prenatal diagnosis and genetic analysis of type i and type ii thanatophoric dysplasia

Prenatal diagnosis and genetic analysis of type I and type IIthanatophoric dysplasia

Chih-Ping Chen1,2*, Schu-Rern Chern2, Jin-Chung Shih3, Wayseen Wang2, Li-Fan Yeh1, Tung-Yao Chang1 andChin-Yuan Tzen4

1Department of Obstetrics and Gynecology, Mackay Memorial Hospital, Taipei, Taiwan, ROC2Department of Medical Research, Mackay Memorial Hospital and National Yang-Ming University, Taipei, Taiwan, ROC3Department of Obstetrics and Gynecology, National Taiwan University, Taipei, Taiwan, ROC4Department of Pathology, Mackay Memorial Hospital, Taipei, Taiwan, ROC

Thanatophoric dysplasia (TD) is one of the most common neonatal lethal skeletal dysplasias. Prenatalsonographic and molecular genetic diagnoses of three cases of TD type I (TD1) and one case of TD type II(TD2) are presented here. Two fetuses of TD1 were characterized by polyhydramnios, macrocephaly, shortlimbs, a narrow thoracic cage and curved short femora, but without a cloverleaf skull at 27 and 31 weeks'gestation, respectively. The third fetus with TD1 was, however, not associated with macrocephaly,polyhydramnios, chest narrowing and severe femoral bowing on prenatal ultrasound at 18 weeks' gestation.The TD2 fetus was characterized by polyhydramnios, short limbs, a narrow thoracic cage, straight shortfemora, hydrocephalus and a cloverleaf skull at 24 weeks' gestation. Three-dimensional ultrasound was ableto enhance the visualization of thickened, redundant skin folds and craniofacial and limb deformitiesassociated with TD. Molecular analysis of the ®broblast growth factor receptor 3 (FGFR3) gene byrestriction enzyme digestion analysis and direct sequencing using cultured amniotic ¯uid cells or cord bloodcells revealed a missense mutation of 742CpT (Arg248Cys) in all cases with TD1 and a missense mutationof 1948ApG (Lys650Glu) in the case with TD2. The present report shows that adjunctive applications ofmolecular genetic analysis of the FGFR3 gene and three-dimensional ultrasound are useful for prenataldiagnosis of TD. Copyright # 2001 John Wiley & Sons, Ltd.

KEY WORDS: thanatophoric dysplasia; prenatal diagnosis; gene mutation; three-dimensional ultrasound;FGFR3 gene

INTRODUCTION

Thanatophoric dysplasia (TD) is one of the mostcommon neonatal lethal skeletal dysplasias with anestimated prevalence rate of 0.2±0.5/10 000 births(Orioli et al., 1986). Maroteaux et al. (1967) ®rstdescribed thanatophoric dwar®sm and differentiated itfrom achondroplasia as a de®nite discrete entity. In1977, at the Second International Conference on theNomenclature of Skeletal Dysplasias, the term thana-tophoric dwar®sm was changed to thanatophoricdysplasia. TD is subdivided into two types accordingto clinical features. TD type I (TD1) is characterizedby short limbs, a narrow thoracic cage and curvedfemora with or without a cloverleaf skull, whereas TDtype II (TD2) is characterized by short limbs, a narrowthoracic cage, straight femora and a cloverleaf skull(Langer et al., 1987; Spranger and Maroteaux, 1990;Norman et al., 1992). These distinct features allowreliable radiologic and prenatal two-dimensionalsonographic assessments (Schild et al., 1996; Cohen,1998; Wilcox et al., 1998). However, a de®nitivediagnosis still requires molecular analysis. Recently,both TD1 and TD2 have been found to be caused by

mutations in the ®broblast growth factor receptor 3(FGFR3) gene (Rousseau et al., 1995, 1996a; Tavor-mina et al., 1995; Cohen, 1998; Wilcox et al., 1998;Passos-Bueno et al., 1999; Tavormina et al., 1999).With the advent of molecular technology and three-dimensional ultrasound, prenatal visualization of grossanomalies and prenatal molecular diagnosis arepossible for both TD1 and TD2. The present reportdetails our experience in this regard.

MATERIALS AND METHODS

Clinical subjects

Case 1

A 28-year-old Chinese woman, gravida 2, para 0, wasreferred for genetic counselling at 27 weeks' gestationbecause of fetal hydrocephalus, short-limbed dwar®smand polyhydramnios. A level II sonographic examina-tion of the fetus revealed mild ventriculomegaly,macrocephaly with a biparietal diameter (BPD) of8.2 cm and a head circumference of 26.3 cm, compa-tible with 32 gestational weeks. It also showed markedshortening of the long bones (the long bone measur-ements<5th percentile with the femur measuring1.98 cm, tibia 1.77 cm, ®bula 1.80 cm, humerus2.3 cm, radius 1.48 cm, ulna 1.44 cm, compatible

*Correspondence to: C.-P. Chen, Department of Obstetricsand Gynecology, Mackay Memorial Hospital, 92, Section 2,Chung-Shan North Road, Taipei, Taiwan, ROC.E-mail: [email protected]

PRENATAL DIAGNOSIS

Prenat Diagn 2001; 21: 89±95.

Copyright # 2001 John Wiley & Sons, Ltd. Received: 17 July 2000Revised: 24 October 2000

Accepted: 29 October 2000

with 15 weeks) and a narrow thorax. The thoraciccircumference (TC) measured 15.7 cm (<5th percen-tile), the abdominal circumference (AC) measured21.8 cm (27 weeks) and the TC/AC ratio was 0.72(Figure 1). Genetic amniocentesis was performed andrevealed a 46,XY karyotype. In addition, molecularstudies showed a heterozygous 742CpT (R248C)mutation in the FGFR3 gene. The mother chose toterminate the pregnancy. A 1062 g baby was deliveredwith a body length of 26 cm and characteristic featuresof TD1 (Figure 2). Postmortem cytogenetic andmolecular studies using fetal tissues con®rmed theprenatal diagnosis. Radiographs showed severe platy-spondyly, short curved long bones and telephonereceiver-like short femora (Figure 2). Histopathologyof the cartilage revealed a severely retarded anddisorganized epiphyseal growth zone.

Case 2

A 28-year-old Chinese woman, gravida 2, para 1, wasreferred for genetic counselling at 31 weeks' gestationbecause of fetal hydrocephalus, short-limbed dwar®smand polyhydramnios. A level II sonographic examina-tion revealed a fetus with macrocephaly (BPD 9.2 cm,compatible with 38 weeks), marked shortening of thelong bones (the long bone measurements<5th percen-tile with the femur measuring 2.11 cm, tibia 2 cm,humerus 2.2 cm, compatible with 17 weeks), bowing ofthe long bones and a narrow thorax. Three-dimensional ultrasound showed frontal bossing, adepressed nasal bridge, rhizomelic shortening oflimbs, a narrow chest, redundant skin folds andhydrocele. Genetic amniocentesis and cordocentesisrevealed a 46,XY karyotype. Molecular studiesshowed a heterozygous 742CpT (R248C) mutationin the FGFR3 gene. A 2170 g baby was subsequentlydelivered with a body length of 37 cm and character-istic features of TD1. Radiographs showed severeplatyspondyly, short curved long bones and telephonereceiver-like short femora. The baby died soon after

birth. Permission to conduct an autopsy was notgranted.

Case 3

A 30-year-old Chinese primigravida woman wasreferred for genetic counselling at 18 weeks' gestationbecause of fetal short-limbed dwar®sm. A level IIsonographic examination revealed a normal amount ofamniotic ¯uid, a normal head (BPD 4.54 cm, compa-tible with 18 weeks), marked shortening of long bones(the long bone measurements<5th percentile with thefemur measuring 1.29 cm, tibia 1.2 cm, ®bula 1.03 cm,humerus 1.06 cm, radius 0.78 cm, ulna 0.86 cm,compatible with 13 weeks), and normal thoracic andabdominal circumferences (TC 11.6 cm, AC 12.6 cm,TC/AC 0.92). The bowing of the long bones was notprominent on prenatal ultrasound. Genetic amniocent-esis was performed and a 46,XY karyotype wasrevealed. Molecular studies showed a heterozygous742CpT (R248C) mutation in the FGFR3 gene. Themother opted to terminate the pregnancy at 21 weeks'gestation. A 292 g fetus was delivered with a bodylength of 19.5 cm, mild frontal bossing and a depressednasal bridge. Postmortem molecular analysis usingfetal tissues con®rmed the prenatal diagnosis. Radio-graphs showed less prominent platyspondyly, and mildcurved long bones without apparent telephonereceiver-like short femora. Histopathology of thecartilage showed a retarded and disorganized epiphy-seal growth zone.

Case 4

A 30-year-old Chinese woman, gravida 4, para 3, wasreferred for genetic counselling at 24 weeks' gestationbecause of fetal hydrocephalus, short-limbed dwar®smand polyhydramnios. Her previous three pregnanciesresulted in normal children. A level II sonographicexamination revealed a fetus with severe ventriculo-megaly, hydrocephalus, a cloverleaf skull, macroce-phaly (Figure 3) and a BPD of 8 cm, compatible with31 weeks. There was also a small chest and

Figure 1ÐTwo-dimensional ultrasonographic images of case 1 at 27 weeks' gestation show (A) mild ventriculomegaly, (B) a curved shortfemur and (C) a narrow thorax

C.-P. CHEN ET AL.90

Copyright # 2001 John Wiley & Sons, Ltd. Prenat Diagn 2001; 21: 89±95.

marked shortening of the long bones (the longbone measurements<5th percentile with the femurmeasuring 1.97 cm, humerus 1.89 cm, compatible with

15 weeks). However, there was no bowing of the longbones. Three-dimensional ultrasound showed a largetrilobed head with low-set dysmorphic ears, shortlimbs, a narrow chest and redundant skin folds(Figures 4 and 5). Cordocentesis revealed a 46,XXkaryotype and molecular studies revealed a hetero-zygous 1948ApG (K650E) mutation in the FGFR3gene. The mother elected to terminate the pregnancy.A 1276 g fetus was delivered with a body length of30 cm and characteristic features of TD2. Radiographsshowed a trilobed head with straight short femora.

Figure 3ÐTwo-dimensional ultrasonographic images of case 4 at 24weeks' gestation show (A) a trilobed skull on the transverse sectionand (B) an abnormal longitudinal section of the head withhydrocephalus, frontal bossing and a depressed nasal bridge

Figure 5ÐThe frontal view of the face of case 4 and its three-dimensional ultrasonographic image with heterotopia of thetemporal lobes and low-set dysmorphic ears (arrows)

Figure 4ÐThree-dimensional ultrasonographic images of case 4 at24 weeks' gestation show (A) heterotopia of the temporal lobe witha low-set dysmorphic ear (e), frontal bossing and a depressed nasalbridge and (B) a straight short femur (f)

Figure 2ÐThe whole-body view and radiograph of the TD1proband case 1

PRENATAL DIAGNOSIS OF THANATOPHORIC DYSPLASIA 91


Histopathology of the cartilage showed a severelyretarded and disorganized epiphyseal growth zonewith prominently ossi®ed cartilage canals verticallyintersecting the epiphysis.

Molecular genetic analysis of the FGFR3gene

Genomic DNA was extracted from either culturedamniotic ¯uid cells, cord blood or fetal tissue of theprobands by the standard method. To detect muta-tions in the FGFR3 gene, two pairs of primers wereused to amplify exons 7 and 15. Primers e7-F (5k-CGGCAG TGA CGG TGG TGG TGA-3k) and e7-R(5k-CCA AAT CCT CAC GCA ACC C-3k) (Belluset al., 1996; PeÂrez-Castro et al., 1997) were used toamplify a 341 bp amplicon encompassing exon 7 andits ¯anking intron sequences. Primers TK2-F (5k-TTCGAC ACC TGC AAG CCG CC-3k) and TK2-R(5k-CCT CAG GCG CCA TCC ACT TC-3k) (Keeganet al., 1991; Tavormina et al., 1995) were used toamplify a 452 bp amplicon, encompassing the secondhalf of tyrosine kinase (TK) region. PCR was carriedout in 50 ml volumes which contained 50 ng DNA,10% DMSO, 50 mM KCl, 10 mM Tris (pH 8.4),1.5 mM MgCl2, 200 mM of each dNTP, 0.1 mM ofeach primer and 1.5 U Taq DNA polymerase (Perkin-Elmer, Norwalk, USA). For 35 cycles, the cyclingparameters were 94uC for 30 s, 55uC for 30 s, and 72uCfor 45 s. PCR products were then digested with 5 UBsiHKAI and BbsI (New England Biolabs, Beverly,USA), and then analyzed on a 4% agarose gel (PeÂrez-Castro et al., 1997). The ampli®ed DNA was thenpuri®ed using a PCR puri®cation kit (QIAGEN,Hilden, Germany) and used as templates in cyclesequencing. Sequencing reactions were performedusing the BigDye Terminator Cycle SequencingReady Reaction Kit with AmpliTaq DNA PolymeraseFS (Perkin-Elmer, Foster City, USA). Fluorescent dyelabeled DNA fragments were then analyzed on a semi-automated DNA sequencer (ABI PRISM 377 DNASequencer; ABI, Palo Alto, USA).

RESULTS

Molecular characterization of the FGFR3gene at nucleotides 742 and 1948

Mutations in the FGFR3 gene were identi®ed in allcases of TD. All three TD1 cases had a heterozygous742CpT (R248C) mutation. It was caused by a CpTtransition at nucleotide 742 in exon 7, leading toArg248Cys (CGCpTGC) substitution. In contrast,the unaffected individual was homozygous for theBsiHKAI digestion site, allowing the cleavage of the341 bp fragment into 244 and 97 bp fragments. The742CpT point mutation created a second BsiHKAIsite that cleaved the 244 bp fragment into 181 and63 bp fragments. All three TD1 probands wereheterozygous for the BsiHKAI digestion site withinthe PCR fragment derived from the linker regionbetween the immunoglobulin-like (Ig) II and IgIIIdomains (restriction fragments 244, 181, 97 and 63 bp)(Figure 6). The TD2 case had a heterozygous1948ApG (K650E) mutation that was caused by anApG transition at nucleotide 1948 in exon 15, leadingto a Lys650Glu (AAGpGAG) substitution. Theunaffected individual was homozygous for the BbsIdigestion site that cleaved the 452 bp fragment into332 and 120 bp fragments. The 1948ApG pointmutation destroyed a BbsI digestion site and resultedin no cleavage. The TD2 proband was heterozygousfor the BbsI site within the PCR fragment derivedfrom the second TK domain (restriction fragments452, 332 and 120 bp) (Figure 6). These mutations werecon®rmed by direct sequencing.

DISCUSSION

Sawai et al. (1999) ®rst described prenatal diagnosis ofTD1 by mutational analysis of the FGFR3 gene. Thepresent results further extend molecular applicationsto TD2 and show that the restriction enzyme digestionanalysis of the FGFR3 gene allows rapid de®nitivediagnosis of common TD1 and TD2.

FGFR3 belongs to a family of four ®broblast

Figure 6ÐDetection of FGFR3 mutations by restriction enzyme digestion analysis. PCR products are digested with BsiHKAI (lanes 2±5) orBbsI (lanes 7, 8). Lanes 1 and 6 are undigested controls; lanes 2 and 7 are unaffected controls; lanes 3±5 are TD1 probands (cases 1±3); lane 8is TD2 proband (case 4); and lane M is a 100 bp molecular weight marker. The 61 bp fragment is not shown in this gel electrophoresisexperiment

C.-P. CHEN ET AL.92


growth factor receptors (FGFRs 1±4), which interactwith ®broblast growth factors and activate intracel-lular signal transduction. These receptors consist ofthree main components: an extracellular region withthree Ig domains, a transmembrane segment, and anintracellular region with two TK domains. Recently,mutations in the FGFR3 gene have been identi®ed inseveral skeletal dysplasias and craniosynostoses(Table 1). TD1 is caused by as many as 12 distinctmissense FGFR3 mutations (Table 1). Five mutations

result in speci®c cysteine substitutions in FGFR3(R248C, S249C, G370C, S371C, Y373C), six muta-tions eliminate the stop codon and result in 141-aminoacid C-terminal extension (J807G, J807R, J807L,J807C l[2421ApT], J807C [2421ApC], J807W), andone mutation leads to a K650M substitution inFGFR3 (Rousseau et al., 1995; Tavormina et al.,1995; Bonaventure et al., 1996; Rousseau et al., 1996a;Bellus et al., 1997; Kitoh et al., 1998; Wilcox et al.,1998; Passos-Bueno et al., 1999). The majority of cases

Table 1ÐMutations of skeletal dysplasias and craniosynostoses on FGFR3 gene

Phenotype Amino acid substitution Clinical features

Skeletal dysplasiasTD1 Short limbs, a narrow thoracic cage, and curved femora with or

without a cloverleaf skullMost common R248C (Arg248Cys)S249C (Ser249Cys)G370C (Gly370Cys)S371C (Ser371Cys)Y373C (Tyr373Cys)K650M (Lys650Met)J807G (Stop807Gly)J807R (Stop807Arg)J807L (Stop807Leu)J807C (Stop807Cys)(2421ApT)J807C (Stop807Cys)(2421ApC)J807W (Stop807Trp)

TD2 Short limbs, a narrow thoracic cage, straight femora, and a cloverleafskullAll cases K650E (Lys650Glu)

ACH Short limbs, a low nasal bridge, and caudal narrowing of spinal canalG346E (Gly346Glu)G375C (Gly375Cys)

Most common G380R (Gly380Arg)(1138GpA)G380R (Gly380Arg)(1138GpC)

HCH Short limbs, near-normal craniofaces, and caudal narrowing of spinalcanalI538V (Ile538Val)

N540T (Asn540Thr)Most common N540K (Asn540Lys)

(1620CpA)N540K (Asn540Lys)(1620CpG)

PLSD-SD Short limbs, platyspondyly, metaphyseal ¯aring, bowed long bones,and large dilated loops of rough endoplasmic reticulum in thechondrocytes

Most common R248C (Arg248Cys)S249C (Ser249Cys)Y373C (Tyr373Cys)J807R (Stop807Arg)J807W (Stop807Trp)

SADDAN Severe achondroplasia, developmental delay, and acanthosis nigricansK650M (Lys650Met)

CraniosynostosesNSC Coronal synostosis, speci®c bone anomalies of the hands and feet, but

without any of classical craniosynostosis syndromesP250R (Pro250Arg)C-AN Shallow orbits, proptosis, craniosynostosis, maxillary hypoplasia, and

acanthosis nigricansA391E (Ala391Glu)

TD1, thanatophoric dysplasia type I (Rousseau et al., 1995; Tavormina et al., 1995; Bonaventure et al., 1996; Rousseau et al., 1996a; Belluset al., 1997; Kitoh et al., 1998; Wilcox et al., 1998; Passos-Bueno et al., 1999); TD2, thanatophoric dysplasia type II (Tavormina et al., 1995;Wilcox et al., 1998); ACH, achondroplasia (Rousseau et al., 1994; Bellus et al., 1995a; Ikegawa et al., 1995); HCH, hypochondroplasia (Belluset al., 1995b; Prinos et al., 1995; Rousseau et al., 1996b; Deutz-Terlouw et al., 1997; GrigelionieneÂ et al., 1998; Prinster et al., 1998); PLSD-SD,platyspondylic lethal skeletal dysplasia, San Diego type (Brodie et al., 1999; Passos-Bueno et al., 1999); SADDAN, severe achondroplasia withdevelopmental delay and acanthosis nigricans (Tavormina et al., 1999); NCS, non-syndromic craniosynostosis (Bellus et al., 1996; Muenkeet al., 1997); C-AN, Crouzon with acanthosis nigricans (Meyers et al., 1995; Wilkes et al., 1996).



with TD involve mutations that result in new cysteineresidues in the linker region between the IgII and IgIIIdomains (R248C, S249C) or in the transmembranedomain (G370C, S371C, Y373C) (Passos-Bueno et al.,1999). Of these, the missense mutation 742CpTcausing an arginine to cysteine substitution (R248C)occurs most often. To date, at least 102 cases ofmutation R248C in association with TD1 or platy-spondylic lethal skeletal dysplasia, San Diego typehave been reported (Passos-Bueno et al., 1999).Conversely, all reported cases with TD2 are associatedwith a single missense mutation, i.e. A to G transitionat nucleotide 1948, causing a lysine to glutamic acidsubstitution (K650E) in the second TK domain ofFGFR3 (Tavormina et al., 1995; Wilcox et al., 1998)(Table 1). So far, at least 24 cases of mutation ofK650E with TD2 have been reported (Passos-Buenoet al., 1999). Molecular analyses of the FGFR3 geneby restriction enzyme digestion and DNA sequencingin the present study revealed a 742CpT (R248C)mutation in all cases with TD1 and a 1948ApG(K650E) mutation in the case with TD2. Since R248Cis the most common mutation in TD1 as is K650E inTD2, PCR-based restriction enzyme digestion analysisof the FGFR3 gene is useful for rapid molecularscreening of fetuses with sonographic ®ndings sugges-tive of TD.

In the present study, three-dimensional ultrasoundwas able to enhance the visualization of fetal structuralabnormalities and was helpful in the investigation ofTD. Currently, prenatal ultrasonography can reliablydetect TD and its associated anomalies. However, insmall fetuses with TD1 before 20 weeks' gestation, thefemoral bowing and the telephone receiver-likecon®guration may not be prominent (Yang, 1998).In addition, TD1 variants that lack characteristicfacial abnormalities or telephone receiver-like femorahave been reported (Nerlich et al., 1996; Camera et al.,1997). As demonstrated by case 3, TD1-associatedabnormalities such as macrocephaly, polyhydramnios,chest narrowing and severe femoral bowing were notapparent by ultrasound in the second trimester.Signi®cant phenotypic variability among patients ofTD with the same mutation in FGFR3 has been welldescribed by Wilcox et al. (1998) who suggested thatvariable presence of radiologic and histologic ®ndingsin each TD mutation might be due to genetic,environmental and stochastic factors. In conclusion,the present report demonstrates that the adjunctiveapplications of molecular genetic analysis of theFGFR3 gene and three-dimensional ultrasound areuseful for the prenatal diagnosis of TD.

ACKNOWLEDGEMENTS

This work was supported by Research Grants NSC-86-2314-B-195-006 and NSC-89-2314-B-195-011 fromthe National Science Council, Taiwan, ROC.

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prenatal diagnosis and genetic analysis of type i and type ii thanatophoric dysplasia

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