_JMed Genet1996;33:873-87687

Isolated autosomal dominant type Bbrachydactyly: exclusion of linkage to candidateregions 2q37 and 20q13

M E M Oude Luttikhuis, D K Williams, R C Trembath

AbstractType E brachydactyly is a digital mal-formtration which characteristically causesan asymmnetrical shortening of one ormore metacarpals or metatarsals or both.Although commnonly seen as part of a syn-drome, it can be inherited as an autosomaldominant characteristic, the gene actingwith variable expressivity, but completepenetrance. As an Albright hereditaryosteodystrophy (AHO)-like syndrome in-cluding brachydactyly type E and mentalretardation may be caused by (micro)deletions at chromosome 2q37, thisregion together with the AHO locus atchromosome 20q13 were considered as

candidate loci for brachydactyly type E.In this paper we describe a famifly withisolated autosomal dominant type E bra-chydactyly inwhom molecular analysis ex-cludes linkage to these regions, providingsupport for further genetic heterogeneityof this trait.(J7Med Genet 1996;33:873-876)

Key words: brachydactyly type E; Albright hereditaryosteodystrophy; Gsot gene.

The brachydactylies are a heterogeneous groupof digital anomalies classified by Bell in 195 1.1Bell reviewed 124 pedigrees containing 1336

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Departmrents ofGenetics,Medicine, andTherapeutics,University ofLeicester,Leicester LEl 7RH,UKM E M Oude LuttikhuisR C Trembath

Clinical GeneticsDepartmrent,Leicester RoyalInfirmary NHS Trust,Leicester LEI 5WW,UKD K WilliamsR C Trembath

Correspondence to:Dr Trembath.Received 27 February 1996Revised version acceptedfor publication 24 May 1996

11.1 11.2 1113

*DNA *DNA

Iv.i1 IV.2Examined Examined

11.2 11.3 11.4115 1

111.4 11.5 11.6 111.7 111.8 119 111.10

*DNA

IV3 IV4 IV.5 V.6Examined

jDNA

V.5 JVt63imined

Figure 1 Brailsford~s originaljfamily with autosomal dominant brachydactyly type E, pedigree updated 1995.

DNA V*DNA ~DNA 6DNA

V.1 V.2 V.3 V.4Examined Not Examined Examined Exe

examined

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11-1 11.2

Oude Luttikhuis, Williams, Trembath

Table 1 Summary of clinical features described

Metacarpal shortening Metatarsal shortenzing

Subject Left hand Right hand Left foot Right foot

IV.2 3, 4 3, 4 1, 4 1, 4IV.3 3 3 1, 4 1, 4V.1 3 3 3,4 3,4V.3 3 3 1, 4 1V.5 3,4 3 3,4,5 4,5

people with this malformation and on ana-tomical grounds defined seven clear groups.Type E brachydactyly is distinguished by short-ening of one or more of the metacarpals ormetatarsals or both. A striking feature is thecommon lack of symmetry and lack of short-ening of corresponding bones of the hands andfeet, together with intrafamilial variation. Atthat time Bell' also recognised that this type ofbrachydactyly was heterogeneous, featuring inseveral genetic conditions (notably Turner syn-drome and Albright hereditary osteodystrophy)in addition to being inherited as a distinctautosomal dominant characteristic. In 1995,chromosome 2q37 was suggested as a can-didate region for type E brachydactyly,2 basedon the presence of (micro)deletions in fivepatients with short metacarpals, mental re-tardation, and other dysmorphic features sim-ilar to that observed in Albright hereditaryosteodystrophy, which is caused by mutations

in the Gec gene located on chromosome20q13.3-5 The purpose of this study was toevaluate these chromosomal loci as possiblecandidate regions for familial brachydactylytype E, and in particular to assess whethermicrodeletions on chromosome 2q37 mightrepresent a contiguous gene syndrome in-volving a gene for brachydactyly type E.2 Mo-lecular analysis was therefore performed ineight members of a family with autosomal dom-inant brachydactyly type E using six micro-satellite markers and a single VNTR onchromosome 2q37, together with three markerson chromosome 20q13.

Patients, materials, and methodsWe recontacted surviving members of a bra-chydactyly type E family originally describedby Brailsford6 (fig 1). The clinical features ofthose with the characteristic are summarisedin table 1. Hand x rays of IV.3 showing bilateralshortening of the third metacarpals and x raysof the feet showing bilateral shortening of thefirst and fourth metatarsals are shown in fig 2.Other congenital malformations, short stature,and mental retardation were not observed.

DNA STUDIESGenomic DNA was isolated from peripheralleucocytes using standard methods. Genotypes

Figure 2 X rays of hands and feet of IV3.

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Isolated autosomal dominant type E brachydactyly

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Markers AlielesD20S93 1,2,3,",5.6.7GNASin3 1,2GNASex5 1,2

111 1

1 1- 2

1 4 2 71 1 2 21 2 1 2

V.2Vi1.V 3 v 4I V.o5

1 7 1 2 4 2 4 2 21 2 1 2 1 2 1 2 1 21 2 1 1 1 1 2 1 1

Figure 3 Chromosome 20q13 haplotype data.

for chromosome 20 markers were determinedusing an endonuclease restriction site poly-morphism (FokI) in exon 5 of Gsoc,37 a dinucleo-tide repeat in intron 3 of the same gene,8 anda highly polymorphic tetranucleotide repeat(D20S93) which maps to a maximum distanceof 4 cM distal to the Gsct gene.9 PCR am-

plification of D20S93 was performed in thebuffer described by Wilson et al 2 for 23 cyclesat an annealing temperature of 62°C. The di-nucleotide repeat markers D2S125, D2S395,D2S345, D2S336, and D2S338 were PCRamplified in the presence of [&C32P]-dCTP atannealing temperatures of 55°C, 62°C, 60°C,60°C, and 60°C respectively. The first fourmarkers were amplified in the same buffer asthat used in the D20S93 study while markerD2S338 was amplified in the buffer used forexon 5 amplification in the presence of 1 mmol/lMgCl2. The PCR products of a further marker,D2S 140, were radiolabelled using a reverse

primer end labelled with [y33P]-ATP in a re-

action with the D20S93 buffer at an annealingtemperature of 56°C. All PCR reactions were

performed for 23 cycles, subjected to electro-phoresis on 6% denaturing polyacrylamide

112

gels, and visualised by autoradiography.'0 Theprimer sequences were described by Gyapay etal." The genotypes for the VNTR D2S90 weredetermined as previously described by Wilsonet al.2

ResultsThe family analysed in this study contained sixpeople with classical type E brachydactyly. Theresults of the chromosome 20q13 study aresummarised in fig 3. The family was un-informative for both polymorphisms in Gsa

113 11.4 and fully informative for the flanking markerD20S93. Direct inspection showed that nocommon haplotypes are shared by all affectedpeople and the data therefore exclude the Gsagene in the development of the brachydactylyE phenotype in this family. The genotypes foreach marker on terminal chromosome 2q weredetermined and haplotypes inferred (fig 4).Inspection failed to show a common haplotypeshared by all affected subjects, excluding link-

IV3 age of autosomal dominant brachydactyly typeE in this family to this region of the genome.

6 3 Two point linkage analysis of disease locusagainst each marker at 2q confirmed the ab-sence of linkage for all markers using MLINK,with exclusion (lod < -2) to recombinationfractions (0) of 0.15 or more (data not shown).

DiscussionWe chose to study two candidate loci becauseof the known association of brachydactyly typeE with Albright hereditary osteodystrophy,caused by mutations in the Gsot gene on chro-mosome 20q13 and an AHO-like syndromerecently defined by (micro)deletions at chro-mosome 2q37, which could be a contiguousgene defect involving a locus for brachydactylytype E.2 These results show no evidence oflinkage to these two regions of the genome andgive no evidence of support for a contiguousgene defect in the AHO-like syndrome. Othersyndromes featuring brachydactyly type E in-clude Biemond syndrome I and Ruvalcaba syn-drome and their localisation will enable furthercandidate loci for familial isolated bra-chydactyly type E to be considered. In addition,homeobox containing genes and genes en-coding growth factors, both classes of genesimplicated in the control of distal limb de-velopment, should be considered. Indeed thegenes HOXD, MSX1, MSX2, FGF-1, andFGF-2 have recently been excluded in twofamilies with type Al brachydactyly.'2 Thesegenes should clearly be considered in othersubtypes of brachydactyly.

The first two authors contributed equally to this work.

1 Bell J. The treasury of human inheritance. Volume V. Onhereditary digital anomalies. Part I. On brachydactyly andsymphalangism. Cambridge: Cambridge University Press,1951.

2 Wilson LC, Leverton K, Oude Luttikhuis MEM, et al.Brachydactyly and mental retardation: an Albright her-editary osteodystrophy-like syndrome localised to 2q37.Am 7Hum Genet 1995;56:400-7.

3 Wilson LC, Oude Luttikhuis MEM, Clayton PT, FraserWD, Trembath RC. Parental origin ofGsot gene mutationsin Albright's hereditary osteodystrophy. J7Med Genet 1994;31:835-9.

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4 Oude Luttikhuis MEM, Wilson LC, Leonard JV, TrembathRC. Characterization of a de novo 43-bp deletion of theGs gene (GNAS1) in Albright hereditary osteodystrophy.Genonuics 1994;21:455-7.

El 5 Yu S, Yu D, Hainline BE, et al. A deletion hot-spot in exon7 of the Gsoz gene (GNAS 1) in patients with Albrighthereditary osteodystrophy. Hunm Mol Genet 1995;4:2001-2.

6 Brailsford JF. Familial brachydactyly. Br _7 Radiol 1945;XVIII: 167-72.

7 Gejman PV, Weinstein LS, Martinez M. Genetic mappingof the GsY subunit gene (GNAS 1) to the distal long armof chromosome 20 using a polymorphism detected bydenaturing gradient gel electrophoresis. Genoniics 1991 ;9:782-3.

8 Granqvist M, Xiang K, Seino M, Bell GI. Dinucleotiderepeat polymorphism in Gs-alpha subunit gene (GNAS 1)on chromosome 20. Nucleic Acids Res 1992;19:4569.

9 Melis R, Bradley P, Elsner T, et al. Polymorphic SSR(simple-sequence-repeat) markers for chromosome 20.Genomics 1993;16:56-62.

U- 10 Sambrook J, Fritsch EF, Maniatis T. Molecular cloning. Alaboratory nianuaL 2nd ed. New York: Cold Spring HarborLaboratory Press, 1989.

11 Gyapay G, Morissette J, Vignal A, et al. The 1993-94Genethon human genetic linkage map. Nat Geniet 1994;7:246-339.

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