Lethal contractural syndrome type 3 (LCCS3) is caused by a mutation in PIP5K1C, which encodes PIPKI gamma of the phophatidylinsitol pathway

Abstract

Lethal congenital contractural syndrome (LCCS) is a severe form of arthrogryposis. To date, two autosomal recessive forms of the disease (LCCS and LCCS2) have been described and mapped to chromosomes 9q34 and 12q13, respectively. We now describe a third LCCS phenotype (LCCS3)--similar to LCCS2 yet without neurogenic bladder. Using 10K single-nucleotide-polymorphism arrays, we mapped the disease-associated gene to 8.8 Mb on chromosome 19p13. Further analysis using microsatallite markers narrowed the locus to a 3.4-Mb region harboring 120 genes. Of these genes, 30 candidates were sequenced, which identified a single homozygous mutation in PIP5K1C. PIP5K1C encodes phosphatidylinositol-4-phosphate 5-kinase, type I, gamma (PIPKI gamma ), an enzyme that phophorylates phosphatidylinositol 4-phosphate to generate phosphatidylinositol-4,5-bisphosphate (PIP(2)). We demonstrate that the mutation causes substitution of aspartic acid with asparagine at amino acid 253 (D253N), abrogating the kinase activity of PIPKI gamma . Thus, a defect in the phosphatidylinositol pathway leading to a decrease in synthesis of PIP(2), a molecule active in endocytosis of synaptic vesicle proteins, culminates in lethal congenital arthrogryposis.

Figure 1.

Israeli-Bedouin kindreds affected with LCCS3. Blackened and unblackened symbols represent affected and unaffected individuals, respectively. Numbers denote individuals whose DNA samples were analyzed. A, Pedigree of family A. B, Pedigree of family B.

Figure 2.

LCCS3-affected fetus at 16 wk gestation

Figure 3.

Linkage results for chromosome 19 for a partial pedigree of family A. The haplotype showing the homozygosity region is boxed. Arrows indicate crossing-over events. The upper and lower boundaries were determined according to individuals 14 and 8, respectively.

Figure 4.

The G757A mutation in exon 7 of PIP5K1C. A, Sequence analysis of the A→G substitution in an unaffected individual, obligate carrier, and affected individual. chr = Chromosome. B, PIP5K1C exon 7 mutation scanned with TaqI restriction enzyme, with schematic diagram illustrating the Taq1 fragments. C, Mutation analysis of family A members. PCR amplification products were digested with Taq1 restriction enzyme and were separated on 2% agarose gel. C = unrelated healthy control. Individuals 7, 8, 11, and 14 were homozygous for the mutated allele. Individuals 1, 3, 4, 6, 12, and 13 were heterozygous for the wild-type and mutated alleles. Individuals 2 and 5 were homozygous for the wild-type allele.

Figure 5.

The D253N PIP5K1C mutation in LCCS3, which abolishes the PIP5K1C kinase activity. A, Prediction of the structural and functional importance of the aspartic acid residue at position 253, done using the ConSeq server. The ConSeq conservation grade was 9 (highly conserved). B, The aspartic acid at position 253, which is within a conserved DLKGS motif in the PIPK domain of the PIP5K1C protein. C, Purified wild-type (WT) and mutant (M) recombinant GST-PIP5K1C fusion proteins (SDS-PAGE). D, Kinase activity of wild-type (WT) and mutant (M) PIP5K1C recombinant proteins, tested in a reaction mixture containing PI4P (80 μM) as substrate and [γ³²P]ATP. The labeled PIP₂ product was separated using thin-layer chromatography and was quantified by phosphor-imaging analysis. PIP₂ (mean±SD of three independent experiments) synthesized by mutant recombinant PIP5K1C was 1.7%±0.56% of PIP₂ synthesized by equal amount of wild-type PIP5K1C. GST = glutathione S-transferase (negative control).