Homozygous WNT3 mutation causes tetra-amelia in a large consanguineous family

Abstract

Tetra-amelia is a rare human genetic disorder characterized by complete absence of all four limbs and other anomalies. We studied a consanguineous family with four affected fetuses displaying autosomal recessive tetra-amelia and craniofacial and urogenital defects. By homozygosity mapping, the disease locus was assigned to chromosome 17q21, with a maximum multipoint LOD score of 2.9 at markers D17S931, D17S1785, D17SS1827, and D17S1868. Further fine mapping defined a critical interval of approximately 8.9 Mb between D17S1299 and D17S797. We identified a homozygous nonsense mutation (Q83X) in the WNT3 gene in affected fetuses of the family. WNT3, a human homologue of the Drosophila wingless gene, encodes a member of the WNT family known to play key roles in embryonic development. The Q83X mutation truncates WNT3 at its amino terminus, suggesting that loss of function is the most likely cause of the disorder. Our findings contrast with the observation of early lethality in mice homozygous for null alleles of Wnt3. To our knowledge, this is the first report of a mutation in a WNT gene associated with a Mendelian disorder. The identification of a WNT3 mutation in tetra-amelia indicates that WNT3 is required at the earliest stages of human limb formation and for craniofacial and urogenital development.

Figure 1

Pedigree, haplotype, and WNT3-mutation status of tetra-amelic family. Individuals’ numbers are listed below the pedigree symbols. Genotyped markers from the chromosome 17q21 region are shown to the left of generations III and IV, and individuals’ allele numbers for each marker are given next to the bar. Black bars represent haplotype segregating with the tetra-amelia gene. WNT3 is located between markers D17S1299 and D17S1834. The mutation genotypes of WNT3 are shown below individuals’ numbers (generations I and II) or below haplotypes (generations III and IV). A plus sign (+) indicates mutation Q83X; a minus sign (−) indicates wild-type allele.

Figure 2

Complete limb agenesis, urogenital defects, and other anomalies in affected fetuses. Postmortem photographs (A–C) and radiographs (D–F) demonstrate complete absence of all four limbs, without defects of scapulae and clavicles. A and D, Tetra-amelic fetus IV:1 (female) at 19 wk of gestation, crown-to-rump length 17 cm. Note cleft lip with cleft palate. The separation of the head from the trunk and the multiple lesions on the fetus were iatrogenic. The poorly preserved caudal region did not allow for assessment of external genitalia. B and E, Tetra-amelic fetus IV:7 (female) at 16 wk of gestation, crown-to-rump length 10.5 cm. Note protrusion and cataract of the left eye, microphthalmia of the right eye, malformed nose, hypoplasia of the pelvis, and undefined vaginal and anal regions, with atresia of the urethra, vagina, and anus. Parts of the head were damaged during abortion. C and F, Tetra-amelic fetus IV:7 (male) at 20 wk of gestation, crown-to-rump length 15.5 cm. Note hypoplasia of the pelvis, persistence of the cloaca, and no external genitalia.

Figure 3

WNT3 nonsense mutation associated with tetra-amelia. A, Location of WNT3 on chromosome 17q21.31-32 and genomic organization of WNT3. The dark boxes represent exons, with the coding region illustrated in dark blue and the 5′ and 3′ UTRs in light blue. Positions of the initiation codon, the termination codon, and the nonsense mutation in exon 2 are indicated. B, Electropherograms of WNT3 exon 2 in a control individual (top), one parent (middle), and an affected fetus (bottom). DNA sequence analysis revealed a homozygous C→T substitution at nt 366 (from the translation start site) in the tetra-amelic fetus, causing a Q83X mutation. The parent is heterozygous with respect to the Q83X mutation.