Identification of a novel gene and a common variant associated with uric acid nephrolithiasis in a Sardinian genetic isolate

Abstract

Uric acid nephrolithiasis (UAN) is a common disease with an established genetic component that presents a complex mode of inheritance. While studying an ancient founder population in Talana, a village in Sardinia, we recently identified a susceptibility locus of approximately 2.5 cM for UAN on 10q21-q22 in a relatively small sample that was carefully selected through genealogical information. To refine the critical region and to identify the susceptibility gene, we extended our analysis to severely affected subjects from the same village. We confirm the involvement of this region in UAN through identical-by-descent sharing and autozygosity mapping, and we refine the critical region to an interval of approximately 67 kb associated with UAN by linkage-disequilibrium mapping. After inspecting the genomic sequences available in public databases, we determined that a novel gene overlaps this interval. This gene is divided into 15 exons, spanning a region of approximately 300 kb and generating at least four different proteins (407, 333, 462, and 216 amino acids). Interestingly, the last isoform was completely included in the 67-kb associated interval. Computer-assisted analysis of this isoform revealed at least one membrane-spanning domain and several N- and O-glycosylation consensus sites at N-termini, suggesting that it could be an integral membrane protein. Mutational analysis shows that a coding nucleotide variant (Ala62Thr), causing a missense in exon 12, is in strong association with UAN (P=.0051). Moreover, Ala62Thr modifies predicted protein secondary structure, suggesting that it may have a role in UAN etiology. The present study underscores the value of our small, genealogically well-characterized, isolated population as a model for the identification of susceptibility genes underlying complex diseases. Indeed, using a relatively small sample of affected and unaffected subjects, we identified a candidate gene for multifactorial UAN.

Figure 1

Physical and transcriptional maps. Markers of the physical map are indicated above the solid bar representing genomic DNA; orientations of genes are indicated (arrows). a, Physical and transcriptional map of the 1.1-Mb region corresponding to the UAN critical region. The known microsatellites markers are D10S1719, D10S1652, and D10S1640. All genes and ESTs located in this region are shown and their mapping position is in scale. b, SNP map of the region around marker D10S1652. Allele frequencies in the Talana population for all SNPs are available from the authors.

Figure 2

Haplotype frequencies in the whole sample (all sample) and in the subsample obtained controlling for close relatedness among subjects (selected sample). Haplotype-association tests performed in the selected sample showed that the haplotype between SNP AV70 and pSNP 3A (dark gray) was significantly associated with the disease. The haplotype flanked by SNP NG3 and AFM214zb6 (light gray) was more frequent in cases than in controls. Microsatellite markers were not considered in the haplotype-association tests. Microsatellite alleles are shown in italics. The same allele at marker D10S1652 was always present in the associated haplotype (Ombra et al. 2001).

Figure 3

Genomic structure of ZNF365, showing alternative splicing and alternative start sites that generate four different transcripts. Initiation codons (ATG) and stop codons (TAA or TGA) are shown for all isoforms. Two different promoters, CpG island (P1) and TATA box (P2) are indicated (arrows). Exons used for each transcript (colored boxes), together with the associated 67-kb interval, are indicated. The Ala62Thr missense variant found in the ZNF365D isoform is shown. Exons 1–5 and 10–13 identified the KIAA0844 transcript and the EST 603040095F1, respectively.

Figure 4

Alignment of the amino acid sequences of ZNF365 protein isoforms. Amino acids are indicated by single-letter codes. Identical amino acids are indicated (gray boxes). The C2H2 zinc finger domain (solid line) and the coiled-coil segments (dotted line) are indicated. The predicted transmembrane domains are framed. ZNF365D has eight consensus sites for N/O-glycosylation (asterisks). The Ala62Thr missense variant found in the ZNF365D isoform is shown (arrow).

Figure 5

RT-PCR analysis of the expression of ZNF365A, -B, -C, and- D transcripts in human heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. G3PDH (bottom panel) was used as a control gene. The amplified fragments of the ZNF365 transcripts and the size of the amplifications are shown (right).

Figure 6

Results of the haplotype-association analysis performed in the selected sample comprising 64 case haplotypes and 82 control haplotypes. The curves represent the extent of association, expressed as −log(p), between UAN and multilocus haplotypes. For two-locus haplotypes, results are shown for the pair of adjacent markers, and points are drawn at the midpoint between the two. For three-locus haplotypes, results are shown for combinations of adjacent markers, and points are drawn at the position of the middle marker. Empirical significance was assessed through Monte Carlo simulations. Only common haplotypes (frequency >5% in the combined sample) were compared.