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, G. S; Hove, Hanne; Kjærsgaard Hansen, Lars; Fagerberg, ChristinaRingmann; Tajir, Mariam; Wilkie, Andrew O MPublished in:Clinical Genetics
DOI:10.1111/cge.12721
Publication date:2016
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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
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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: [email protected]
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
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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
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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,
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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
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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.).
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