Tetra‐amelia and lung aplasia syndrome: report of a new family and exclusion of candidate genes

To the Editor: Amelia, the complete absence of a limb, is the most severe limb reduction defect with a prevalence of 0.4–1.5 per 100,000 births (1, 2). A distinct multiple congenital anomaly syndrome including tetra-amelia, craniofacial malformations and pulmonary hypoplasia/aplasia was reported in consanguineous families. We report a new family of seemingly autosomal recessive tetra-amelia with pulmonary hypoplasia, in which we excluded mutations in the human WNT3, FGF10,HS6ST1 andHS6ST3 genes, and the existence of an abnormal FGFR2 isoform. A 27-year-old Moroccan woman presented at 16 weeks of gestation of her third pregnancy with recurrent severe limb deficiency in the foetus. She did not consume alcohol or medication during her pregnancies, and had no diabetes. Physical examination was unremarkable for both parents, who are first cousins. The first and third pregnancies were terminated, respectively, at 18 and 28 weeks of gestation because foetal ultrasonography showed absent limbs. Both foetuses had a normal karyotype on cultured amniocytes (respectively 46,XY and 46,XX), and clinical examination (Fig. 1a) was consistent with the prenatal findings. Foetal x-ray confirmed the complete absence of limb bones with normal clavicles and scapulae in the second foetus (Fig. 1b). The second child of this couple was a normal boy. Genomic DNA was extracted from cultured amniocytes and paraffin-embedded material from the second affected foetus, and from peripheral blood lymphocytes from the parents. Primers were specifically designed to separately amplify all exons and intron – exon boundaries of the WNT3 and FGF10 genes, exon 9 of the FGFR2 gene and the coding regions of HS6ST1 and HS6ST3 (Table 1). Standard PCRs were set, except for amplification of HS6ST1 exon 1, and HS6ST3 exon 1, for which we used 2.5 U AmpliTaqGold (Applied Biosystems, Courtaboeuf, France) and added, respectively, 10 and 5%DMSO. PCR products were directly sequenced, and results were compared with the respective genomic reference sequences. Our cases fit the clinical description of ‘tetraamelia with pulmonary hypoplasia’ [MIM273395] (3–5), reported initially by Rosenak et al. in two foetuses with total absence of limbs, absent lungs and a cleft lip in one, without other internal malformations. In the Zimmer type phocomelia [MIM301090] (6–8), limb abnormalities range from phocomelia to amelia and include multiple additional defects (i.e. absent nose and ears; ocular, cerebral and anal anomalies). Recently, Niemann et al. (9) reported homozygous mutations in the WNT3 gene in two consanguineous foetuses from the same sibship who presented with tetra-amelia and complex craniofacial and urogenital malformations, but without pulmonary hypoplasia. This description clinically resembles to the Zimmer type phocomelia. In the case of the family reported in this study, direct sequencing of all five exons of the WNT3 gene in both parents excluded any pathogenic mutation. The father carries an heterozygous sequence variation (c. 29G>A, not referenced as SNP) in the 50 UTR, which was not found in the affected foetus. This result strongly suggests genetic heterogeneity of hereditary tetra-amelia. Other candidates include genes encoding proteins involved in Fgf signalling. Fgf-10-deficient mice display a complete absence of limbs and lungs (10, 11). However, sequencing of the entire three exons of the FGF10 gene in the foetus failed to find any mutation. Mice, in which the IIIb isoform of Fgfr2 is excluded by targeted disruption of Fgfr2 exon The authors wish it to be known that, in their opinion, the first two authors M Krahn and S Julia should be regarded as joint first authors. Clin Genet 2005: 68: 558–560 Copyright # Blackwell Munksgaard 2005 Printed in Singapore. All rights reserved CLINICAL GENETICS doi: 10.1111/j.1399-0004.2005. 00531.x