Syddansk Universitet

Acromelic frontonasal dysostosis and ZSWIM6 mutation

Twigg, Stephen R F; Ousager, Lilian Bomme; Miller, Kerry A; Zhou, Yan; Elalaoui, Siham C;Sefiani, Abdelaziz; Bak, Geske Sidsel; Hove, Hanne; Kjærsgaard Hansen, Lars; Fagerberg,Christina Ringmann; Tajir, Mariam; Wilkie, Andrew O MPublished in:Clinical Genetics

DOI:10.1111/cge.12721

Publication date:2016

Document versionFinal published version

Document licenseCC BY

Citation for pulished version (APA):Twigg, S. R. F., Ousager, L. B., Miller, K. A., Zhou, Y., Elalaoui, S. C., Sefiani, A., ... Wilkie, A. O. M. (2016).Acromelic frontonasal dysostosis and ZSWIM6 mutation: phenotypic spectrum and mosaicism. Clinical Genetics,90(3), 270-275. DOI: 10.1111/cge.12721

General rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright ownersand it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.

• Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ?

Take down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.

Download date: 19. Apr. 2017

http://dx.doi.org/10.1111/cge.12721

Clin Genet 2016: 90: 270–275Printed in Singapore. All rights reserved

CLINICAL GENETICSdoi: 10.1111/cge.12721

Short Report

Acromelic frontonasal dysostosis and ZSWIM6mutation: phenotypic spectrum and mosaicism

Twigg S.R.F., Ousager L.B., Miller K.A., Zhou Y., Elalaoui S.C., Sefiani A.,Bak G.S., Hove H., Hansen L.K., Fagerberg C.R., Tajir M., Wilkie A.O.M.Acromelic frontonasal dysostosis and ZSWIM6 mutation: phenotypicspectrum and mosaicism.Clin Genet 2016: 90: 270–275. © 2015 The Authors. Clinical Geneticspublished by John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.,2015

Acromelic frontonasal dysostosis (AFND) is a distinctive and rarefrontonasal malformation that presents in combination with brain and limbabnormalities. A single recurrent heterozygous missense substitution inZSWIM6, encoding a protein of unknown function, was previously shown tounderlie this disorder in four unrelated cases. Here we describe fouradditional individuals from three families, comprising two sporadic subjects(one of whom had no limb malformation) and a mildly affected female witha severely affected son. In the latter family we demonstrate parentalmosaicism through deep sequencing of DNA isolated from a variety oftissues, which each contain different levels of mutation. This has importantimplications for genetic counselling.

Conflict of interest

All authors declare no conflict of interest.

S.R.F. Twigga,†, L.B. Ousagerb,†,K.A. Millera, Y. Zhoua, S.C.Elalaouic,d, A. Sefianic,d, G.SBake, H. Hovef, L.K. Hanseng,C.R. Fagerbergb, M. Tajirc,d andA.O.M. Wilkiea

aClinical Genetics Group, WeatherallInstitute of Molecular Medicine, Universityof Oxford, Oxford, UK, bDepartment ofClinical Genetics, Odense UniversityHospital, Odense, Denmark, cHumanGenomics Center, Faculty of Medicineand Pharmacy of Rabat, Rabat, Morocco,dDepartment of Medical Genetics,National Institute of Health, Rabat,Morocco, eDepartment of Obstetrics andGynecology, Odense University Hospital,Odense, Denmark, fDepartment ofClinical Genetics, Copenhagen UniversityHospital Rigshospitalet, Copenhagen,Denmark, and gDepartment ofPaediatrics, Hans Christian AndersenChildren’s Hospital, Odense UniversityHospital, Odense, Denmark

†These authors contributed equally to thiswork.

Key words: frontonasal malformation –mosaicism – preaxial polydactyly –ZSWIM6

Corresponding author: Dr StephenTwigg, Clinical Genetics Group,Weatherall Institute of MolecularMedicine, University of Oxford, Oxford,UK.Tel.: +44 1865 222353fax: +44 1865 222500e-mail: stephen.twigg@imm.ox.ac.uk

Received 24 November 2015, revisedand accepted for publication 22December 2015

270 © 2015 The Authors. Clinical Genetics published by John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and

reproduction in any medium, provided the original work is properly cited.

Acromelic frontonasal dysostosis and ZSWIM6 mutation

Acromelic frontonasal dysotosis (AFND; MIM 603671)is characterized by a combination of characteristic fron-tonasal malformation (FNM) with limb defects andanomalies of the brain and usually occurs as a spo-radic disorder. Following initial description in a reviewof the diverse presentations of FNM (1), Verloes et al.proposed AFND as a distinct entity (2); subsequentreports have highlighted characteristic features of severehypertelorism, ptosis, median cleft face with distinc-tive nasal bifurcation and widely separated nasal alae,parietal foramina, variable brain abnormalities includ-ing dysgenesis of the corpus callosum, hydrocephalusand interhemispheric lipoma, limb anomalies with preax-ial polydactyly of the feet, tibial aplasia or hypopla-sia, and talipes equinovarus (3–5). Although mainlyarising sporadically, possible vertical transmission (5)suggested a dominant mechanism, subsequently con-firmed by identification of the underlying heterozy-gous mutation in four AFND cases (6). These fourcases were found to carry an identical mutation ofZSWIM6 (MIM: 615951; c.3487C>T; p.Arg1163Trp),all apparently de novo in origin. In one subject, areduced ratio of mutant to wild-type allele indicatedthat the mutation was present in mosaic state andhad likely arisen post-zygotically; in another family,mild phenotypic features in the father were speculatedto be caused by mosaicism, although no evidence ofZSWIM6 mutation was found in the blood from thisindividual (6).

We describe four additional cases of AFND carryingthe ZSWIM6 c.3487C>T variant. Limb anomalies, usu-ally part of the AFND phenotype, were absent in onecase with a constitutive de novo mutation, establishingthat this is not an absolute requirement for diagnosis.In a mildly affected parent we demonstrate mosaicism,confirming that this mechanism can result in a milderphenotype within the FNM spectrum.

Materials and methods

Subjects

The study was approved by Oxfordshire ResearchEthics Committee B (reference C02.143) and RiversideResearch Ethics Committee (reference 09/H0706/20);written informed consent was obtained from all par-ticipants by the referring clinicians. Karyotyping ofall subjects was normal; although array comparativehybridisation (aCGH; Agilent 244 K) in Subjects 1-1and 1-2 showed a 3.2 Mb duplication of 16p12.3-p13.1that had arisen de novo in the mother, this appears tobe coincidental to the AFND phenotype. DNA wasextracted from peripheral blood samples (Subjects 1-1,1-2, 2 and parents of Subjects 2 and 3), an aminiocen-tesis sample (Subject 3), and buccal brushings, saliva,urine and skin (Subject 1-1). The resequencing panelconsisted of 27 individuals with mild to severe FNMs,with or without extracranial abnormalities, and lackinga molecular diagnosis.

Molecular analysis

A 370 bp fragment covering the ZSWIM6 (RefseqNT_034772.7) exon 14 c.3487C>T variant was ampli-fied using primers E14F 5′-GCTATAATACCTCTGGTGGTCAAGAGTG-3′ and E14R 5′-CCCGAACCAACATCATCAGTTTC-3′. Amplification was carried with0.5 U of FastStart polymerase (Roche Diagnostics,Burgess Hill, UK) in a total volume of 20 μl contain-ing 15 mM Tris–HCl (pH 8.0), 50 mM KCl, 2.5 mMMgCl2, 100 μM each dNTP and 0.4 μM primers. Cyclingconditions consisted of an 8 min denaturation step at94∘C, followed by 33 cycles of 94∘C for 30 s, 63∘Cfor 30 s and 72∘C for 30 s, with a final extension at72∘C for 10 min. This product was sequenced usingBigDye Terminator v3.1 (Applied Biosystems, FosterCity, CA, USA). Deep sequencing on the Ion TorrentPGM platform was used to quantify the proportionsof wild-type to mutant allele in genomic DNA. Frag-ments of 220 bp spanning the c.3487C>T variantwere generated (ZSWIM6-specific primers: Exon14F5′-GCCTACATCAACACAACGCACTCACGG-3′ andExon14R 5′-CATACAAGATCTATCAACCAAACCTCTCCC-3′ with a 10 bp barcode incorporated into eitherthe reverse or forward oligonucleotide and flanked byIon Torrent P1 and A adapter sequences). The P1 andA adapter sequences were flipped so that Ion Torrentsequencing could be carried out in both directions. Thehigh-fidelity Taq polymerase Q5 (NEB, Hitchin, UK)was used for amplification (0.02 U/μl) in a reactionvolume of 25 μl containing 0.5 μM primers, 25 mMTap-HCl (pH 9.3), 50 mM KCl, 2 mM MgCl2, 1 mMβ-mercaptoethanol and 200 μM each dNTP. Cyclingwas carried out as described above except the cyclenumber was reduced to 30 and the annealing temper-ature was 60∘C. Amplification products were purifiedwith AMPure beads (Beckman Coulter, High Wycombe,UK) and emulsion polymerase chain reaction (PCR)and enrichment performed with the Ion PGM TemplateOT2 200 Kit (Life Technologies) according to themanufacturer’s instructions. Sequencing of enrichedtemplates was performed on the Ion Torrent PGM (Lifetechnologies, Carlsbad, CA, USA) for 125 cycles usingthe Ion PGM Sequencing 200 kit v2 on an Ion 316 chip.Data were processed with Ion Torrent platform-specificpipeline software v4.2.1. As the variant must be presentat a level of 50% in a heterozygous individual, and at0% in a normal control, the forward and reverse deepsequencing read counts were separately normalizedusing the data from Subject 1-2 and a control, and theaverage of the two corrected percentages calculated.

Results

Clinical description

The clinical features of Subjects 1-1, 1-2, 2 and 3 aresummarized in Table 1 and shown in Figure 1. Sub-ject 1-2 was born at 32 weeks’ gestation (birth weight:1580 g) and diagnosed with AFND due to the associationof severe FNM and limb abnormalities. Currently aged7 years, he has severe neurocognitive and motor delay

271

Twigg et al.

Tabl

e1.

Clin

ical

feat

ures

ofsu

bjec

tsw

ithZS

WIM

6c.

3487

C>

T;p.

Arg

1163

Trp

Cra

niof

acia

lB

rain

Sub

ject

#G

ende

raE

yes

Nos

eM

outh

Sku

llM

orph

olog

yD

evel

opm

ent

Lim

bsO

ther

1-1b

FH

yper

telo

rism

Wid

ena

salb

ridge

,sh

ortn

asal

ridge

,bi

fidna

salt

ip

Nor

mal

Nor

mal

Nor

mal

Nor

mal

Nor

mal

–

1-2

MS

ever

ehy

pert

elor

ism

,do

wns

lant

ing

palp

ebra

lfiss

ures

Wid

ena

salb

ridge

,w

idel

ysp

aced

nasa

lala

e,w

idel

yse

para

ted

slit-

like

nare

s

Car

p-sh

aped

mou

th,m

idlin

eno

tch

inup

per

lip,

clef

tpal

ate

Bon

yde

fect

ofan

terio

rcr

ania

lfo

ssa,

parie

tal

fora

min

a

Inte

rhem

isph

eric

lipom

a,pa

rtia

lag

enes

isof

the

corp

usca

llosu

m

Sev

ere

mot

oran

dne

uroc

ogni

tive

dela

y

Nor

mal

uppe

rlim

bs,b

ilate

ralt

ibia

lhe

mim

elia

,bila

tera

lbi

fidfir

stto

e,bi

late

ralc

lubf

oot

–

2M

Hyp

erte

loris

m,

bila

tera

lpto

sis,

dow

nsla

ntin

gpa

lpeb

ralfi

ssur

es,

bila

tera

lcat

arac

t

Wid

ena

salb

ridge

,sh

ortn

asal

ridge

,bi

fidna

salt

ip,

wid

ely

spac

edna

sala

lae,

wid

ely

sepa

rate

dsl

it-lik

ena

res

Car

p-sh

aped

mou

th,l

ong

philt

rum

,mid

line

notc

hin

uppe

rlip

,cl

eftp

alat

e

Bon

yde

fect

ofan

terio

rcr

ania

lfo

ssa

Ant

erio

rin

terh

emis

pher

iclip

oma

Sev

ere

psyc

hom

otor

dela

y,ab

senc

eof

spee

ch,

does

notw

alk

aged

8ye

ars

Nor

mal

Mic

rope

nis,

cryp

torc

hidi

sm,

scol

iosi

s

3cF

Hyp

erte

loris

m,

dow

nsla

ntin

gpa

lpeb

ralfi

ssur

es

Apl

asia

/hyp

opla

sia

ofth

ena

salb

ones

,w

ide

nasa

lbrid

ge,

bifid

nasa

ltip

Mid

line

notc

hin

uppe

rlip

––

–N

orm

alup

per

limbs

,bila

tera

ltib

ial

hypo

plas

ia,b

ilate

ral

club

foot

–

aF,

fem

ale,

M,m

ale.

bM

osai

cfo

rth

em

utat

ion.

cP

regn

ancy

term

inat

edat

20w

eeks

’ges

tatio

n.

272

Acromelic frontonasal dysostosis and ZSWIM6 mutation

Fig. 1. Clinical features of individuals with ZSWIM6 c.3487C>T mutations. (a) Subject 1-1 showing facial features at about 1 year of age. Notehypertelorism and bifid nasal tip. (b,c) Neonatal appearance of Subject 1-2, with severe FNM, hypertelorism, carp-shaped mouth with notch in upperlip (b), and bifid great toes and clubfoot (c). (d) Brain magnetic resonance imaging of Subject 2 showing interhemispheric lipoma (white arrow),and severe hypertelorism. (e,f) Ultrasound images of Subject 3 showing orbital hypertelorism (e, arrows indicate the eyes) and hypoplastic nose (f,arrowhead). (g) Clinical appearance of Subject 3 with FNM, hypertelorism and clubfoot. Polydactyly is absent.

and is unable to walk and communicate with words. Hismother, Subject 1-1, who had undergone numerous sur-gical procedures to reshape the frontonasal region, wassuspected to have a milder form of the same disorder. Shehas hypertelorism with a short, broad nose and bifid nasaltip, but normal intelligence and no extracranial features.Subject 2, the third of four children born to unrelated andunaffected parents, is a 12-year-old boy with severe psy-chomotor delay who was not diagnosed with AFND dueto absence of limb abnormalities. Extracranially he hadscoliosis, cryptorchidism and micropenis. Subject 3 wasa female fetus with abnormalities detected by ultrasoundat 19+ 3 weeks’ gestation, including facial malformationwith hypertelorism and broad glabella, nasal hypoplasiaand bilateral talipes equinovarus. Following elective ter-mination of pregnancy, postnatal examination revealedfeatures in keeping with a diagnosis of AFND, includ-ing median facial cleft and bilateral tibial hypoplasia,although polydactyly was absent. No other family mem-bers had similar abnormalities.

Molecular analysis

Screening for ZSWIM6 c.3487C>T in a cohort of 27FNM cases revealed the presence of this variant in threeindividuals, Subjects 1-2, 2 and 3 (Fig. 2a). The ratio

of mutant to wild-type allele was approximately 50:50in each case. As the variant was not detected in bloodfrom Subject 1-1, the mildly affected mother of Subject1-2 and in whom mosaicism was suspected, we ana-lyzed DNA from four other tissues (buccal brushings,saliva, skin and urine). Sanger sequencing was incon-clusive although a subtle drop in peak height of thewild-type allele was evident in the two buccal samples,suggesting the presence of the variant allele (data notshown). This prompted us to undertake more sensitiveIon Torrent-based deep sequencing, which identified themutant variant in all five tissue samples from Subject 1-1(Fig. 2b). The average sequencing depth obtained was>152,800 with a lowest read number of 55,173. The per-centage of variant allele was highest at ∼11% in the buc-cal scrapings (equivalent to ∼22% mutant cells), at 3% insaliva, around 2% in urine and blood and lowest at under1% in skin. The sensitivity of mutation detection usingSanger sequencing is around 6% (7) providing an expla-nation for why testing of DNA from peripheral blood ofSubject 1-1 was negative.

Discussion

AFND is an extremely rare FNM with fewer than20 recognizable cases described in the literature (1,

273

Twigg et al.

Fig. 2. ZSWIM6 sequence analysis. (a) Sequence chromatograms showing ZSWIM6 c.3487C>T in Subjects 1-2, 2 and 3 (red arrows). The C>T variantis absent in Subject 1-1 (DNA from peripheral blood) and the parents of Subjects 2 and 3. (b) Deep sequence analysis for ZSWIM6 c.3487C>T. Theleft hand panel shows the percentage of the variant T allele detected in Subject 1-1 and 1-2. The value for Subject 1-2 has been corrected to 50% andall other figures adjusted accordingly. The T allele is shown in red and the C allele in blue. The right hand panel shows the uncorrected read depthsachieved for each sample.

2, 4–6, 8–10). The most consistent clinical featuresare FNM accompanied by preaxial polydactyly of thelower limbs. The nasal deformity is usually severe,with symmetrical clefting and widely separated slit-likesnares, while limb anomalies can also include tibialhypoplasia and clubfoot. Recently, a recurrent mutationof ZSWIM6, c.3487C>T encoding p.Arg1163Trp, wasidentified in four AFND individuals (6). ZSWIM6 is amember of a group of proteins, found in bacteria, archaeaand eukaryotes, that all contain a SWIM Zn-finger-likedomain that could function both as a DNA bindingdomain or in protein–protein interaction (11). Verylittle is known about the role of ZSWIM6, althoughthe missense substitution identified in AFND is likelyto disrupt the function of a highly conserved sin3-likedomain at the C-terminus of the protein (6). Expressionappears to be ubiquitous although higher in the brain,and analysis of AFND patient cells suggests an effecton hedgehog signaling (6). A molecular-developmentalexplanation for the specific pattern of malformationsoccurring in AFND is currently lacking.

In this report we screened a phenotypically diverseFNM cohort for this variant and identified three positive

individuals, all of whom shared the characteristic nosewith symmetrical, widely separate nostril openings andsevere hypertelorism (Fig. 1b,e). This included a previ-ously undiagnosed patient with severe FNM but normallimbs. A confident diagnosis of AFND with normal limbshas only been possible in one previous case, one of thetwo half-sisters reported by Warkany et al. (10), wherea diagnosis could be made because of the classicallyaffected relative. Our findings imply that similar caseswith isolated severe symmetrical FNM should undergoZSWIM6 screening. Interestingly, although Subject 3 hadlower limb abnormalities, polydactyly was absent, high-lighting that this feature may not always be present either.

Although mosaicism had been suspected in the mildlyaffected parent of a classical AFND patient (6), it was notmolecularly confirmed. We prove, through next genera-tion deep sequencing of DNA from multiple tissues, thatmosaicism can occur in the mildly affected parents ofAFND cases. Notably, the low level of mosaicism foundcould not be convincingly detected by Sanger sequenc-ing, even of multiple tissues. The use of PCR-based orcapture techniques combined with next generation deep

274

Acromelic frontonasal dysostosis and ZSWIM6 mutation

sequencing is an effective method to identify low fre-quency mosaic mutations that are missed by conventionaltechniques (12, 13). In our analysis deep sequencingallowed the convincing detection of mutations at lessthan a 2% level. The finding of mosaicism has importantcounselling implications for AFND families and the pos-sibility of mosaicism in one of the parents of a child witha germline mutation, whether they are mildly affected orappear normal, should be considered. The phenotype ofSubject 1-1 shows similarities to frontorhiny, a distinctFNM caused by biallelic mutations of ALX3 (14). Wepropose that for patients thought to have frontorhiny, butwith a negative ALX3 mutation screen, the possibility oflow-level mosaicism for the ZSWIM6 mutation shouldbe sought by deep sequencing of multiple tissues.

Acknowledgements

We are very grateful to the families for their participation in thisstudy. We thank Sanjena Mithra and Emily Taylor for their assistanceand Sue Butler, John Frankland and Tim Rostron for help withcell culture and DNA sequencing. This work was supported by theNIHR Biomedical Research Centre, Oxford and the Wellcome Trust(Project Grant 093329 to A. O. M. W. and S. R. F. T., and SeniorInvestigator Award 102731 to A. O. M. W.) and Newlife Foundationfor Disabled Children (10-11/04 to A. O. M. W. and S. R. F. T.).

References1. Sedano HO, Cohen MM Jr, Jirasek J, Gorlin RJ. Frontonasal dysplasia.

J Pediatr 1970: 76: 906–913.2. Verloes A, Gillerot Y, Walczak E, Van Maldergem L, Koulischer L.

Acromelic frontonasal "dysplasia": further delineation of a subtype with

brain malformation and polydactyly (Toriello syndrome). Am J MedGenet 1992: 42: 180–183.

3. Sueldo G, Fernandes MC. Fronto-nasal dysostosis, callosal agenesis,crossed-fused ectopia, tibial hemimelia, and preaxial polydactyly of feet:severe expression of the acrocallosal syndrome? Am J Med Genet 1993:46: 355–357.

4. Slaney SF, Goodman FR, Eilers-Walsman BLC et al. Acromelic fron-tonasal dysostosis. Am J Med Genet 1999: 83: 109–116.

5. Hing AV, Syed N, Cunningham ML. Familial acromelic frontonasaldysostosis: autosomal dominant inheritance with reduced penetrance.Am J Med Genet 2004: 128A: 374–382.

6. Smith JD, Hing AV, Clarke CM et al. Exome sequencing identifiesa recurrent de novo ZSWIM6 mutation associated with acromelicfrontonasal dysostosis. Am J Hum Genet 2014: 95: 235–240.

7. Ihle MA, Fassunke J, Konig K et al. Comparison of high resolution melt-ing analysis, pyrosequencing, next generation sequencing and immuno-histochemistry to conventional Sanger sequencing for the detection ofp.V600E and non-p.V600E BRAF mutations. BMC Cancer 2014: 14:13.

8. Edwards WC, Askew W, Weisskopf B. Median cleft face syndrome. AmJ Ophthalmol 1971: 72: 202–205.

9. Calli LJ Jr. Ocular hypertelorism and nasal agenesis (midface syndrome)with limb anomalies. Birth Defects Orig Artic Ser 1971: 7: 268.

10. Warkany J, Bofinger MK, Benton C. Median facial cleft syndromein half-sisters. Dilemmas in genetic counseling. Teratology 1973: 8:273–285.

11. Makarova KS, Aravind L, Koonin EV. SWIM, a novel Zn-chelatingdomain present in bacteria, archaea and eukaryotes. Trends Biochem Sci2002: 27: 384–386.

12. Xu X, Yang X, Wu Q et al. Amplicon resequencing identified parentalmosaicism for approximately 10% of "de novo" SCN1A mutations inchildren with Dravet syndrome. Hum Mutat 2015: 36: 861–872.

13. Hasegawa K, Ohno S, Kimura H et al. Mosaic KCNJ2 mutation inAndersen-Tawil syndrome: targeted deep sequencing is useful for thedetection of mosaicism. Clin Genet 2015: 87: 279–283.

14. Twigg SRF, Versnel SL, Nürnberg G et al. Frontorhiny, a distinctivepresentation of frontonasal dysplasia caused by recessive mutations inthe ALX3 homeobox gene. Am J Hum Genet 2009: 84: 698–705.

275