Am. J. Hum. Genet.,63:711-716, 1998

0002-9297/98/6303-0009$02.00

© 1998 by The American Society of Human Genetics. All rights reserved.

MutationsinFibroblastGrowth-FactorReceptor3inSporadicCasesofAchondroplasiaOccurExclusivelyonthePaternallyDerivedChromosome

DouglasJ.Wilkin,¹JinnyK.Szabo,¹RhodaCameron,²ShirleyHenderson,³GaryA.Bellus,^1,4,*MichelleL.Mack,^1,5IlkkaKaitila,⁶JohnLoughlin,³ArnoldMunnich,²BryanSykes,³
JackyBonaventure,²andClairA.Francomano<sup>1,4


1</sup>MedicalGeneticsBranch,NationalHumanGenomeResearchInstitute,NationalInstitutesofHealth,Bethesda;²INSERMU393,InstitutNecker,Paris;³UniversityofOxford,DepartmentofCellularScience,InstituteofMolecularMedicine,TheJohnRadcliffeHospital,Oxford;⁴CenterforMedicalGenetics,JohnsHopkinsUniversitySchoolofMedicine,Baltimore;⁵GeorgeWashingtonUniversity,InstituteforBiomedicalSciences,WashingtonDC;and⁶DepartmentofClinicalGenetics,HelsinkiUniversityCentralHospital,Helsinki

Received December 10, 1997; accepted for publication July 15, 1998; electronically published August 7, 1998.

Summary

     More than 97% ofachondroplasia cases are causedby one of twomutations (G1138A and G1138C)in the fibroblast growthfactor receptor 3 (FGFR3)gene, which results ina specific amino acidsubstitution, G380R. Sporadic casesof achondroplasia have beenassociated with advanced paternalage, suggesting that thesemutations occur preferentially duringspermatogenesis. We have determinedthe parental origin ofthe achondroplasia mutation in40 sporadic cases. Threedistinct 1-bp polymorphisms wereidentified in the FGFR3gene, within close proximityto the achondroplasia mutationsite. Ninety-nine families, eachwith a sporadic caseof achondroplasia in achild, were analyzed inthis study. In thispopulation, the achondroplasia mutationoccurred on the paternalchromosome in all 40cases in which parentalorigin was unambiguous. Thisobservation is consistent withthe clinical observation ofadvanced paternal age resultingin new cases ofachondroplasia and suggests thatfactors influencing DNA replicationor repair during spermatogenesis,but not during oogenesis,may predispose to theoccurrence of the G1138FGFR3 mutations.

     Achondroplasia (MIM 100800) isthe most common formof dwarfism in humans.The disorder is inheritedin an autosomal dominantmanner, with full penetrance.Two mutations, G1138A andG1138C, in exon 10of the FGFR3 generesult in a specificamino acid substitution (G380R)(Rousseau et al. 1994;Shiang et al. 1994)and account for morethan 97% of allreported cases (Bellus etal. 1995a). On thebasis of the incidenceof achondroplasia, FGFR3 nucleotide1138 is among themost highly mutable singlenucleotides known in thehuman genome. Specific FGFR3mutations also have beenidentified in disorders relatedto achondroplasia, including thanatophoricdysplasia types I andII (Tavormina et al.1995; Rousseau et al.1996), hypochondroplasia (Bellus etal. 1995b), and severeachondroplasia with developmental delayand acanthosis nigricans dysplasia,a recently described skeletaldysplasia (Francomano et al.1996; G. A. Bellus,M. J. Bamshad, andC. A. Francomano, unpublisheddata). In addition, FGFR3mutations have been identifiedin several craniofacial disorders,including nonsyndromic craniosynostosis andCrouzon syndrome with acanthosisnigricans (Meyers et al.1995; Bellus et al.1996).

     Sporadic cases of achondroplasiaand other dominant geneticdisorders have been associatedwith advanced paternal age,suggesting that these mutationsoccur preferentially during spermatogenesis(Penrose 1957; Thompson etal. 1986). Using single-base-pairpolymorphisms in the FGFR3gene that are proximateto the site ofthe achondroplasia mutation, wehave determined the parentalorigin of the achondroplasiamutation in 40 sporadiccases. Heteroduplex analysis wasused to screen forFGFR3 intron polymorphisms, identifyingpolymorphisms in FGFR3 introns9 and 10. DNAfrom 99 families withsporadic occurrences of achondroplasiawas analyzed for heterozygosityof these polymorphisms, withinformative probands heterozygous forat least one ofthe polymorphisms. The amplification-refractorymutation system (ARMS) (Moloneyet al. 1996) orsequencing was used todetermine the phase ofthe polymorphism, together withthe achondroplasia mutation ornormal allele, in theprobands. Determination of thepolymorphic variant status inthe parents allowed theidentification of the parentfrom whom the mutantallele had been inherited.This analysis revealed thatthe achondroplasia mutation occurredon the paternal chromosomein all 40 informativeprobands. The paternal originof the mutant achondroplasiaallele suggests that factorsinfluencing DNA replication or<
UP> repair during spermatogenesis, butnot during oogenesis, maypredispose to the occurrenceof achondroplasia.

     Since the recurrent achondroplasiamutation is in exon10 of the FGFR3gene, regions of thegene that are closeto this site wereexamined for polymorphisms. Primerpairs (int10-F and int10-R,961-F and DT-R, orDT-F and 1395-R) (table 1)were used to amplifyFGFR3 intron 9 orFGFR3 intron 10. Polymorphismswere identified by eitherSSCP or heteroduplex analysisand/or by sequencing. Primers961-F, I9A-F, and I9B-Fwere used as sequencingprimers for intron 9,and primers 1395-R andI10B-R were used tosequence intron 10. Sequencingwas performed either directly,by use of DynabeadsM-280 streptavidin (Dynal) (Theinand Hinton 1991), orby use of anABI 377 automated DNAsequencer (Applied Biosystems).

Table 1     FGFR3 Oligonucleotide Primer Sequences

     Polymorphisms thataltered a restriction sitewere analyzed by meansof the ARMS method(Moloney et al. 1996).In brief, in theprobands, allele-specific PCR reactionswere designed to amplifyan FGFR3 gene regionthat included both thesite of the polymorphismand the site ofthe common achondroplasia mutation.Allele-specific PCR was performedon either the mutantor normal chromosome, bymeans of oligonucleotide primersspecific for either thenormal allele (1138 wt-For 1138wt-R) or theachondroplasia allele (1138mut-F or1138mut-R), and the appropriatecorresponding primer (CD5-F orint10-R) (table 1). Allele-specific primerswere designed to createa mismatch at thenucleotide adjacent to the3' nucleotide, with the3' nucleotide corresponding toFGFR3 nucleotide 1138 (Kwoket al. 1990) (fig. 1).The normal-allele PCR primers(1138wt-F and 1138wt-R) containedthe wild-type nucleotide G(or C, on thereverse primer) as the3' base, maximizing themismatch to the mutantallele (fig. 1). The mutant-allelePCR primers (1138mut-F and1138mut-R) contain the achondroplasia-mutationnucleotide A (or T,on the reverse primer)as the 3' base,maximizing the mismatch tothe normal allele (fig. 1).Digestion of the PCRproduct, with either PflMIor PmlI, distinguished thephase of the polymorphismand mutat
on. For theintron 9 PflMI polymorphism,primers CD5-F and either1138wt-R or 1138mut-R wereused to amplify a619-bp fragment (fig. 2). PCRof the intron 10Pml1 polymorphism used primersint10-R and either 1138wt-For 1138mut-F to amplifya 473-bp fragment (fig. 2).Parental genomic DNA wasamplified with either oligonucleotideprimers CD5-F and 1138wt-Ror int10-R and 1138wt-F(table 1). PCR reactions wereperformed with an initialdenaturation step of 2min at 94°C, followedby 35 cycles ofdenaturation for 30 sat 94°C, 30 sat an annealing temperatureof 60°C or 62°C,and an extension stepfor 30 s at72°C. After amplification, thePCR products were digestedwith the appropriate restrictionenzyme (PmlI or PflMI)and were analyzed byagarose-gel electrophoresis.

Figure 1     FGFR3 achondroplasia mutantand wild-type allele-specific oligonucleotideprimers, designed to createa mismatch at thenucleotide adjacent to the3' nucleotide, with the3' nucleotide corresponding toFGFR3 nucleotide 1138, thebase mutated in thecommon achondroplasia mutation analyzedin this study. Thenormal-allele PCR primers (1138wt-Fand 1138wt-R) contained thewild-type nucleotide G (orC on the reverseprimer) as the 3'base, maximizing the mismatchwith the mutant allele.The mutant-allele PCR primers(1138mut-F and 1138mut-R) containthe achondroplasia-mutation nucleotide A(or T on thereverse primer) as the3' base, maximizing themismatch with the normalallele.

Figure 2     PmlI (A) and PflMI(B) restriction digestion ofallele-specific PCR products, asrecommended by the manufacturer.The sizes of productsof restriction digestion areshown. Analyses of fourfamilies are presented. Allele-specificPCR of genomic DNAwas performed with eithera normal-sequence (wt) oligonucleotideprimer or an oligonucleotideprimer specific for thecommon achondroplasia mutation (mut)(see fig. 1). The allele-specificprimer used in thePCR reactions is depictedas wt or mut.Reactions using DNA fromthe probands are shownin lanes 2, 3,6, and 7. PCRusing genomic DNA fromthe affected child wasperformed with either awt oligonucleotide primer ora mut primer. ParentalDNAs are shown inlanes 1, 4, 5,and 8. PCR ofparental genomic DNA wasperformed with a wtprimer. In each caseshown, the allele amplifyingwith the mut primeris inherited from thefather.

     The polymorphic stretch ofguanosine residues was analyzedby either of twomethods: by direct sequencingof PCR products orby cloning of thePCR products and thensequencing (TA cloning; Invitrogen).Allele-specific PCR was usedto amplify probands' genomicDNA corresponding to thenormal or FGFR3 G1138Amutant allele, by useof both primer 1138wt-Ror primer 1138mut-R andthe CD5-F corresponding primer(table 1). Products from amplificationsusing the allele-specific primerswere purified (Quiquick PCRPurification columns; Qiagen) andwere sequenced. Parental genomicDNA was amplified byuse of primers 1138wt-rand CD5-F (table 1). PCRproducts were cloned intothe pCR-3 vector (Invitrogen),plasmid DNA was isolated,and the insert wassequenced directly to determinethe phase of thepolymorphic allele and themutation.

Determination of Polymorphisms

     Three distinct polymorphisms wereidentified in the FGFR3gene, within close proximityto the achondroplasia mutationsite

two in intron 9and one in intron10. The polymorphisms includeda G C transition towardthe 5' end ofintron 9, which createda PflMI site; aC T transversion in intron10, which created aPmlI site; and asingle G deletion ina stretch of 11consecutive guanosine residues inintron 9. To determinethe frequency of thePflMI and PmlI polymorphismsin the general population,chromosomes of normal individualswere genotyped. Ten (4.5%)of 224 chromosomes carriedthe PflMI polymorphism, and30 (10.4%) of 288chromosomes carried the PmlIpolymorphism.

     For a family tobe informative for anypolymorphism, the proband mustbe heterozygous at thesite (see results discussedbelow), with only oneparent heterozygous for thepolymorphism or both parentshomozygous for opposing variants(fig. 3). Families in whichthe proband was homozygousfor all three polymorphismsor in which bothparents were heterozygous forthe polymorphisms and inwhich the affected childalso was heterozygous werenoninformative. Ninety-one families weregenotyped for the PmlIpolymorphism, 83 were genotypedfor the PflMI polymorphism,and 82 were genotypedfor the poly(G) variant.Most families were typedfor more than onepolymorphism. Once an informativepolymorphism had been found,no further analysis wasdone on that family.

Figure 3