Prenat. Diagn. 18: 621–625 (1998)

SHORT COMMUNICATION

PRENATAL DIAGNOSIS OF APERT SYNDROME

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1Department of Obstetrics and Gynecology, China Medical College Hospital, Taichung, Taiwan, R.O.C.2Department of Pediatrics, China Medical College Hospital, Taichung, Taiwan, R.O.C.

Received 5 June 1997Revised 29 September 1997

Accepted 2 October 1997

SUMMARY

Apert syndrome (AS) is clinically characterized by typical facial features and symmetrical syndactyly of the digits.AS is inherited as an autosomal dominant trait. Recently, a fibroblast growth factor receptor 2 (FGFR2) mutation,either C934G or C937G, was identified in exon IIIa. Our report documents an affected mother and son in whom oneof the two mutations in AS had occurred sporadically in the mother. The diagnosis of AS was based on associatedabnormal physical features and on molecular genetic analysis. A C-to-G transversion at position 937 of the cDNAresulting in a proline-to-arginine substitution at codon 253 was found in the mother. In her second pregnancy,prenatal diagnosis by both restriction analysis and direct sequencing was undertaken and this showed that the femalefetus had not inherited the mutation. ? 1998 John Wiley & Sons, Ltd.

: Apert syndrome; prenatal diagnosis; fibroblast growth factor receptor; polymerase chain reaction

INTRODUCTION

The incidence of AS in the general populationhas been estimated to be between 15·5 per millionand 6·25 per million live births, and the mutationrate has been calculated to be 7·8#10"6 per geneper generation (Cohen et al., 1992; Blank, 1960).Some studies have shown an association of ASwith advanced paternal age (Erickson and Cohen,1974), however, reproductive fitness is low andmore than 98 per cent of cases are the result of newmutations (Moloney et al., 1996). Although chro-mosomal abnormalities have been reported in AS,the majority of patients have normal karyotypes

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CCC 0197–3851/98/060621–05 $17.50? 1998 John Wiley & Sons, Ltd.

(Kaplan, 1991). Either of two mutations in ad-jacent FGFR2 residues have been were demon-strated to be responsible for this disorder in mostcases. Prenatal diagnosis can be achieved success-fully by polymerase chain reaction (PCR) withrestriction enzyme for pregnant patients with ASor in pregnancies where AS is suspected inthe fetus due to for abnormal ultrasonographicfindings.

*Correspondence to: C.-H. Tsai, Department of Pediatrics,China Medical College Hospital, 2 Yuh-Der Road, Taichung,Taiwan, R.O.C. Tel: +886-4-205-2121, ext. 2408. Fax: +886-4-205-2247. E-mail: sib01323@tptsl.seed.net.tw

Contract grant sponsor: National Science Council ofRepublic of China; Contract grant number: NSC 86-2314-B-

CASE REPORT

We report the case of a 28-year-old gravida 2,para 1, Chinese woman in whom AS had beendiagnosed. She had the typical features of acro-cephaly and symmetrical syndactyly with no majorvisceral abnormalities and without apparent intel-lectual impairment (Fig. 1). Her family memberswere all unaffected and were without advancedpaternal age or parental consanguinity. Her first

pregnancy was complicated by preterm labour.

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She gave birth to a similarly affected male infant.She became pregnant again the following year andwas advised to receive prenatal diagnosis. At termshe delivered a female infant with no discernibleabnormalities.

Obstetric history

The patient was admitted to our hospital forantepartum vaginal bleeding at 25 weeks’ gesta-tion. Real-time ultrasound evaluation revealed asingle fetus with placenta previa partialis andpolyhydramnios. Congenital fetal anomalies werehighly suspected because of prominent lateralventricles, irregular skull outline and inability toclearly identify the fingers. An emergency caesar-ean section was performed due to signs of fetal

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distress. At birth, the male infant weighed 540grams. His physical anomalies were almost identi-cal to those of the mother. Post-mortem examin-ation confirmed cleft palate and a ventricularseptal defect. Radiographic examination revealeddistal phalangeal fusion bilaterally. The mother,her husband and the affected infant all had normalkaryotypes. In her second pregnancy in 1996,ultrasound evaluation revealed separate fingermotion, and no abnormalities were detected.

MATERIALS AND METHODS

Amniocentesis was performed at 16 weeks’ ges-tation in the second pregnancy, and genomic DNAwas extracted from the amniocytes. The selectiveprimers were 5*-GGTCTCTCATTCTCCCAT

Fig. 1—Photograph of the patient’s face reveals characteristic facial elongation with midfacial hypoplasia, high forehead, ocularhypertelorism and proptosis, down-slanting palpebral fissures, strabismus, malocclusion, and depressed nasal bridge, prominentlower jaw and shortened maxilla. (b) Photograph of the patient’s left hand reveals subcutaneous and osseous syndactyly of thesecond, third and fourth digits forming a distal-digital bony mass with a single common nail similar to mitten-like hands and feet(right hand had previously received reconstructive surgery)

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CCC-3* (forward primer) and 5*-GCTGGGCATCACTGTAAAC-3* (reverse primer). GenomicDNA was extracted from aminocytes as well asfrom maternal peripheral blood leukocytes andamplified by PCR under the following conditions:an initial denaturation of two minutes at 94)C,followed by 29 cycles at 94)C for one minute,annealing at 50)C for one minute and extension at72)C for two minutes, and a terminal extensionstep at 72)C for seven minutes. The PCR wascarried out in a reaction volume of 50 ìl, contain-ing 200 ìmol/l of each dNTP, 40 pmol of eachprimer, approximately 250 ng of genomic DNA,1·5 mmol/l MgCl2, PCR buffer (Amersham,Takara, Japan) and one unit of Taq DNA poly-merase (Amersham, Takara, Japan) in a PerkinElmer Cetus 480 thermo-cycler. The PCR productwas digested with the restriction endonucleaseBstUI (Biolabs, U.S.A.) and SfiI (Biolabs, U.S.A.)for two hours.

Digested products were analysed by gel electro-phoresis on 4·5 per cent agarose gels (FMC bio-products) and visualized by staining with ethidiumbromide. The diagnosis was confirmed by directautomatic sequencing (ABI PRISM 377 DNAsequencer).

RESULTS

Using PCR-based restriction analysis, anFGFR2 mutation was detected in the mother. Theamplified fragment was 159 base pairs (bp) inlength. A P253R mutation was detected by BstUIdigestion which yielded fragments at 100 bp and59 bp in addition to the normal 159 bp fragment(Fig. 2), while the SfiI digestion showed normalbands. Amniocentesis of the second pregnancydemonstrated no C-to-G transversion in therestriction analysis and DNA sequencing whichconfirmed the fetus to be unaffected (Fig. 3). Inaddition, fetal ultrasonography revealed a normalfacial profile with clearly identifiable fingers andtoes. At term, the mother delivered a female infantwith no discernible anomalies.

DISCUSSION

AS is frequently associated with mental retard-ation. The severity of the skeletal malformationsand the 50 per cent risk of transmitting the mu-tation to offspring creating life-long handicap

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Fig. 2—PCR and enzyme-digestion analysis. Detection ofmutation by direct sequencing of PCR products and restrictionenzyme of exon IIIa. Lane M: DNA ladder (2000, 1500, 1000,700, 500, 400, 300, 200, 100 and 50 bp; Amersham); lane 1:normal control; lane 2: patient; lane 3: fetus. Digestion of thePCR product from fetal amniocytes without a mutationresulted in fragment 159 bp, whereas additional fragments (59and 100 bp) were detected from the maternal serum

makes prenatal diagnosis a reasonable consider-ation. Fetoscopy was used successfully to demon-strate AS by Leonard et al. (1982), however, theinvasive nature of fetoscopy limits its use. Fetalultrasonography is a valuable non-invasive toolfor the prenatal detection of AS. When ultra-sonographic findings show oxycephaly, prominentlateral ventricles, flat facial profile, dextracardia,syndactyly and even the non-specific finding ofthickened nuchal folds, AS should be suspected(Chenoweth-Mitchell and Cohen, 1994).

AS was previously classified solely on the basisof clinical presentation, but has now been definedat the molecular level (Cohen and Kreiborg, 1995;Osada et al., 1996). Mutations of human FGFRshave been identified to be causative for severalskeletal dysplasias and a number of cranio-synostosis syndromes (Crouzon, Jackson–Weiss,Pfeiffer, Apert syndromes) (Mulvihill, 1995; Meyeret al., 1996). Wilkie et al. (1995) demonstratedmutations in the FGFR2 exon IIIa in AS patients.There are two specific C-to-G transversions atadjacent amino acids on chromosome 10 (Parket al., 1995). The first mutation is a C934G trans-version, which changes the codon TCG to TGG,resulting in a serine-to-tryptophan substitution atamino acid 252 (S252W). The second mutation is a

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C937G transversion which changes the codonCCT to CGT, producing a proline-to-argininesubstitution at amino acid 253 (P253R). TheP253R mutation cuts, with BstUI, into 100-bp and59-bp fragments; the S252W mutation cuts, withSfiI, into 96-bp and 63-bp fragments (Slaney et al.,1996). Chinese patients with AS revealed the samemutations (Tsai et al., 1997). To date, there hasbeen only one reported patient with AS withoutone of these two mutations (Park et al., 1995). It ispossible that other rare mutations will be identifiedeither in the same functional region in otherFGFRs or other functional regions of FGFR2that have similar biochemical consequences. The

Fig. 3—DNA sequencing: (Bottom). Maternal DNA demon-strated the mutation P253R (C-to-G transversion) by BstUIdigestion (long arrow), with normal Serine 252 sequence (5*-TCG-3*) with SfiI digestion (short arrow). (Top). In the fetalamniocytes, the positions of both 934 (short arrow, 5*-TCG-3*)and 937 (long arrow, 5*-CCT-3*) appeared normal

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same C-to-G transversion in FGFR1 (P252R) hasbeen identified as Pfeiffer syndrome (PS). PS isobviously genetically heterogenous (FGFR1 andFGFR2) as Muenke et al. (1994) emphasized. Thetwo common mutations in AS can easily bedetected by PCR-based restriction analysis usingthe same PCR products. However, both geneticanalysis and fetal ultrasonography should beused in sequence to increase the probability ofdiagnosis.

This study was supported by a grant fromNational Science Council of Republic of China(NSC86-2314-B-039-003).

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