Molecular, Radiologic, and HistopathologicCorrelations in Thanatophoric Dysplasia

William R. Wilcox,1,2* Patricia L. Tavormina,5 Deborah Krakow,1,3 Hiroshi Kitoh,1Ralph S. Lachman,1,4 John J. Wasmuth,5† Leslie M. Thompson,5 and David L. Rimoin1,2

1Medical Genetics Birth Defects Center, Steven Spielberg Pediatrics Research Center, Cedars-Sinai Burns and AllenResearch Institute, Los Angeles, California

2Department of Pediatrics, UCLA School of Medicine, Los Angeles, California3Department of Obstetrics and Gynecology, UCLA School of Medicine, Los Angeles, California4Department of Radiology, UCLA School of Medicine, Los Angeles, California5Department of Biological Chemistry, University of California at Irvine, Irvine, California

Various mutations in the fibroblast growthfactor receptor 3 (FGFR3) gene have re-cently been reported in thanatophoric dys-plasia (TD). We examined the clinical, radio-graphic, and histologic findings in 91 casesfrom the International Skeletal DysplasiaRegistry and correlated them with the spe-cific FGFR3 mutation. Every case of TD ex-amined had an identifiable FGFR3 muta-tion. Radiographically, all of the cases withthe Lys650Glu substitution demonstratedstraight femora with craniosynostosis, andfrequently a cloverleaf skull (CS) was dem-onstrated. In all other cases, the femorawere curved, and CS was infrequently pre-sent but was occasionally as severe as TDwith the Lys650Glu substitution. Histopath-ologically, all of the cases shared similar ab-normalities, but cases with the Lys650Glusubstitution had better preservation of thegrowth plate. Cases with the Tyr373Cys sub-stitution tended to have more severe radio-graphic manifestations than the Arg248Cyscases, but there was overlap in the pheno-typic spectrum between them. One commonclassification of TD distinguishes affectedinfants based on the presence or absence ofCS. In contrast, and as originally proposedby Langer et al. [1987: Am J Med Genet 3:167–179], our data suggest that TD can be

divided into at least two groups (TD1 andTD2) based on the presence of straight orcurved femora. The variable presence of CSand severity of the radiologic and histologicfindings in the other substitutions may bedue to other genetic, environmental, or sto-chastic factors. Am. J. Med. Genet. 78:274–281, 1998. © 1998 Wiley-Liss, Inc.

KEY WORDS: thanatophoric dysplasia; fibro-blast growth factor receptor3; craniosynostosis; dwarfism

INTRODUCTIONThanatophoric dysplasia (TD) is one of the most com-

mon lethal skeletal dysplasias with a birth incidence of1/35,000 [Martı́nez-Frı́as et al., 1988; Stoll et al., 1989]to 1/50,000 [Orioli et al., 1986]. Affected individualshave severe short-limb dwarfism, relative macro-cephaly, and a small thorax. Most affected newborninfants succumb in the first few days of life. Prolongedsurvival in TD is unusual and is associated with poorgrowth and development and chronic respiratory insuf-ficiency [Moir and Kozlowski, 1976; Stensvold et al.,1986; Tonoki, 1987; MacDonald et al., 1989; Pokharelet al., 1996; Baker et al., 1997]. Radiologically, TD ischaracterized by rhizomelic shortening of the limbs,platyspondyly, short ribs, and a variety of other abnor-malities [Taybi and Lachman, 1996]. Morphologically,the chondrocyte columns are disorganized and short,there is lateral overgrowth of metaphyseal bone aroundthe physis, mesenchymal cells extend inward from theperichondrium as a narrow band at the periphery ofthe physeal zone (the so-called fibrous band), and thereis increased vascularity of the resting cartilage [Ornoyet al., 1985; Langer et al., 1987; Horton et al., 1988].The other organs are unaffected, with the exception ofthe brain, where various abnormalities have been de-scribed, particularly neuronal migration abnormalitiesof the temporal lobe [Wongmongkolrit et al., 1983; Hoet al., 1984; Knisely and Ambler, 1988].

Contract grant sponsor: National Institutes of Health; Contractgrant number: HD22657; Contract grant sponsor: HumanGrowth Foundation; Contract grant sponsor: UCLA Child HealthResearch Center; Contract grant number: P30 HD34610; Con-tract grant sponsor: National Institutes of Health, Clinical Asso-ciate Physician Award; Contract grant number: PA 90-30.

†Deceased.*Correspondence to: William R. Wilcox, M.D., Ph.D., Medical

Genetics, Cedars-Sinai Medical Center, 8700 Beverly Boulevard,SSB-3, Los Angeles, CA 90048. E-mail: wwilcox@mailgate.csmc.edu

American Journal of Medical Genetics 78:274–281 (1998)

© 1998 Wiley-Liss, Inc.

TD has been divided into two radiologic groups basedon two different criteria. One classification scheme di-vides cases by the presence or absence of cloverleafskull (CS) [Taybi and Lachman, 1996]. Langer et al.[1987] analyzed the radiographic findings in 116 casesof TD and the chondroosseous histology in 8 cases (6with straight femora, 2 with curved) and found bettercolumn preservation and less mesenchymal ingrowthin the cases with straight femora. The cases withstraight femora had less platyspondyly and CS,whereas the cases with curved femora infrequently hadcraniosynostosis, and if it was present, it was milder.Langer et al. proposed that TD be divided into the morecommon type 1, consisting of curved femora with vari-able but milder craniosynostosis, and type 2 withstraight femora and CS.

After genetic linkage of achondroplasia to chromo-some band 4p16.3 [Francomano et al., 1994; Le Merreret al., 1994; Velinov et al., 1994], a Gly380Arg substi-tution in the transmembrane domain of the fibroblastgrowth factor receptor 3 gene (FGFR3) was identifiedin more than 98% of cases of achondroplasia [Rousseauet al., 1994; Shiang et al., 1994; Bellus et al., 1995]. Thesimilarity between TD and the homozygous form ofachondroplasia suggested that both disorders may re-sult from mutations in the same gene. Subsequently,our group [Tavormina et al., 1995a,b] and others [Rous-seau et al., 1995, 1996; Bonaventure et al., 1996;Nerlich et al., 1996; Pokharel et al., 1996] describedseveral mutations in the FGFR3 gene in TD. Whereasall cases with straight femora were due to a Lys650Glusubstitution, the more common form with curvedfemora was due to a variety of mutations. In this work,we extend our findings with 49 additional cases of TDfrom the International Skeletal Dysplasia Registry andmore carefully analyze the range of phenotypes foreach mutation.

MATERIALS AND METHODSMutational Analysis

Ninety-one cases of TD collected from 1972 to 1995by the International Skeletal Dysplasia Registry wereanalyzed for mutations in the coding region of theFGFR3 gene by a combination of denaturing gradientgel electrophoresis, restriction enzyme analysis, andsequencing. In general, for each patient sample or cellline, either total RNA or genomic DNA was isolated forsubsequent analysis. Oligonucleotide primers specificfor the region of interest were then used for polymerasechain reaction (PCR) amplification of reverse tran-scription products derived from the isolated RNA or ofgenomic DNA. Screening methods for the Arg248Cys,Ser249Cys, Ser371Cys, Lys650Glu, and Lys650Metmutations from either total RNA or genomic DNA havebeen described in detail elsewhere [Tavormina et al.,1995a,b] (Tavormina et al., in press). For the Gly370Cys(1108G→T) and Tyr373Cys (1118A→G) mutations,fragments of 164 bp were amplified from genomic DNAusing two primers. These primers are 58-AGGAGCTG-GTGGAGGCTGA-38 and 58-GGAGATCTTGTGCACG-GTGG-38. Reactions were done in 1× PCR buffer (Boeh-ringer Mannheim, Indianapolis, IN) with 10% dimethyl

sulfoxide, 125 mM each deoxynucleotide triphosphate,and 25 pmol of each primer. Taq polymerase (1 unit[U]; Boehringer Mannheim) was added immediatelybefore cycling. Cycling parameters were: 30 cycles of94°C, 65°C, and 72°C for 30 sec each, with an initial3-min 94°C denaturation step and a 7-min, 72°C finalextension step. Both mutations create a new BsgI re-striction site, resulting in fragments of 117 and 47 bp(1108G→T) and 106 and 58 bp (1118A→G). Approxi-mately 5 U of BsgI (New England Biolabs, Beverly,MA) was used to digest 2-5 ml of PCR product as rec-ommended by the manufacturer. Dideoxy DNA se-quencing using Sequenase (United States BiochemicalCorp., Cleveland, OH) was done in each case to confirmmutation identification.

Radiographic and Histologic Analysis

Gestational age was usually based on the last men-strual period. We also estimated the gestational age byexamining the radiographs for epiphyseal centers us-ing published normal fetal skeletal X-rays for compari-son [Ornoy et al., 1988]. In general, skeletal matura-tion and the appearance of centers of ossification werenot altered in TD and correlated well with other esti-mates of gestational age. Ultrasonographic data, whenavailable, were also used. Gestational ages estimatedby the abdominal circumference [Tamura and Sab-bagha, 1980] correlated well with the gestational agebased on the last menstrual period (r 4 0.97, P <0.00001). Skeletal radiographs were evaluated for thedegree of craniosynostosis (mild, moderate, severe, orCS), platyspondyly (mild, mild/moderate, moderate,moderate/severe, or severe), curvature of the femora(straight, mild, mild/moderate, moderate, moderate/severe, or severe) and shortening of the femora. Histo-logic sections of long bones, when available, were ex-amined for the extent of column preservation (normal,disordered with some shortening, disordered and short,poor, very poor, or absent columns), mesenchymalbanding (none, peripheral only, peripheral and patchy,almost complete, or complete), and bony abnormalities(normal, mildly, moderately, and very widened trabec-ulae) (Fig. 1). This study was approved by the HumanSubjects Institutional Review Board at Cedars-Sinai.

Statistical Analysis

Pairwise comparisons for different radiographic andhistologic traits were analyzed using the Mann-Whitney U rank-sum test, and two-tailed probabilitiesare reported. For examining the pairwise correlation oftraits, Pearson’s correlation coefficient (r) and a two-tailed probability were computed. All calculations wereperformed with the TRUE EPISTAT™ software pack-age (Epistat Services, Richardson, TX).

RESULTSFGFR3 Mutations

Mutations in the FGFR3 gene were identified in all91 cases of TD (Table I). All of the mutations can beeasily screened using standard molecular biologicaltechniques. The most common mutations result in cys-teine substitutions in the extracellular domain. Five

Thanatophoric Dysplasia 275

Fig. 1. Scored phenotypic features in cases of TD. Skeletal radiographs were evaluated for the degree of craniosynostosis, platyspondyly, andcurvature and shortening of the femora. Histologic sections of long bones, when available, were examined for the extent of column preservation,mesenchymal banding, and bony abnormalities. Femur: straight (A), moderate curve (B), severe curve (C). Column preservation: normal (D), disorderedand short (E), very poor (F). Mesenchymal banding: peripheral only (G), peripheral and patchy (H), complete (I).

276 Wilcox et al.

Fig. 1. Continued.

mutations were identified in the stop codon and arepredicted to result in an abnormal protein with an ad-ditional 141 amino acids at the carboxy-terminal end.Eighteen cases had mutations in the tyrosine kinasedomain: 17 cases with a Lys650Glu and 1 with aLys650Met substitution.

Radiology

There were a total of 91 cases, 73 with curved femoraand 14 with straight femora, and the radiographs wereinadequate to evaluate the femur in 4 cases. The ges-tational age of the cases varied from 17 to 42 weeks(31 ± 7). Qualitatively, the straight femora were thelongest, and among the curved femur cases, the greaterthe femoral curvature, the shorter the length. Therewere 31 cases with some degree of craniosynostosis evi-dent, and 11 cases with a complete CS. The skull couldnot be evaluated in 15 cases because of inadequate ra-diographs. In the milder cases, craniosynostosis waspresent only inferiorly in the coronal and lambdoid su-tures. There was more sclerosis of rostral sutures asthe craniosynostosis became more advanced.

Chondro-osseous Morphology

Although abnormalities are present in all the growthplates in TD, the physes of the long bones were themost useful for analysis, because they have the longest,most regular columns and are the widest. Adequatehistologic sections of the long bones were available in57 cases. All of the cases shared similar findings: thechondrocyte columns were disorganized and short,there was lateral overgrowth of metaphyseal bonearound the physis, the metaphyseal trabeculae wereshort and widened, mesenchymal cells extended in-ward from the perichondrium and periosteum as a nar-row band at the periphery of the physeal zone (theso-called fibrous band), and there was increased vas-cularity of the resting cartilage. However, there wassignificant variability in the organization and length ofthe columns, the extent of the fibrous band, and theabnormalities of the metaphyseal bone. In some cases,the columns were relatively well preserved, the fibrousband was present only at the periphery, and the bony

trabeculae were fairly normal. At the other end of thespectrum, there was no column formation, the fibrousband extended across the entire physis, and the me-taphyseal trabeculae were quite thick. In general, theextent of the fibrous band and metaphyseal bone ab-normalities was directly proportional to the abnormali-ties of the chondrocyte columns (r 4 0.7, P < 0.00001).

Phenotype/Genotype Analysis

The severity of craniosynostosis in the skull, platy-spondyly of the spine, curvature of the femur, and his-tologic abnormalities are graphed in Figure 2 for caseswith the three most common substitutions (Arg248Cys,Tyr373Cys, and Lys650Glu). In the skull, craniosynos-tosis was infrequently present in the Arg248Cys cases,whereas it was almost always present and significantlymore severe in the Lys650Glu cases (P < 0.001). TheTyr373Cys mutation produced an intermediate pheno-type: craniosynostosis was present more frequentlythan in the Arg248Cys group (P 4 0.02), but it wasusually less severe than in the Lys650Glu group (P <0.001). In contrast, the degree of platyspondyly wasmuch more severe in the cysteine substitution casesthan in the Lys650Glu cases (P < 0.001). TheTyr373Cys mutation was generally associated withmore platyspondyly than the Arg248Cys mutation (P4 0.02). Similarly, the amount of shortening and cur-vature of the femur was more severe with a Tyr373Cysthan an Arg248Cys substitution (P 4 0.01). All caseswith straight femora were heterozygous for aLys650Glu substitution; two cases with the Lys650Glusubstitution had mild curvature of the femora.

Histologically, the chondrocyte columns were betterpreserved in the Lys650Glu cases than in the cysteinesubstitution cases (P < 0.001). Similarly, the ingrowthof mesenchymal cells was less extensive with theLys650Glu mutation than either Arg248Cys (P 40.002) or Tyr373Cys (P 4 0.01). In some cases, themesenchymal band transversed the entire physis, andthere was no proliferative or hypertrophic zone pre-sent. Concordant with the preservation of the physis,the trabecular bone was more normal in the Lys650Glucases than in the cysteine substitution cases (P <0.001). Although the radiographic appearance of theTyr373Cys mutations was generally more severe thanthe Arg248Cys cases, there was no significant differ-ence in the chondroosseous morphology between them.

Although there were too few cases of the Ser249Cys,Gly370Cys, and stop codon mutations to analyze sepa-rately, the spectrum of phenotypes was similar to theArg248Cys and Tyr373Cys cases. Even though our casewith the Lys650Met substitution had typical radiologicand histologic findings (in a specimen of the costochon-dral junction) of TD, we know of three other patientswith this same mutation who are alive with a pheno-type between achondroplasia and TD and developedacanthosis nigricans (Bellus et al., submitted). It is in-teresting to note that an individual who survived withTD due to an Arg248Cys mutation also developed ac-anthosis nigricans [Baker et al., 1997].

Except for platyspondyly, the severity of the featuresdid not depend on the gestational age of the fetus. We

TABLE I. FGFR3 Mutations in Thanatophoric Dysplasia

Substitution This seriesa Other reportsb

TD 1Arg248Cys 45 23Ser249Cys 4 3Gly370Cys 1 1Ser371Cys 1Tyr373Cys 18 11Lys650Met 1 3c

Stop codon mutation 5 7TD 2

Lys650Glu 17Total 91 48

aIncludes 42 Registry cases from Tavormina et al. [1995a].bTavormina et al. [1995a,b (excluding Registry cases)], Rousseau et al.[1995, 1996], Bonaventure et al. [1996], Nerlich et al. [1996], Pokharel etal. [1996], Bellus et al. [1997].cThe phenotype of these cases is between achondroplasia and thanato-phoric dysplasia with acanthosis nigricans. These cases are the subject ofanother report (Bellus et al., submitted).

278 Wilcox et al.

Fig. 2. A–F: Phenotype/genotype correlations in TD. Skeletal radiographs were evaluated for the severity of craniosynostosis, platyspondyly, andcurvature and shortening of the femora. Histologic sections of long bones, when available, were examined for the extent of column preservation,mesenchymal banding, and bone abnormalities. For the three most common mutations, Arg248Cys, Tyr373Cys, and Lys650Glu, the percentage of caseswith each feature were graphed. Pairwise comparisons for different traits were analyzed using the Mann-Whitney U rank-sum test, and two-tailedprobabilities are reported.

separately analyzed cases at <34 weeks and ù34 weeksgestation (Fig. 2). The conclusions, in general, were thesame no matter which group was examined, exceptthat, in those cases at <34 weeks gestation, platyspon-dyly was scored as more severe compared with cases atù34 weeks gestation with the Arg248Cys mutation (P4 0.05).

DISCUSSIONMolecular Pathology of TD

Most, if not all, cases of TD are due to mutations inFGFR3. We were able to identify mutations in all 91cases of TD studied. A total of eight different substitu-tions have now been identified in TD (Table I).Arg248Cys is the most common, accounting for 45% ofthe identified mutations and 57% of the curved femurcases. In our series, there was some ascertainment biasin favor of obtaining specimens with straight femoraand CS; hence, they are overrepresented in our series.Compared with cases with curved femora, straight fe-mur cases tend to be uncommon in our experience. Themost frequent substitutions, accounting for 85% of allcases of TD (Arg248Cys, Tyr373Cys, and Lys650Glu),were the result of transition mutations. The most fre-quent substitution, Arg248Cys, is due to a mutation ata CpG dinucleotide, possibly explaining its frequency.The percentage of mutations due to transitions ishigher than the average of 68% [Cooper et al., 1995],perhaps because of the high GC content of the FGFR3gene.

Classification of TD

There has been some controversy over the years re-garding the relationship of CS to TD. TDs with andwithout CS have sometimes been classified as differententities. However, identical twins with TD discordantfor CS have been described [Horton et al., 1983;Corsello et al., 1992]. Based on radiologic and histologiccriteria, Langer et al. [1987] proposed that TD be di-vided into two groups by the presence (type 1; TD1) orabsence (type 2; TD2) of femoral curvature. They wereonly able to examine the chondroosseous morphology ofeight cases. With the radiographic and morphologicanalysis of the additional cases here and elucidation ofthe molecular defects, the classification system ofLanger et al. [1987] is the most compelling. Most, if notall, cases of TD2 are due to a Lys650Glu substitution.Except for the findings in the skull, the radiologic andhistologic abnormalities are less severe in TD2. In con-trast, TD1 can result from cysteine substitutions in theextracellular domain, stop codon mutations, and a me-thionine substitution at position 650.

There is significant phenotypic variability for eachmutation, even allowing for differences because of dif-ferent gestational ages, although the phenotype for theLys650Glu mutation appears to be more narrowly de-fined. Cases with the Tyr373Cys substitution tended tohave more severe manifestations than the Arg248Cyscases, but there was significant overlap in the pheno-typic spectrum between them. Some of the phenotypicvariability for each TD mutation is no doubt due toother genetic, environmental, or stochastic factors,

similar to the multiple determinants of normal adultstature.

Chondro-osseous Morphology in TD

The abnormalities in the growth plate correlate wellwith the radiologic abnormalities in the spine and longbones; i.e., the more abnormal the growth plate, thegreater the platyspondyly and micromelia. The shortand irregular chondrocyte columns can be easily ex-plained by decreased chondrocyte proliferation. Theorigin of the mesenchymal band is not entirely clear,but it appears to arise from the perichondrium andperiostium [Ornoy et al., 1985]. The function of themesenchymal tissue is also unclear, but some evidencesuggests that it is involved not only in a peculiar formof ossification with aspects of membranous ossification,but also in the synthesis of cartilaginous matrix mol-ecules [Horton et al., 1988]. Its origins, therefore, maybe from pluripotential mesenchymal stem cells. Themesenchymal band is not unique to TD, but it is no-table in other skeletal dysplasias in which there is rela-tive overgrowth of metaphyseal bone, such as the platy-spondylic lethal skeletal dysplasias and short-rib poly-dactyly syndromes, but it is generally more peripheralin those disorders. Why the growth of the mesenchymaltissue is less affected than the proliferation of chondro-cytes is not clear, but it may be due to a different rep-ertoire of expressed FGFRs in the two cell types.

Cloverleaf Skull in TD

There have been many pathologic examinations ofthe CS in TD [Bonucci and Nardi, 1972; Kokich et al.,1982; Kremens et al., 1982; Isaacson et al., 1983;Dambrain et al., 1987], but the pathologic basis is stillnot understood. The younger TD fetuses show cranio-synostosis only near the cranial base, suggesting thatthat is where the deformity begins. Although thismight be a developmental problem with formation ofthe synchondroses of the cranial base, it could also oc-cur because of inadequate growth at the sutural edge orexcessive proliferation of cells. Why CS is more com-mon with the Lys650Glu mutation than with the oth-ers is not clear. A variety of mutations in FGFRs 1, 2,and 3 are associated with craniosynostosis syndromes[Wilkie, 1997].

ACKNOWLEDGMENTSWe gratefully acknowledge the donation of speci-

mens to the International Skeletal Dysplasia Registryby referring physicians and families. We thank Mary-ann Priore and Sheilah Levin for administering theRegistry; Betty Mekikian for technical support; andMichael Cohen, John Graham, and Hui-Ying Yang foruseful discussions. This work is supported by NIH Proj-ect Grant HD22657, a grant from the Human GrowthFoundation (to WRW), UCLA Child Health ResearchCenter Grant P30 HD34610; (to WRW), and an NIHClinical Associate Physician Award Grant PA 90-30 (toWRW).

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