Carpal and tarsal synostoses and transverse reduction defects of the toes in two brothers heterozygous for a double de novoNOGGIN mutation

Carpal and tarsal synostoses and transverse reduction defects of the toes in two brothers heterozygous for a double de novoNOGGIN mutation
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   American Journal of Medical Genetics 134A:318–320 (2005) Clinical Report Carpal and Tarsal Synostoses and Transverse ReductionDefects of the Toes in Two Brothers Heterozygous for aDouble De Novo  NOGGIN   Mutation Philippe Debeer, 1,2 * Christel Huysmans, 3  Wim J.M. Van de Ven, 3 Jean-Pierre Fryns, 1 and Koen Devriendt 1 1 Centre for Human Genetics, Herestraat 49, Leuven, Belgium  2  Department of Orthopedics, Weligerveld 1, Pellenberg, Belgium 3  Laboratory for Molecular Oncology, Herestraat 49, Leuven, Belgium  We describe two siblings with carpal and tarsalsynostoses associated with transverse deficien-cies of the toes. Mutation analysis of the  NOG gene revealed a double missense mutation in both boys resulting in Pro42Ala and Pro50Arg.The parents were clinically unaffected, and thesetwo mutations were not detected in their white blood cells or buccal mucosa. This indicatesthe presence of gonadal mosaicism or a low levelofsomaticmosaicisminoneoftheparents.  2005 Wiley-Liss, Inc. KEY WORDS: BMP antagonist; mosaicismINTRODUCTION Noggin,anantagonistofmembersoftheTGF b family,playsanimportantroleinjointmorphogenesisinmiceandhumans.Homozygous Noggin mouse mutants have severe defects inribs,vertebrae,andlimbs.Limbsareshorterandbroaderwithfusionsattheelbowandabsenceofdigitaljoints[Brunetetal.,1998]. In humans,  NOG  mutations are responsible forproximal symphalangism (SYM1, OMIM 185800), multiplesynostosis syndrome (SYNS1, OMIM 186500), tarsal/carpalcoalition syndrome (TCC, OMIM 186570), and autosomaldominant stapes ankylosis [Gong et al., 1999; Dixon et al.,2001; Brown et al., 2002].Here,wedescribetwobrotherswithsynostosesofthecarpalandtarsalbones,andseveretransversedefectsofthetoes.Oneoftheboysalsohasahearingimpairment.Mutationanalysisof the  NOG  gene revealed the presence of two novel mutationsthat were not present in the unaffected parents. PATIENT REPORTS Patient 1 and Patient 2 are the second and third children of healthy non-consanguineous parents. Both were born after anuneventfulpregnancy.Malformationsoffingersandtoeswerenoted immediately at birth. At the age of 9 years, Patient 1 had thin lips, small mouth,andbroadnose(Fig.1a).Skeletalanomaliesincludedproximalsymphalangism of the third fingers, proximal and distalsymphalangismofthefourthandfifthfingers,andclinodactylyofthefifthfingers(Fig.1b).Thethumbswereshortwithamoreadducted position than normally. A transverse reductiondefect of the third and fourth toe with absence of the middleand distal phalanges was noted (Fig. 1c). Radiographs of thehands (at 17 months) showed brachydactyly of the firstmetacarpal with an irregular epiphysis. There was no obvioussynostosis of the interphalangeal joints but there was clearfusionofthecarpalbones(Fig.2a).Boneagewasadvanced(28monthsforbiologicalageof17months).Alsonotedwerefusionbetween the calcaneus and the cuboid and fusion between thecuboidandthecuneiformbone(Fig.2b).Ahearingdeficitof35dB was present at the right side and 12 dB on the left. Thestapedial reflexes were absent on both sides.Patient 2, at 6 years, had the same facial appearance as hisbrother and similar abnormalities of the hands. He also had atransversereductiondefectofthefourthtoewithabsenceofthemiddle and distal phalanges. Radiographs of the feet at 17months showed more pronounced fusion of the tarsal bones:therewasfibrousfusionofthelefttalusandnavicularboneandbilateral fusion of the cuboid, calcaneus, and navicular bones.The second metatarsal was longer than normal (Fig. 3).Radiographs of the hands were not available. Both parentswere normal. METHODS Chromosome analysis on peripheral blood lymphocytesshowed a normal male karyotype in both. DNA was extractedfrom peripheral blood leukocytes according to standardtechniques. The single exon gene,  NOG , was amplified inone PCR round using primers Nog1 and Nog2 as describedby Takahashi et al. [2001]. An allele specific PCR wasperformed using primers Pro42Ala-up (5 0 -GGCACCCAGC-GACAACCTGG-3 0 ) and Pro42Ala-low (5 0 -TGGCCATGA- AGCCTGGGTCG-3 0 ) to detect the Pro42Ala mutation andprimers Pro50Arg-up (5 0 -CAGCCTAGGGGTCACCCTCT-3 0 )and Pro50Arg-low (5 0 -CTTGGGGTCAAAGATAGGGTCTC-3 0 ) for the Pro50Arg mutation. The correct length of the PCRproducts was evaluated by agarose-gel electrophoresis andPCR products were directly sequenced. Amplified fragmentswere subcloned into pGEM-T-Easy and sequenced using standard techniques. Paternity was confirmed usingstandardtechniques (data not shown). RESULTS Sequencing of the  NOG  gene revealed heterozygousc.124C ! G and a c.149C ! G base substitutions in the *Correspondence to: Philippe Debeer, Centre for HumanGenetics, Herestraat 49, B-3000 Leuven, Belgium.E-mail: 15 April 2004; Accepted 1 October 2004DOI 10.1002/ajmg.a.30645   2005 Wiley-Liss, Inc.  coding sequence of   NOGGIN   (GenBank accession numberNM_005450) (Fig. 4). Cloning of the amplified PCR productsand sequencing confirmed the presence of both wild-type andmutant alleles in the population of subclones. Moreover, bothmutations were present on the same chromosome in bothpatients.TheidentifiedbasechangesresultedinPro42AlaandPro50Arg. The de novo origin of both mutations was con-firmed by their absence in peripheral blood leukocytes of theunaffected parents and this finding indicates that bothmutations arise from gonadal or a restricted form of somaticmosaicism in one of the parents. An allele-specific PCRamplifying only the mutant allele was established to detectlowlevelofmosaicismintheparents.Mutantalleleswereonlypresentinperipheralbloodleukocytesofthetwoboysbutcouldnot be demonstrated in the peripheral blood leukocytes or inthe buccal mucosa of either parents (Fig. 5). DISCUSSION Both siblings have characteristic manifestations of SYNS1but with severe reduction defects of the feet, which have not Fig. 1.  a : Facial appearance of Patient 1.  b : Clinical appearance of thehands. Flexion creases at the interphalangeal joints are absent.  c : ClinicalappearanceofthefeetofPatient1.Atransversereductiondefectofthethirdand fourth toe with absence of the middle and distal phalanges is noted.Fig. 2.  a :RadiographsofthehandsofPatient1.Therearecarpalfusionsand brachydactyly of the first metacarpal with an irregular epiphysis.  b :RadiographsofthefeetofPatient1.Thereisafusionbetweenthecalcaneumand the cuboid and between the cuboid and one of the cuneiform bones.Fig. 3. Radiographs of the feet of Patient 2 demonstrating a fusion be-tweenthecuboid,thecuneiformbones,thecalcaneum,andthenavicularbone. Carpal and Tarsal Synostoses 319  been previously described. We found de novo double hetero-zygous mutations in the  NOG  gene: Pro42Ala and Pro50Arg pointing to gonadal or low-grade somatic mosaicism in one of the parents. Gong et al. [1999] described another family withmultiple  NOG  mutations. They identified three base substitu-tions:c.1470T !  A,c.1471C ! T,andc.1475T ! G,resultinginIle120Asn and Tyr222Asp in their family of four.  ACKNOWLEDGMENTS PhilippeDebeerisaSeniorClinicalInvestigatoroftheFundfor Scientific Research, Flanders, Belgium. REFERENCES BrownDJ,KimTB,PettyEM,DownsCA,MartinDM,StrousePJ,MoroiSE,Milunsky JM, Lesperance MM. 2002. Autosomal dominant stapesankylosis with broad thumbs and toes, hyperopia, and skeletalanomaliesiscausedbyheterozygousnonsenseandframeshiftmutationsin NOG, the gene encoding noggin. Am J Hum Genet 71:618–624.Brunet LJ, McMahon JA, McMahon AP, Harland RM. 1998. Noggin,cartilage morphogenesis, and joint formation in the mammalianskeleton. Science 280:1455–1457.Dixon ME, Armstrong P, Stevens DB, Bamshad M. 2001. Identicalmutations in NOG can cause either tarsal/carpal coalition syndrome orproximal symphalangism. Genet Med 3:349–353.Gong Y, Krakow D, Marcelino J, Wilkin D, Chitayat D, Babul-Hirji R,Hudgins L, Cremers CW, Cremers FP,BrunnerHG,Reinker K,RimoinDL, Cohn DH, Goodman FR, Reardon W, Patton M, Francomano CA, Warman ML. 1999. Heterozygous mutations in the gene encoding noggin affect human joint morphogenesis. Nat Genet 21:302–304.Takahashi T, Takahashi I, Komatsu M, Sawaishi Y, Higashi K, NishimuraG, Saito H, Takada G. 2001. Mutations of the NOG gene in individualswith proximal symphalangism and multiple synostosis syndrome. ClinGenet 60:447–451.Fig. 4. Sequencing of the coding region of   NOG  reveals that both brothers (Patients 1 and 2) are heterozygous for a c.124C ! G and c.149C ! G basesubstitution resulting, respectively, in a P42A and a P50R amino acid change in the NOG protein.Fig. 5. AnallelespecificPCRcouldonlydemonstratethemutantallelesinthe peripheral blood leukocytes ofthe twoboys(Patients1and 2)and notin the peripheral blood leukocytes or the buccal mucosa of both parents. Panel a : It shows the results of the allele-specific PCR using the P42A primerset. Panelb :ItdemonstratestheresultsoftheP50Rprimerset;andin panelc ,acontrolPCRwithactinisshown.(M,marker; lane1 ,Patient2blood; lane2 ,Patient1blood; lane3 ,mother’sblood; lane4 ,mother’sbuccalmucosa;  lane 5 , father’s blood;  lane 6 , father’s buccal mucosa;  lane 7 ,unaffected control person (blood); and  lane 8 , negative control). 320 Debeer et al.
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