a wide range of phenotypes. We have analyzedGLI3 in these patients to determine the frequency and type ofmutations and assessed whether the mutation positions correlatedwith the phenotypes.

Methods

Patients

This study was reviewed and approved by the institutionalreview board of the National Human Genome Research Institute.The overall GLI3 project included 174 probands with features ofPHS or GCPS. Ninety-three probands were the focus of thisreport, and they were subdivided into the following groupsaccording to inclusion criteria in Table 1. Eighty-one probands(174–93) have been reported previously [Galasso et al., 2001;Johnston et al., 2005; Killoran et al., 2000; Kos et al., 2008; Nget al., 2004; Turner et al., 2003] and details on these probands arenot included in this report.

Probands with features of GCPS or PHS insufficient to meetclinical criteria

These probands had one or more features of GCPS or PHS butdid not meet clinical criteria for either disorder. Detailed clinicaldata are reported for these 53 probands (plus 9 relatives) who didnot fulfill clinical criteria for either GCPS or PHS. Anomalies weredefined according to the recently published standard terminology[Biesecker et al., 2009; Hall et al., 2009]. This pool of 53 probandswas subdivided into three groups based on phenotypic manifesta-tions. The first group (28 probands and six affected familymembers) (Tables 2 and 3) was designated as sub-GCPS andcomprised patients with one or more features of GCPS, includingpreaxial polydactyly, cutaneous syndactyly, widely spaced eyes, ormacrocephaly, but who did not meet the suggested clinical criteriafor GCPS. The second group comprised patients who had one ormore features of PHS, polydactyly, bifid epiglottis, and/orhypothalamic hamartoma, but who did not meet the publishedcriteria for PHS. We refer to this group as sub-PHS patients(20 probands and 3 affected family members) (Tables 4 and 5). Weplaced individuals with isolated postaxial polydactyly (PAP-A) intoa separate group that could overlap with PHS or GCPS as PAP-A isa manifestation of both GCPS and PHS (five probands).

Probands with features that overlappedwith the oral– facial– digital syndromes (OFDS)

Key features of the OFDS include tongue and other oralhamartomas, multiple buccal–oral frenula, cleft lip and/or cleftpalate, polydactyly, tibial hypoplasia, or cerebellar vermishypoplasia [Gurrieri et al., 2007]. There are 13 clinical types ofOFDS but only OFDS type 1 has a known molecular etiology[Ferrante et al., 2001]. We delineated this group because therehave been reports of patients with manifestations that overlappedPHS, OFDS, and other disorders [Muenke et al., 1991]. We

HUMAN MUTATION, Vol. 31, No. 10, 1142–1154, 2010 1143

identified 21 probands who had polydactyly and one or morefeatures of an OFDS (Tables 6 and 7).

Probands with typical GCPS or PHS

Nineteen probands who fulfilled diagnostic criteria for GCPS[Johnston et al., 2005] (17 probands) or PHS [Biesecker et al.,1996] (2 probands) were included in this report as they have notbeen reported previously. Detailed clinical data are reported forthese 19 probands (plus 4 relatives) (Tables 8–11). The clinicaldiagnostic criteria for PHS require the presence of mesoaxialpolydactyly and a hypothalamic hamartoma in the proband[Biesecker et al., 1996]. Suggested clinical criteria for GCPSinclude (1) preaxial polydactyly in at least one limb or broad greattoes or thumbs, and (2) cutaneous syndactyly, macrocephaly, andwide-spaced eyes [Biesecker, 2001]. For this study we set GCPSeligibility criteria of preaxial polydactyly and the presence of atleast one additional feature (cutaneous syndactyly, macrocephaly,wide-spaced eyes, postaxial polydactyly).

DNA Isolation, PCR, and Sequencing

DNA was isolated from whole blood using the salting out method(Qiagen, Valencia, CA) following the manufacturer’s instructions.PCR of GLI3 exons and flanking intron sequences was performedusing standard methods and primers as described [Johnston et al.,2005]. Sequencing of the GLI3 coding exons was performed withv3.1 BigDye terminator cycle sequencing kit (Applied Biosystems,Foster City, CA) and either the ABI 377 (Applied Biosystems) or ABI3100 (Applied Biosystems) per the manufacturer’s protocol.Sequence data were compared with the published GLI3 sequence(GenBank reference number NM_000168.5) using Sequencher 4.9

(Gene Codes Corp., Ann Arbor, MI). Nucleotide numbering reflectscDNA numbering with 11 corresponding to the A of the ATGtranslation initiation codon in the reference sequence, according tojournal guidelines (www.hgvs.org/mutnomen). The initiation codonis codon 1. The entire coding region was analyzed for all probandsexcept OFD2 due to insufficient DNA.

DHPLC Analysis

For some probands, screening of exons 3 through 12 and thelast third of exon 15 was performed using dHPLC as described inJohnston et al. [2005].

Classification of Sequence Variants

We classified sequence variants as causative mutations if they were:a nonsense or frameshift variant or, (2) a missense variant thatpredicted a nonconservative amino acid change and segregated withthe phenotype in multiple family members or was de novo in apatient with a GLI3-related phenotype and unaffected parents.

qPCR Analysis

qPCR was performed in a subset of individuals to identifydeletions and duplications of GLI3 exons. qPCR analysis of the GLI3coding exons was performed with the Platinum SYBR Green qPCRSuperMix UDG kit (Invitrogen) and the ABI PRISM 7000 (PEApplied Biosystems) as described in Johnston et al. [2005] (Table 3).

Array Hybridization

Zoom-in comparative genomic hybridization (CGH) forchromosome 7p14 was performed as described previously

Table 1. Inclusion Criteria for Patient Groups

Column A All required Column B Minimum of one required Column C Confirming featuresa

OFD-overlap Polydactyly Oral frenulae

Oral hamartoma

Clef lip/palate

Cerebellar vermis hypoplasia

Tibial hypoplasia

PHS Mesoaxial polydactyly

Hypothalamic hamartoma

GCPS Preaxial polydactyly Syndactyly

Macrocephaly

Hypertelorism

Postaxial polydactyly

Sub-PHS Mesoaxial polydactyly Bifid epiglottis

Hypothalamic hamartoma Imperforate anus

Oligodactyly Small nails

OR Hypopituitarism

Postaxial polydactyly plus one feature from column C Growth hormone deficiency

Genital hypoplasia

Sub-GCPS Preaxial polydactyly Hypoplasia of the corpus callosum

Broad thumbs or great toes

Syndactyly

Macrocephaly

Hypertelorism

OR

Postaxial polydactyly plus one feature from column C

aConfirming features were used to place individuals into sub-PHS or sub-GCPS groups when their only feature from column B was postaxial polydactyly. Probands were evaluatedsequentially for inclusion in the OFD-overlap group, then the PHS or GCPS groups, and last the sub-PHS or sub-GCPS groups. Probands were placed into the first group where theyfulfilled the inclusion criteria. Individuals who fulfilled the criteria for both sub-PHS and sub-GCPS were placed based upon the number of features they demonstrated for each group.

1144 HUMAN MUTATION, Vol. 31, No. 10, 1142–1154, 2010

[Johnston et al., 2007] in a subset of individuals to identify largedeletions and duplications on chromosome 7 including GLI3(Tables 2, 3, 5–9 and 11).

FISH Analysis

FISH analysis was performed in a subset of individuals toidentify large deletions on chromosome 7 in the vicinity of GLI3.FISH analysis was performed as described in Johnston et al. [2003](Tables 3 and 7).

Results

The cohort delineated in this study included 93 probands andwas drawn from a pool of 174 probands who were referred to ourresearch protocol because they had one or more manifestationsconsistent with either (or both) GCPS or PHS. In addition, someof these probands were from multiplex families and clinical dataon some of those affected family members are included in thiscohort. These 93 probands were divided into several groups (seeinclusion criteria) (Table 1) and each group is described in turn.

GLI3 mutations in probands with features of GCPS or PHSinsufficient to meet clinical criteria

The first group included 53 probands with features thatoverlapped with GCPS or PHS, but these probands did not havesufficient features to warrant a clinical diagnosis of either disorder.Of these 53 probands, 28 were categorized in the sub-GCPS groupand 8 of them had mutations. Of these eight mutations, five wereframeshift or nonsense mutations, one was a splice mutation, onewas a missense mutation, and one was a large genomic deletion.Four of the truncation or termination mutations were in thepredicted domains (either 50 of position 1998 or 30 of 3481);c.4240C4T, which predicts p.Q1414X; c.4430_4431delCT, whichpredicts p.S1477X; c.4432G4T, which predicts p.E1478X; andc.4594_4596delTCCinsA, which predicts p.S1532TfsX2. The fifthwas at the 30 border of the PHS region; c.3474delG, which predictsp.I1160FfsX46. The splice site alteration, c.149711G4C, IVS10,has been identified previously [Kalff-Suske et al., 1999]. Themissense alteration, c.2708C4T, which predicts p.S903L, was alsoidentified in this study in a proband who fulfilled the clinicalcriteria for GCPS.

We noted that all five of the frameshift or nonsense mutationsin the sub-GCPS group were located in the 30 region of the gene.Overall, the mutation yield for patients with typical GCPS was 39of 57 (68%), compared to 8 of 28 (29%) for the sub-GCPS group(P 5 0.0006, Fisher’s exact test). The distribution of mutations fortypical GCPS with frameshift or nonsense mutations is as follows;31 were in the 50 region, 9 were in the PHS region, and 14 were inthe 30 region [Fujioka et al., 2005; Furniss et al., 2009; Johnstonet al., 2005]. Interestingly, all five of the patients with sub-GCPSwho have frameshift or nonsense mutations have those mutationsin the 30 region (P 5 0.0023 Fisher’s exact test).

Of the 20 probands in the sub-PHS group, 8 had mutations(40%). Of these eight mutations, all were nonsense or frameshiftmutations and all but one of these mutations were in thepreviously defined PHS region (between cDNA positions 1998and 3481). One proband had a c.3887_3894del mutation thatpredicts p.L1297SfsX4. As this mutation would be predicted tocause GCPS, some clinical details are provided here. The probandhad bilateral mesoaxial polydactyly of the hand, isolated growthTa

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mil

dD

D

G16

FIS

HH

B,

FB

1P

rom

inen

tve

ntr

icle

sB

road

fore

hea

d,

hyd

ron

eph

rosi

s,in

guin

alh

ern

ias,

DD

G17

Arr

ayH

BH

BM

icro

cep

hal

y1

Age

nes

iso

fC

CF

ron

talb

oss

ing,

infa

nti

lesp

asm

s,ex

tra

rib

,DD

,hea

rin

g

loss

,co

nst

ipat

ion

,co

ntr

actu

res

G18

Arr

ayH

BH

B3

Bro

adh

and

s

wit

hu

nu

sual

crea

ses

11

Hyp

op

lasi

ao

fth

eC

CP

rom

inen

tfo

reh

ead

,d

epre

ssed

nas

alb

rid

ge,

do

wn

slan

ted

pal

peb

ral

fiss

ure

s,d

isti

nct

ive

ears

,m

ild

sixt

h

cran

ial

ner

vep

alsy

,tr

ach

eom

alac

iaw

ith

on

en

arro

w

bro

nch

us,

shaw

lsc

rotu

m,

elb

ow

dim

ple

s,lo

wto

ne

G19

QP

CR

HL

2,3

toe

Hyp

op

lasi

ao

fth

eC

CB

ilat

eral

clu

bfe

et,

VSD

/ASD

,ig

uin

alh

ern

ia

G20

Arr

ayF

BA

gen

esis

of

CC

,ce

reb

ella

ran

db

rain

stem

hyp

op

lasi

a,sc

hiz

ence

ph

aly

Hig

hp

alat

e,fi

fth

fin

ger

clin

od

acty

ly,

left

tib

ial

bo

win

g,

dec

ease

dat

8d

ays

G21

QP

CR

FB

G22

Arr

ayH

B,

FL

Sho

rtd

ista

lph

alan

ges,

abse

nt

fin

ger

nai

ls,s

ho

rth

um

eri

G23

Arr

ayH

RIn

crea

sed

CSF

spac

eo

nu

ltra

sou

nd

Hem

iver

teb

rae,

10ri

bs

bil

ater

ally

,m

ild

pla

gio

cep

hal

y,

dep

ress

edn

asal

bri

dge

,fr

on

tal

bo

ssin

g

G24

Arr

ayB

ifid

seco

nd

toe

FL

FB

3N

orm

alC

om

ple

xca

rdia

can

ato

my,

bel

lsh

aped

rib

cage

,h

ip

dys

pla

sia,

smal

lp

enis

,re

tin

ald

ysp

lasi

a,fo

veal

hyp

op

lasi

a,p

reau

ricu

lar

skin

tag,

dia

ph

ram

atic

her

nia

,su

per

nu

mer

ary

nip

ple

,sh

ort

met

acar

pal

s

and

met

atar

sals

,cr

ypto

rch

idis

m,

sco

lio

is

G25

Arr

ayC

om

ple

te2,

3to

e1

1N

orm

alA

nte

vert

edn

ares

,sh

ort

no

se,

hyp

erex

ten

sib

lejo

ints

,le

g

len

gth

dis

crep

ancy

,D

D

G26

Arr

ayH

LA

gen

esis

of

CC

,ce

reb

ella

ran

d

bra

inst

emh

ypo

pla

sia,

mid

lin

ecy

st

Ora

lfr

enu

la,

hyd

ron

eph

rosi

s,D

D

G27

QP

CR

11

G28

HB

,F

BP

on

toce

reb

ella

rh

ypo

pla

sia,

hyp

op

lasi

a

of

the

CC

Fac

ial

dys

mo

rph

ism

,re

du

nd

ant

ton

gue

tiss

ue,

ruff

led

gum

s,h

ors

esh

oe

kid

ney

,d

ecea

sed

at5

mo

nth

s,

1af

fect

edsi

bli

ng

HB

,h

and

sb

ilat

eral

;H

R,

han

dri

ght;

HL

,h

and

left

;H

B3,

wid

eth

um

bs;

FB

,fo

ot

bil

ater

al;

FR

,fo

ot

righ

t;F

L,

foo

tle

ft;

FB

3,w

ide

grea

tto

es;

CC

,co

rpu

sco

llo

sum

;C

SF,

cere

bra

lsp

inal

flu

id;

GH

,gr

ow

thh

orm

on

e;SZ

,se

izu

res;

DD

,d

evel

op

men

tal

del

ay;

VSD

/ASD

,ve

ntr

icu

lar/

atri

alse

pta

ld

efec

t;1

,p

rese

nce

of

fin

din

g.

1146 HUMAN MUTATION, Vol. 31, No. 10, 1142–1154, 2010

Tabl

e4.

Sub

-PH

SP

atie

nts

Wit

hM

utat

ions

Fin

din

gsan

dsy

mp

tom

s

Ind

ivid

ual

Mu

tati

on

Mes

oax

ial

po

lyd

acty

ly

Po

stax

ial

po

lyd

acty

ly

Pre

axia

l

po

lyd

acty

ly

Cu

tan

eou

s

syn

dac

tyly

Cra

nio

faci

al

feat

ure

s

Bif

id

epig

lott

isM

RI

fin

din

gsA

dd

itio

nal

fin

din

gs

PH

1c.

2149

C4

T,

p.Q

717X

HB

2,3

toe

FB

Dee

pse

tey

es,

smal

ln

ose

,

dia

stem

a,sm

all

ears

1H

Hw

ith

mu

ltic

ysti

c

exte

nsi

on

Clo

aca

(man

ysu

rger

ies)

,u

nil

ater

alre

nal

agen

esis

,

dec

reas

edre

nal

fun

ctio

n,

thro

mb

ocy

top

enia

,

SZ,

seve

reM

R

PH

2c.

2437

C4

T,

p.Q

813X

HB

,F

BN

AH

HSm

all

nai

ls,

PD

A,

ASD

,tr

icu

spid

regu

rgit

atio

n,

abse

nt

pit

uit

ary

and

adre

nal

glan

ds,

pu

lmo

nar

y

hyp

erte

nsi

on

,ab

no

rmal

lun

glo

bu

lati

on

,

imp

erfo

rate

anu

s,ge

nit

alh

ypo

pla

sia,

dec

ease

d

at1

day

PH

3c.

2466

del

G,

p.M

824X

Oli

god

acty

lyH

L,

fusi

on

of

met

acar

pal

s

FL

HR

,2,

3to

eF

L,

2,3,

4F

R

NA

HH

Smal

ln

ails

,d

ecea

sed

at3

mo

nth

s

PH

4c.

2542

del

G,

p.D

848T

fsX

12H

RM

acro

cep

hal

y,

smal

lte

eth

1H

HV

isu

alp

rob

lem

s,h

eari

ng

pro

ble

ms,

ecto

pic

righ

t

kid

ney

,G

Hd

efic

ien

cy,

ob

esit

y,SZ

,D

D

PH

5c.

2621

_26

24d

el,

p.R

874P

fsX

15B

ilat

eral

NA

HH

Smal

ln

ails

,p

ulm

on

ary

hyp

op

lasi

a,ab

sen

t

pit

uit

ary

glan

d,

adre

nal

hyp

op

lasi

a,th

yro

id

hyp

op

lasi

a,va

gin

alat

resi

a,ve

sico

vagi

nal

fist

ula

,

hyd

roco

lpo

s,b

ilat

eral

ren

alh

ypo

pla

sia,

dec

ease

dan

tep

artu

mat

41w

eeks

PH

6c.

3004

del

G,

p.V

1002

XO

ligo

dac

tyly

HR

NA

HH

Oss

eou

ssy

nd

acty

lyo

fm

etac

arp

als

and

met

atar

sals

,sh

ort

stat

ure

,gr

ow

thh

orm

on

e

def

icie

nt,

lau

ghin

gsp

ells

PH

7c.

3302

du

pA

,p

.N11

01K

fsX

28H

B1

HH

Smal

ln

ails

,h

ypo

pla

stic

toes

,p

oin

ted

teet

h,

mid

lin

efr

enu

la,

lary

nge

alcl

eft,

GH

def

icie

nt,

gen

ital

hyp

op

lasi

a,n

euro

sen

sory

hea

rin

glo

ss,

gela

stic

SZ

PH

8-1

c.38

87_

3894

del

,p

.L12

97Sf

sX4

HB

FB

1E

nla

rged

cere

bel

lar

ton

sils

Gro

wth

ho

rmo

ne

def

icie

nt,

13:1

7tr

ansl

oca

tio

n

PH

8-2

c.38

87_

3894

del

,p

.L12

97Sf

sX4

HB

,F

B1

No

rmal

PH

8-3

c.38

87_

3894

del

,p

.L12

97Sf

sX4

HB

1T

ho

raci

csc

oli

osi

s,n

ysta

gmu

s,D

D

PH

8-4

c.38

87_

3894

del

,p

.L12

97Sf

sX4

HB

,F

BB

road

fore

hea

d1

Sph

eno

idsi

nu

sE

xtra

bo

ne

inri

ght

foo

t,ch

ron

icsi

nu

sp

rob

lem

s,

13:1

7tr

ansl

oca

tio

n

HB

,han

ds

bil

ater

al;H

R,h

and

righ

t;H

L,h

and

left

;FB

,fo

ot

bil

ater

al;F

R,f

oo

tri

ght;

FL

,fo

ot

left

;HH

,hyp

oth

alam

ich

amar

tom

a;SZ

,sei

zure

s;M

R,m

enta

lret

ard

atio

n;P

DA

,pat

ent

du

ctu

sar

teri

osu

s;A

SD,a

tria

lsep

tald

efec

t;G

H,g

row

thh

orm

on

e;D

D,d

evel

op

men

tal

del

ay;1

,pre

sen

ceo

ffi

nd

ing.

Nu

cleo

tid

en

um

ber

ing

refl

ects

cDN

An

um

ber

ing

wit

h1

1co

rres

po

nd

ing

toth

eA

of

the

AT

Gtr

ansl

atio

nin

itia

tio

nco

do

nin

the

refe

ren

cese

qu

ence

,acc

ord

ing

tojo

urn

algu

idel

ines

(ww

w.h

gvs.

org

/m

utn

om

en).

Th

ein

itia

tio

nco

do

nis

cod

on

1.

HUMAN MUTATION, Vol. 31, No. 10, 1142–1154, 2010 1147

Tabl

e5.

Sub

-PH

SP

atie

nts

Wit

hout

Mut

atio

ns

Fin

din

gsan

dsy

mp

tom

s

Ind

ivid

ual

Del

etio

n

anal

ysis

Mes

oax

ial

po

lyd

acty

ly

Po

stax

ial

po

lyd

acty

ly

Pre

axia

l

po

lyd

acty

ly

Cu

tan

eou

s

syn

dac

tyly

Cra

nio

faci

alfe

atu

res

Bif

id

epig

lott

isM

RI

fin

din

gsA

dd

itio

nal

fin

din

gs

PH

9H

BF

ine

scal

ph

air

1D

isru

pti

on

bet

wee

nh

ypo

thal

amu

s

and

pit

uit

ary

Nu

chal

fold

s,h

ypo

ton

ia,

pan

hyp

op

itu

itar

ism

,D

D

PH

10H

BM

icro

cep

hal

y,p

oin

ted

teet

hN

orm

alSm

all

nai

ls,

pit

uit

ary

pro

ble

m,

mic

rop

hal

lus,

SZ,

DD

PH

11H

BD

epre

ssed

nas

alb

rid

ge1

No

rmal

Smal

ln

ails

,p

anh

ypo

pit

uit

aris

m,

vagi

nal

tag,

hyd

ron

eph

rosi

s,u

rin

ary

refl

ux

PH

12H

B,

FB

HB

,F

BL

arge

ante

rio

rfo

nta

nel

le1

Mic

rop

hal

lus,

un

ilat

eral

un

des

cen

ded

test

es

PH

13H

BH

BSm

all

mo

uth

and

ton

gue

1H

HA

trio

ven

tric

ula

rca

nal

def

ect,

dec

ease

dat

5m

on

ths

PH

14A

rray

HB

,F

BM

icro

cep

hal

y,fr

on

tal

bo

ssin

g,

mil

dd

oli

cho

cep

hal

y,h

igh

nar

row

pal

ate

Ven

tric

ulo

meg

aly,

per

iven

tric

ula

r

leu

kom

alac

ia

Slig

htl

yh

ypo

pla

stic

left

fift

hm

etac

arp

al,

hyp

oto

nia

,p

ost

nat

algr

ow

th

fail

ure

,p

seu

do

stra

bis

mu

s,m

ild

righ

tes

tro

pia

,b

ilat

eral

acce

sso

ry

nip

ple

s,m

ult

iple

bla

dd

erin

fect

ion

,u

reth

ral

ob

stru

ctio

n,

con

stip

atio

n,

abn

orm

alE

EG

,st

arin

gsp

ells

,D

D

PH

15H

BP

lagi

oce

ph

aly

Sho

rt

epig

lott

is

Po

ssib

leH

H,

abse

nt

ante

rio

r

pit

uit

ary

Lar

ynge

alw

eb,

lary

nge

alcl

eft,

ASD

,m

itra

lva

lve

clef

t,u

reth

ral

refl

ux,

hyp

op

itu

itar

ism

PH

16H

B3,

FB

3H

ypo

telo

rism

,le

ftm

icro

ph

tham

ia,

NA

HH

Pan

hyp

op

itu

itar

ism

,ch

oan

alat

resi

a,d

iap

hra

gmat

ich

ern

ia,

seve

reD

D

PH

17ri

ght

ano

ph

thal

mia

,cl

eft

lip

and

pal

ate

NA

HH

SZ,

DD

PH

18M

icro

cep

hal

y,cl

eft

lip

and

pal

ate

NA

HH

Mic

rop

hal

lus,

DD

PH

19a

Ora

lfr

enu

lae

NA

HH

Hyp

op

last

icfi

fth

fin

ger

PH

20a

Ora

lfr

enu

lae

NA

HH

Hyp

op

last

icm

idd

lep

hal

anx

of

fift

hd

igit

,en

do

crin

ed

efic

ien

cy

HB

,h

and

sb

ilat

eral

;H

B3,

wid

eth

um

bs;

FB

,fo

ot

bil

ater

al;

FB

3,w

ide

grea

tto

es;

NA

,n

ot

asse

ssed

;H

H,

hyp

oth

alam

ich

amar

tom

a;D

D,

dev

elo

pm

enta

ld

elay

;SZ

,se

izu

res;

ASD

,at

rial

sep

tal

def

ect;

1,

pre

sen

ceo

ffi

nd

ing.

a[B

on

nem

ann

,su

bm

itte

d].

Tabl

e6.

OFD

-Ove

rlap

Pat

ient

sW

ith

Mut

atio

ns

Fin

din

gsan

dsy

mp

tom

s

Ind

ivid

ual

Mu

tati

on

Mes

oax

ial

po

lyd

acty

ly

Po

stax

ial

po

lyd

acty

ly

Pre

axia

l

po

lyd

acty

ly

Ora

l

fren

ula

Ora

l

ham

arto

ma

Cle

ftli

p/

pal

ate

Cer

ebel

lar

verm

is

hyp

op

lasi

a

Tib

ial

hyp

op

lasi

a

Cu

tan

eou

s

syn

dac

tyly

MR

Ifi

nd

ings

Oth

erfi

nd

ings

OF

D1a

c.20

77A4

T,

p.K

693X

HB

FR

11

HH

Eso

tro

pia

,am

bly

op

ia,

op

tic

ner

veh

ypo

pla

sia,

pre

coci

ou

s

pu

ber

ty,

sup

ern

um

erar

ym

axil

lary

inci

sor,

gela

stic

seiz

ure

s,D

D

OF

D2

c.29

77C4

T,

p.Q

993X

HL

HL

1Sh

ort

pal

peb

ral

fiss

ure

s,sh

ort

fin

gers

,sm

all

nai

ls,

imp

erfo

rate

anu

s,A

SD,

dec

ease

dat

5d

ays

OF

D3

c.30

02d

elG

,

p.G

1001

Afs

X2

HB

Pal

ate

1H

H,

agen

esis

of

the

CC

Bil

ater

alch

oan

alat

resi

a,sm

all

wid

esp

aced

eyes

,sm

all

mo

uth

,sy

ngn

ath

ia,

dys

pla

stic

kid

ney

,sh

ort

lim

bs,

imp

erfo

rate

anu

s,sh

ort

fin

gers

,sm

all

nai

ls,

pre

gnan

cy

term

inat

edat

22w

eeks

OF

D4

c.30

40G4

T,

p.E

1014

XO

ligo

dac

tyly

HL

HR

1H

RH

ypo

thal

amic

mas

sA

bse

nt

left

kid

ney

,im

per

fora

tean

us,

dec

ease

dat

1w

eek

OF

D5b

c.33

70d

up

C,

p.H

1124

Pfs

X5

HB

,F

B1

1R

HB

,F

BH

H,

left

cere

bra

l

atro

ph

y

Sho

rtle

ftu

lna,

smal

lfi

bu

lae,

hyd

rom

etro

colp

os

wit

ha

vagi

no

-cys

tic

fist

ula

,p

reco

cio

us

pu

ber

ty,

MR

OF

D6

Ch

r7:d

el33

.2-4

7.2

Mb

FB

11

1D

ilat

edve

ntr

icle

s,

du

ral

der

mo

idcy

st

Mac

roce

ph

aly,

hyp

erte

lori

sm,

bif

idep

iglo

ttis

,b

ilat

eral

cho

anal

hyp

op

lasi

a,p

aten

tfo

ram

eno

vale

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ors

esh

oe

kid

ney

,ac

cess

ory

sple

en,

bil

ater

alu

nd

esce

nd

edte

stes

,

kyp

ho

sis,

dec

ease

dat

2.5

year

s

HB

,han

ds

bil

ater

al;H

R,h

and

righ

t;H

L,h

and

left

;FB

,fo

ot

bil

ater

al;F

R,f

oo

tri

ght;

FB

3,w

ide

grea

tto

es;H

H,h

ypo

thal

amic

ham

arto

ma;

CC

,co

rpu

sca

llo

sum

;ASD

,atr

ials

epta

ldef

ect;

DD

,dev

elo

pm

enta

ld

elay

;1,p

rese

nce

of

fin

din

g.N

ucl

eoti

de

nu

mb

erin

gre

flec

tscD

NA

nu

mb

erin

gw

ith

11

corr

esp

on

din

gto

the

Ao

fth

eA

TG

tran

slat

ion

init

iati

on

cod

on

inth

ere

fere

nce

seq

uen

ce,

acco

rdin

gto

jou

rnal

guid

elin

es(w

ww

.hgv

s.o

rg/m

utn

om

en).

Th

ein

itia

tio

nco

do

nis

cod

on

1.a[S

tep

han

etal

.,19

94].

b[F

uji

war

aet

al.,

1999

].

1148 HUMAN MUTATION, Vol. 31, No. 10, 1142–1154, 2010

Tabl

e7.

OFD

-Ove

rlap

Pat

ient

sW

itho

utM

utat

ions

Fin

din

gsan

dsy

mp

tom

s

Ind

ivid

ual

Del

etio

n

anal

ysis

Mes

oax

ial

po

lyd

acty

ly

Po

stax

ial

po

lyd

acty

ly

Pre

axia

l

po

lyd

acty

ly

Ora

l

fren

ula

Ora

l

ham

arto

ma

Cle

ftli

p/

pal

ate

Cer

ebel

lar

verm

is

hyp

op

lasi

a

Tib

ial

hyp

op

lasi

a

Cu

tan

eou

s

syn

dac

tyly

MR

Ifi

nd

ings

Oth

erfi

nd

ings

OF

D7

HB

,F

BH

B,

FB

Lip

and

pal

ate

1A

gen

esis

of

CC

,fu

sed

bas

alga

ngl

ia,

abn

orm

alco

rtic

algy

ral

pat

tern

Sho

rtli

mb

s,cl

ub

feet

,A

Vca

nal

,cy

stic

pan

crea

s,

dec

ease

d

OF

D8

FIS

HH

L1

2,3

toe

Hig

han

teri

or

hai

rlin

e,p

ylo

ric

sten

osi

s,

un

der

dev

elo

ped

rib

s,m

icro

ph

allu

s,m

ild

DD

OF

D9

Arr

ayF

BP

alat

eN

orm

alC

TD

epre

ssed

nas

alb

rid

ge,

epil

epsy

,h

ydro

nep

hro

sis,

refl

ux,

seve

reD

D

OF

D10

Arr

ayH

BF

B1

Lip

,to

ngu

e1

HB

,F

BM

ild

hea

rin

glo

ss,

wid

esp

aced

eyes

,cl

efte

d

epig

lott

is,

DD

OF

D12

FL

1H

HM

ild

hyp

osp

adia

s,SZ

,D

D

OF

D12

HB

,F

B1

1E

nd

oca

rdia

lcu

shio

nd

efec

t,

Dan

dy-

Wal

ker

mal

form

atio

n

Smal

lea

rs,

bil

ater

alp

oly

cyst

icki

dn

ey

OF

D13

HB

,F

BL

ip/p

alat

eA

bse

nt

pre

max

illa

and

mid

lin

efr

enu

lum

,V

SD/

ASD

,ab

sen

tp

itu

itar

y,p

anh

ypo

pit

uit

aris

m,

DD

OF

D14

HB

11

1H

H,

agen

esis

of

CC

Wid

esp

aced

eyes

,h

ypo

pla

stic

left

hea

rt,

imp

erfo

rate

anu

s,d

ecea

sed

at4

mo

nth

so

fag

e

OF

D15

FIS

HH

BH

BP

alat

eN

orm

alW

ide

spac

edey

es,

fro

nta

lb

oss

ing,

dep

ress

edn

asal

bri

dge

,m

icro

gnat

hia

,re

tin

ald

ysp

lasi

a,o

pti

c

ner

veh

ypo

pla

sia,

det

ach

edre

tin

a,se

vere

hea

rin

g

loss

OF

D16

Arr

ayH

L,

FB

HB

,F

BL

ip/p

alat

e1

FB

HH

Ab

sen

tfi

bu

lae,

sho

rtri

bs,

sho

rtlo

ng

bo

nes

,sm

all

jaw

,p

regn

ancy

term

inat

edat

20w

eeks

OF

D17

1H

B1

HB

Age

nes

iso

fC

CU

nil

ater

alra

diu

sh

ypo

pla

sia,

bil

ater

alti

bia

hyp

op

lasi

a,gi

ngi

vao

verg

row

th,

cyst

icki

dn

eys

OF

D18

HB

FB

11

Lip

/pal

ate

1M

ola

rto

oth

sign

,H

HM

arke

drh

izo

mel

ican

dm

eso

mel

icsh

ort

enin

gw

ith

smal

lh

and

san

dfe

etan

db

rach

ydac

tyly

,ab

sen

t

epig

lott

is,

op

tic

ner

veco

lob

om

asw

ith

sear

chin

g

nys

tagm

us

and

abse

nt

VE

Rs,

no

tch

edm

idli

ne

smal

lja

w

OF

D19

HL

11

Ham

arto

ma

Mac

roce

ph

aly,

smal

lfi

nge

rn

ails

,sh

ort

5th

fin

ger,

seco

nd

deg

ree

mic

roti

a,ge

last

icse

izu

res,

bif

id

too

th,

abse

nt

too

th,

sup

ern

um

erar

yto

oth

OF

D20

HB

,F

BH

B,

FB

1L

ip/p

alat

eH

HTe

ther

edto

ngu

e,va

gin

alat

resi

a,D

D,

dec

ease

d

OF

D21

11

HH

,h

ypo

pla

sia

of

cere

bel

lum

,

Dan

dy

Wal

ker

cyst

wit

h

mo

lar

too

thsi

gn

Mac

roce

ph

aly,

wid

esu

ture

s,fr

on

tal

bo

ssin

g,b

road

dep

ress

edn

asal

bri

dge

,d

ecea

sed

at2

mo

nth

s

HB

,h

and

sb

ilat

eral

;H

L,

han

dle

ft;

FB

,fo

ot

bil

ater

al;

FL

,fo

ot

left

;C

C;

corp

us

call

osu

m;

HH

,h

ypo

thal

amic

ham

arto

ma;

NA

,n

ot

asse

ssed

;D

D,

dev

elo

pm

enta

ld

elay

;SZ

,se

izu

res;

VSD

/ASD

,ve

ntr

icu

lar/

atri

alse

pta

ld

efec

t;1

,p

rese

nce

of

fin

din

g.

HUMAN MUTATION, Vol. 31, No. 10, 1142–1154, 2010 1149

Tabl

e8.

GC

PS

Pat

ient

sW

ith

Mut

atio

ns

Fin

din

gsan

dsy

mp

tom

s

Ind

ivid

ual

Mu

tati

on

Mes

oax

ial

po

lyd

acty

ly

Po

stax

ial

po

lyd

acty

ly

Pre

axia

l

po

lyd

acty

ly

Cu

tan

eou

s

syn

dac

tyly

Mac

roce

ph

aly

Wid

e-sp

aced

eyes

MR

Ifi

nd

ings

Ad

dit

ion

alfi

nd

ings

G29

c.10

96C4

T,

p.R

366X

HB

HB

3,F

BH

B,

FB

11

Den

tal

cro

wd

ing,

tali

pes

equ

ino

varu

s,u

nd

esce

nd

edte

stis

,

righ

tin

guin

alh

ern

ia

G30

c.15

61_

1576

del

,p

.S52

1Pfs

X9

FB

FB

11

Hyp

op

lasi

ao

fco

rpu

sca

llo

sum

,

calc

ifie

dfa

lx

Lip

om

ao

nfo

reh

ead

,d

elay

eder

up

tio

no

fm

ola

rs

G31

c.17

28C4

A,

p.Y

576X

HB

FB

FB

11

Cra

nio

syn

ost

osi

s,ep

ican

thal

fold

s,d

epre

ssed

nas

alb

rid

ge

G32

c.17

48G4

T,

p.C

583F

HB

FB

FB

No

rmal

U/S

Um

bil

ical

her

nia

G33

-1c.

2374

C4

T,

p.R

792X

HB

FB

FB

1N

orm

alSZ

G33

-2c.

2374

C4

T,

p.R

792X

HB

FB

Bro

adn

asal

bri

dge

G34

-1c.

2708

C4

T,

p.S

903L

FB

FB

No

rmal

Ast

hm

a

G34

-2c.

2708

C4

T,

p.S

903L

HB

,F

BF

B1

1A

gen

esis

of

the

CC

,

mil

dve

ntr

icu

lar

pro

min

ence

Do

lich

oce

ph

aly,

sagi

ttal

cran

iosy

no

sto

sis,

bu

lbo

us

no

se,

um

bil

ical

her

nia

wit

hd

iast

asis

rect

i,D

D

G34

-3c.

2708

C4

T,

p.S

903L

FB

FB

Hig

han

teri

or

hai

rlin

e,

G35

-1c.

2741

del

G,

p.G

914A

fsX

38H

B,

FB

2,3

toe

11

Fam

ily

his

tory

of

pre

axia

lp

oly

dac

tyly

G36

-1c.

4072

C4

T,

p.Q

1358

XH

B,

FB

FB

G36

-2c.

4072

C4

T,

p.Q

1358

XH

B,

FB

FB

11

Um

bil

ical

her

nia

,SZ

,D

D

G37

Ch

r7:d

el37

.1-4

9.3

Mb

HB

,F

BH

B,

FB

1C

CM

,ab

no

rmal

CC

Bil

ater

alh

ydro

nep

hro

sis,

L-u

rete

ral

refl

ux,

cou

rse

live

r,

lary

ngo

mal

acia

,SZ

/DD

G38

Ch

r7:d

el39

.7-4

5.8

Mb

HB

3,F

B2,

3to

e1

1C

CM

,ve

ntr

icu

lom

egal

yD

uan

esy

nd

rom

e,V

SD/A

SD,

SZ/D

D

G39

Ch

r7:d

el41

.0-4

5.1

Mb

FB

HB

FB

,H

B1

Sub

du

ral

effu

sio

nC

ryp

torc

hid

ism

,h

ori

zon

tal

earl

ob

ecr

ease

s,an

tih

elix

pit

,

sin

gle

tran

sver

sep

alm

arcr

ease

of

the

left

han

d,

SZ,

DD

HB

,han

ds

bil

ater

al;H

R,h

and

righ

t;F

B,f

oo

tb

ilat

eral

;CC

,co

rpu

sco

llosu

m;C

CM

,cer

ebra

lcav

ern

ou

sm

alfo

rmat

ion

;SZ

,sei

zure

s;D

D,d

evel

op

men

tal

del

ay;V

SD/A

SD,v

entr

icu

lar/

atri

alse

pta

ldef

ect;

1,p

rese

nce

of

fin

din

g.N

ucl

eoti

de

nu

mb

erin

gre

flec

tscD

NA

nu

mb

erin

gw

ith

11

corr

esp

on

din

gto

the

Ao

fth

eA

TG

tran

slat

ion

init

iati

on

cod

on

inth

ere

fere

nce

seq

uen

ce,

acco

rdin

gto

jou

rnal

guid

elin

es(w

ww

.hgv

s.o

rg/m

utn

om

en).

Th

ein

itia

tio

nco

do

nis

cod

on

1.

1150 HUMAN MUTATION, Vol. 31, No. 10, 1142–1154, 2010

hormone deficiency without a hypothalamic hamartoma, and abifid epiglottis. Her three affected family members have two tofour limb postaxial polydactyly with a bifid epiglottis without ahypothalamic hamartoma. One family member had a broadforehead. The biologic mechanism of how this variant causes asub-PHS phenotype requires further study.

Seven of the eight mutations in the sub-PHS group were novel.One mutation (c.2149C4T, p.Q717X) has been describedpreviously in a patient with typical PHS [Johnston et al., 2005].The overall mutation yield for the sub-PHS probands was 8 of 20(40%), which is significantly lower than for patients with typicalPHS (20 of 22, 91%; P 5 0.0008, Fisher’s exact test).

One of five patients (20%) in the isolated PAP-A group wasfound to have a mutation in GLI3, c.874C4T, p.R292C. Thismutation is upstream of the zinc finger in a conserved region ofthe protein.

GLI3 mutations in probands with features of OFDSs

We identified 21 probands from our cohort who had one ormore features of PHS or GCPS and in addition, one or morefeatures of OFDS. Among these 21 probands we identified fiveframeshift or nonsense mutations that we concluded were

pathologic and one large genomic deletion of 14.0 Mb. All five ofthe frameshift or nonsense mutations were similar in positionwithin GLI3 to other mutations that have been reported to causePHS (Fig. 1). Indeed, several of the probands in this group met theclinical criteria for PHS (OFD1, c.2077A4T, p.K693X; OFD2,c.2977C4T, p.Q993X; OFD3, c.3002delG, p.G1001AfsX2). PatientOFD4 with the c.3040G4T, p.E1014X mutation did not meetclinical criteria for PHS but he had oligodactyly, which we haveobserved in affected relatives of probands with typical PHS(unpublished observations). Similarly, patient OFD5 with thec.3371dupC, p.H1124PfsX5 mutation had postaxial polydactyly anda hypothalamic hamartoma. Although not sufficient for a clinicaldiagnosis of PHS, this combination of features has been observed inaffected relatives of probands with PHS. Six of 21 patients withfeatures that overlap an OFDS had a GLI3 mutation for an overallyield of 29%. This yield of mutations is significantly below that fortypical PHS (20 of 22, 91%, Po0.0001, Fisher’s exact test).

GLI3 mutations in probands with typical GCPS or PHS

The final group included patients with typical manifestations ofGCPS or PHS. These patients were similar in their clinicalmanifestations to patients described previously [Johnston et al.,

Table 9. GCPS Patients Without Mutations

Findings and symptoms

Individual

Deletion

analysis

Mesoaxial

polydactyly

Postaxial

polydactyly

Preaxial

polydactyly

Cutaneous

syndactyly Macrocephaly

Wide-spaced

eyes MRI findings Additional findings

G40 Array FL 1

G41 HB FB, soft

tissue

HB Agenesis of CC, brain cyst,

brain stem hypoplasia

G42 Array HB FB FB

G43 Array HB HR 1 Agenesis of CC Small nose, prominent forehead,

deformed ear, MR

G44 Array FB 1 Porencephaly of left occipital

and left temporal lobes,

absence of septum pellucidum,

hypoplastic optic nerves

Bilateral hernia, midline capillary

vascular malformation,

tetralogy of Fallot

G45 Array HB3, FB HB, FB Trigonocephaly

HB, hands bilateral; HR, hand right; FB, foot bilateral; FL, foot left; HB3, wide thumbs; CC, corpus collosum; MR, mental retardation; 1, presence of finding.

Table 11. PHS Patients Without Mutations

Findings or symptoms

Individual

Deletion

analysis

Mesoaxial

polydactyly

Postaxial

polydactyly

Preaxial

polydactyly

Cutaneous

syndactyly

Craniofacial

features

Bifid

epiglottis

MRI

findings Additional findings

PH22 Array HB 1 HH Small nails, pointed teeth, genital hypoplasia,

microglossia, MR

HB, hands bilateral; HH, hypothalamic hamartoma; MR, mental retardation; 1, presence of finding.

Table 10. PHS Patients With Mutations

Findings and symptoms

Individual Mutation

Mesoaxial

polydactyly

Postaxial

polydactyly Preaxial p

Cutaneous

syndactyly

Craniofacial

features

Bifid

epiglottis

MRI

findings

Additional

findings

PH21 c.2685C4G, p.Y895X HB HB HB, FB NA HH Bilateral renal hypoplasia

HB, hands bilateral; FB, foot bilateral; HH, hypothalamic hamartoma; NA, not assessed. Nucleotide numbering reflects cDNA numbering with 11 corresponding to the A ofthe ATG translation initiation codon in the reference sequence, according to journal guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1.

HUMAN MUTATION, Vol. 31, No. 10, 1142–1154, 2010 1151

2005]. Among the 17 probands with GCPS we identified 11mutations. Of these 11 mutations, 6 were frameshift or nonsensemutations, 2 were missense mutations, and 3 were large genomicdeletions. Of the six frameshift or nonsense mutations, threewere in the 50 segment of GLI3 (between the start codon andcDNA position 1998); c.1096C4T, which predicts p.R366X;c.1561_1576del, which predicts p.S521PfsX9; and c.1728C4A,which predicts p.Y576X. One nonsense mutation (c.4072C4T,p.Q1358X) was in the 30 segment of GLI3 (between cDNA position3481 and the normal stop codon). Two nonsense or frameshiftmutations were in the middle region of GLI3 (between cDNApositions 1998 and 3481), which in most cases is associated with aphenotype of Pallister-Hall syndrome. Mutation (c.2374C4T,p.R792X) represents the eighth report of this variant associatedwith GCPS and this variant has been associated with nonsense-mediated mRNA decay [Furniss et al., 2007]. The secondframeshift or nonsense mutation in the middle region of GLI3 isc.2741delG, which predicts p.G914AfsX38. The proband with thismutation did not have preaxial polydactyly but was included inthe GCPS group based on an extensive family history of preaxialpolydactyly in combination with macrocephaly. Two missensemutations were identified, c.1748G4T, p.C583F, and c.2708C4T,p.S903L. Of these eight mutations, six are novel. There were threeprobands in this group with deletions that included GLI3 andranged from 4.1 Mb to 12.2 Mb. All three of these deletions havenovel breakpoints. These individuals were given a diagnosis ofGCPS contiguous gene syndrome based on their molecularfindings. This phenotype can include microcephaly or normoce-phaly, cognitive impairment, seizures, and other manifestations.

Of the two probands with PHS, one had a mutation in GLI3,c.2685C4G, p.Y895X. This mutation conforms to the previouslydescribed correlation that PHS mutations lie between cDNApositions 1998 and 3481 and is novel.

The overall yield of mutations was 65% for GCPS (11 of 17) and50% for PHS (1 of 2). We previously showed that among patientswith typical GCPS, 28 of 40 patients had a GLI3 mutation (70%)and 19 of 20 probands with PHS had GLI3 mutations (95%)[Johnston et al., 2005]. The results in the current study are similarfor GCPS. Merging these data, the current estimates for GCPS wouldbe 39 of 57 (68%) and for PHS would be 20 of 22 probands (91%).

Discussion

GLI3 mutations have been associated with several phenotypesincluding GCPS [Vortkamp et al., 1991], PHS [Kang et al., 1997]isolated polydactyly types A, A/B, and preaxial polydactyly type 4[Radhakrishna et al., 1997, 1999], and a single case of acrocallosalsyndrome [Elson et al., 2002]. By combining the data in thisreport with those of our prior work [Johnston et al., 2005] wepredict that when an individual manifests features sufficient for

the clinical diagnostic criteria for PHS or GCPS, their chance ofhaving a mutation in GLI3 is high: 91 and 68%, respectively. Thedata presented here extend these observations into several distinctgroups of patients.

In the early phases of gene discovery efforts, it is important tomaximize the likelihood of locus homogeneity by setting strictclinical eligibility criteria. This was done successfully for PHS, andwas likely done for GCPS as well. As noted above, nearly all patientswho met the clinical criteria for PHS had a truncating mutationin the middle third of GLI3. In this study we hypothesized that arelaxation of the clinical criteria would identify additional patientswith GLI3 mutations. By relaxing the criteria to allow subjects witheither mesoaxial polydactyly or hypothalamic hamartoma (but notrequiring both), we show that a substantial proportion (50% or 8 of16) of patients have mutations in GLI3, a substantial and clinicallyuseful yield that is slightly more than half the rate for patients whomeet clinical criteria. When the criteria are relaxed even furtherto allow patients with syndromic postaxial polydactyly withoutmesoaxial polydactyly or hypothalamic hamartoma, no mutationswere identified in four additional probands. Similar to the situationfor PHS, the relaxation of the clinical criteria for GCPS allowed usto identify mutations in 29% of patients in the sub-GCPS category,again about half the yield for patients who meet the former criteria.We had a limited set of probands enrolled in the study who hadnonsyndromic polydactyly, which was mostly postaxial polydactyly.The yield in these patients was one of five or 20% but becausethis cohort is small, we believe that the implications of this findingare limited.

We also identified a cohort of patients who had one or morefeatures of an OFDS. Other than OFDS type 1, there is no knownmolecular etiology for the many types that have been described(up to 13 types have been proposed). We reasoned that some casesof OFDS could be caused by mutations in GLI3 because: (1) therewere a number of clinical reports of patients whose findingsoverlapped OFDS and PHS; (2) OFDS type 1 is a ciliopathy[Ferrante et al., 2006]; and (3) GLI3 requires ciliary function forproper processing [Haycraft et al., 2005]. We selected 21 casesfrom our cohort with one or more features of an OFDS. Some ofthe patients had sufficient features to warrant a diagnosis of PHSor GCPS as well, but some of the patients have been accepted asexamples of an OFDS as evidenced by their publication in theliterature [Fujiwara et al., 1999; Stephan et al., 1994]. Among these21 probands, we identified 6 cases with causative mutations inGLI3, establishing molecular evidence that mutations of this genecan cause phenotypes within the OFDS spectrum.

Taken together, these data suggest that clinicians and moleculardiagnostic laboratories should encourage a relaxation of clinicalcriteria for GLI3 testing for patients with one or more features ofGCPS or PHS. This would include patients with a feature of PHSor GCPS and one or more features of an OFDS. In this way

34811998

sub-PHS

sub-GCPS

OFD-overlap

Figure 1. Diagram of the position within the gene of newly described nonsense and frameshift mutations in probands with sub-GCPS, sub-PHS, and OFD-overlap. Some of the closely spaced mutations have been adjusted for increased visual clarity. Red bars denote the 50 and 30 limitsof the PHS region at nucleotides 1998 and 3481, respectively. The colored bars on the protein show the conserved domains of GLI3 as definedelsewhere [Ruppert et al., 1990].

1152 HUMAN MUTATION, Vol. 31, No. 10, 1142–1154, 2010

additional patients will be diagnosed molecularly, which can bevaluable for directing further clinical evaluations (endocrine andimaging studies), prognostic advice, molecular diagnostics inother family members, and family planning.

Beyond the clinical diagnostic utility, these data further theunderstanding of the biology of this gene and its pathway. Themutational spectra of typical GCPS and PHS are distinct; GCPS iscaused by a wide range of mutations, but PHS is caused essentiallyonly by truncating mutations. The data presented here, combinedwith published cases [Borg et al., 2007; Fujioka et al., 2005; Furnisset al., 2009; Johnston et al., 2005; Mendoza-Londono et al., 2005;Roscioli et al., 2005; Yilmaz et al., 2008], describe 147 mutations inpatients with typical GCPS or PHS. The mutation distribution inthese two phenotypes is distinct. The GCPS mutations includelarge deletions/duplications (n 5 31) and translocations (n 5 5),and a variety of point mutations including missense (n 5 9), inframe deletions (n 5 1), splice (n 5 11), and frameshift ornonsense mutations (n 5 54). The distribution among patientswith PHS is limited to one splice mutation and 35 frameshift ornonsense mutations. The difference in these mutation spectra ishighly statistically significant (frameshift/nonsense vs. all othertypes; Fisher’s exact test o0.0001). We have previously shown thatamong the patients with truncating or frameshift mutations, theposition of the mutations in GLI3 robustly correlates with thephenotype; patients with PHS have mutations only in the middleportion of the gene (cDNA position 1998–3481), whereas patientswith GCPS typically have mutations 50 of position 1998 or 30 of3481. Again, the association between mutation position andphenotype is highly significant (50 or 30 mutation vs. middlesegment, Fisher’s exact test o0.0001).

The data presented here not only strengthen the knownassociation among those with typical GCPS and PHS but alsoshow the same mutation trend in atypical forms of the disorders.Among eight probands with sub-PHS, all eight mutations areframeshift or nonsense, whereas this is the case for slightly morethan half, five of eight, of the sub-GCPS probands. Seven of eightof the PHS truncation or nonsense mutations lie in the middlethird of the gene, whereas this is the case for none of five frameshiftor nonsense mutations among patients with sub-GCPS. These datasupport the notion that the anomalies of GCPS and PHS arespecific to their mutational mechanism, whether those anomaliesare typical (PHS and GCPS) or atypical (sub-PHS and sub-GCPS).

There was no apparent correlation for the type or position offrameshift or nonsense mutations within the sub-PHS group thatexplained or predicted that these mutations caused an atypicalphenotype as distinct from typical PHS, as nearly all were in themiddle third of the gene. However, we did find a correlation ofmutations in sub-GCPS patients that distinguished them fromGCPS. In probands with GCPS, the frameshift or nonsensemutations were distributed among the three segments of the gene;50 segment (n 5 31), middle segment (n 5 9), and 30 segment(n 5 14). In contrast, five of five frameshift or nonsense mutationsin probands with sub-GCPS were in the 30 segment of the gene (30

mutation vs. 50 or middle segment, Fisher’s exact test 5 0.0023).These data suggest that the frameshift and nonsense mutations inthe 30 segment of the gene cause distinct biologic and phenotypicconsequences from those in the other two segments of the gene.

The transition at nucleotide 1998 relates to the position of thesemutations with respect to the zinc-finger domain-encoding regionand the normal proteolytic processing site of the GLI3 protein [Kalff-Suske et al., 1999]. The transition at nucleotide 3481 may relate to thepresence of the transactivation domain [Ruppert et al., 1990; Shinet al., 1999]. There are known exceptions to these correlations. There

is a recurrent c.2374C4T, p.R792X mutation, which lies within thePHS region of the gene, but in eight of eight families (including oneproband in this report) is associated with a typical GCPS phenotype[Debeer et al., 2003; Furniss et al., 2009; Johnston et al., 2005; Kalff-Suske et al., 1999]. A similar mutation, c.2741delG, p.G914AfsX38,has been identified in a single family with a typical GCPS phenotypein this report. The proband in this case manifested postaxialpolydactyly with macrocephaly and hypertelorism and had a familyhistory of preaxial polydactyly. A third exception is a single familywith PHS that has a splice mutation instead of a frameshift ornonsense mutation, although that mutation likely produces atruncated gene product [Johnston et al., 2005].

These data show that the clinical spectrum of phenotypescaused by mutations in GLI3 is wider than previously appreciated.Further, they demonstrate that some mutant alleles of GLI3 cancause malformations that are milder than the typical, clinicallydefined pleiotropic picture of these disorders, in that they do notdemonstrate all of the features required for a clinical diagnosis.The previously reported association of mutation type andphenotype (PHS vs. GCPS) is strengthened by this report and itis extended into milder phenotypes as well. In addition, thedistribution of frameshift and nonsense mutations in patientswith sub-GCPS is distinct from that in those with typical GCPS,which suggests that these mutations are pathogenetically distinct.The data presented here should encourage molecular diagnosticlaboratories to test a wider array of patients and the data should beuseful to further understand the pathogenesis of these distinctpleiotropic developmental anomalies.

Acknowledgments

The authors thank the following genetic professionals for referring patients

to our study: William P. Allen, David J. Aughton, Christopher Cunniff,

Sally Davies, William B. Dobyns, Linda Genen, Daniel Gruskin, Ketil

Heimdal, Gail Herman, Jodi Hoffman, Helen Hughes, LaDonna Immken,

Jeffrey Innis, Ian Krantz, David Manchester, Elizabeth McPherson, Thomas

Morgan, Maximilian Muenke, Tracy Oh, Melissa Parisi, Betsy Peach, Lynda

Pollack, Nazneen Rahman, Miranda Splitt and LuAnn Weik. Grant

sponsors: SHARE’s Childhood Disability Center, the Steven Spielberg

Pediatric Research Center, the NIH/NICHD Program Project Grant; grant

number: HD22657; the Medical Genetics NIH/NIGMS Training Program

Grant; grant number: 5-T32-GM08243 (all to J.M.G). Grant sponsor:

funding from the Intramural Research Program of the National Human

Genome Research Institute of the National Institutes of Health. We also

acknowledge the Manchester NIHR Biomedical research Centre.

Disclaimer: The opinions and assertions contained herein are the views of

the authors and are not to be construed as official or as reflecting the views

of the United States Department of Defense.

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