PMC

Human NPHP4 cDNA sequence and deduced nephroretinin amino acid sequence. Exons are numbered at right. Exon sequence alternates in color. Start codon, stop codon, and the variant polyadenylation signal AACAAA are underlined.

Alignment of human, mouse, and C. elegans deduced nephroretinin sequences. The order from top to bottom is human, mouse, and C. elegans ( accession numbers BankIt 486643, BankIt 486675, and Z81579, respectively). Identities are shown in reverse (white on black), and similarities between all three sequences are shown on gray background. There was 82% amino acid identity between human and mouse sequences and 23% amino acid identity between human and C. elegans sequences. Boxes above the sequence delimit motifs as predicted by the programs given in parenthesis: NLS = nuclear localization domain (); ER = endoplasmic reticulum membrane domain (); E-rich = glutamic acid–rich domain (); S-rich = serine-rich domain (); P-rich = proline-rich domain (); and DUF339 = PFAM (protein families database of alignments) domain of unknown function (). In H. salinarium, the program detected a 30% sequence identity with gas-vesicle protein gvpL of H. salinarium ( accession number P33964).

Northern blot analysis of the NPHP4 expression pattern. A multiple-tissue northern blot with human adult poly(A)⁺ RNA was hybridized with a 584-bp NPHP4 human DNA probe. Expression of a 5.0-kb transcript (arrowhead) is apparent in all tissues studied, with highest expression being in skeletal muscle.

Positional cloning strategy for the NPHP4 gene, on human chromosome 1p36. A, Genetic map position for microsatellites used in linkage mapping of NPHP4 (see ). Sex-averaged genetic distance (in cM) from the map was used. Published flanking markers are underlined (Schuermann et al. ). p-ter = Telomeric; cen = centromeric. B, Physical map distances of critical microsatellites relative to D1S2660. The secure 1.2-Mb critical interval (solid bar) and the 700-kb suggestive critical interval (stippled bar) are delimited by the newly identified secure flanking markers (asterisks) and suggestive flanking markers (double asterisks) defined by haplotype analysis (see ). Below the axis, known genes (green), predicted unknown genes (blue), and the NPHP4 gene (also known as “Q9UFQ2”) are represented as arrows in the direction of transcription. C, Genomic organization of NPHP4, with exons indicated by vertical hatches and numbered. D, Exon structure of NPHP4 cDNA. Blackened and unblackened boxes represent the 30 exons encoding nephroretinin. The number of the first codon of each exon is indicated; exons beginning with the second or third base of a codon are indicated by “b” or “c,” respectively. At bottom, locations of the 11 different mutations identified in eight kindreds with NPHP4 mutations are shown. fs = Frameshift. E, NPHP4 mutations occurring homozygously in affected individuals from five consanguineous families (underlined). Compound heterozygous mutations are not shown. Mutated nucleotides and altered amino acids are depicted on gray background.

Nephrocystin proteins and their protein domains. Domain structure of the nephrocystin proteins. Nephrocystin proteins contain a diverse variety of protein domains and no common pattern can be identified. Nephrocystin-1, encoded by NPHP1, is a 732 amino acid (aa) protein, which possesses an N-terminal coiled-coil domain (CC) and a Src-homology 3 domain (SH3). Nephrocystin-2 (alias inversin), encoded by NPHP2/INVS, is a 1260 aa protein with 16 tandem ankyrin repeats, two IQ calmodulin binding domains (IQ). There are two destruction-box (D-box) regions (the first of which is Apc2 binding) and a bipartite nuclear localization signal (b-NLS) and a putative coiled-coil (CC) domain. Nephrocystin-3, encoded by NPHP3, is a 1330 aa protein, with a coiled-coil domain (CC), a tetratricopeptide-repeat domain (TPR) and a tubulin tyrosine ligase domain (TTL). Within the TTL a STAND (signal transduction ATPases with numerous domains) domain, which may be found in P-loop NTPases, is located. Nephrocystin-4 (alias nephroretinin), encoded by NPHP4 is a 1426 aa protein, which lacks any known domains. There is a central proline rich region. Nephrocystin-5, encoded by NPHP5/IQCB1 is a 598 aa protein. This protein possesses two IQ calmodulin binding sites, which surround a putative coiled-coil (CC) domain. Nephrocystin-6 (alias CEP290) is encoded by NPHP6/CEP290. This is a 2479 aa protein with multiple domains which include 13 coiled-coil (CC) domains; 3 tropomyosin homology domains (TM); 6 RepA/Rep⁺ protein KID motifs (KID); a bipartite nuclear localization signal (b-NLS); a ATP/GTP-binding site motif A (p-loop). The extent of homology with Structural Maintenance of Chromosomes proteins (SMC) is also indicated. AHI1 (alias Jouberin) is encoded by AHI1 and is an 1196 aa protein, which contains a Src-homology 3 domain (SH3), 6 WD40 domains (WD40) and an N-terminal coiled-coil domain. GLIS family zinc finger 2 (alias nephrocystin-7) is a 524 aa protein encoded by GLIS2. It contains 5 zinc finger domains (ZnF). RPGRIP1L (alias nephrocystin-8) is a 1315 aa protein encoded by RPGRIP1L. Protein domains include 6 coiled-coil (CC) domains and two protein kinase C conserved region 2 (C2) domains. The C-terminal C2 domain mediates the interaction with nephrocystin-4. NEK8 (alias nephrocystin-9) is a 692 aa protein with a serine/threonine protein kinases, catalytic domain (S-TKc) and a regulator of chromosome condensation (RCC1) domain, which is highly conserved throughout evolution.

Haplotype results on chromosome 1p36 performed for refinement of the NPHP4 locus in affected offspring from three consanguineous families with NPHP. Family, generation, and individual numbers are indicated above haplotypes. Paternal haplotypes are shown on blue background, and maternal haplotypes are shown on yellow background. A recombination in the maternal haplotype, which was directly observed in parent-to-child transmission in this pedigree, is shown on orange background. At left, 8 published microsatellites are given in italic, and 38 newly generated microsatellites are given in roman. Flanking markers to the published 2.1-Mb critical NPHP4 interval are depicted in red. Informative alleles are underlined. Haplotypes homozygous in continuity are encased in boxes. Homozygosity mapping revealed a p-terminal recombinant for marker D1E23 (asterisks) on the basis of heterozygosity in individuals F30 II-2 and F30 II-3 and an observed recombinant for marker SNP-KIAA0720-Ex19 (asterisks) in individual F30 II-3, thus refining the critical genetic region to a secure interval <1.2 Mb. On the basis of a significant LOD score yielded for F30 alone (Schuermann et al. ), this refinement yields secure borders. Further refinement was achieved through heterozygosity for markers D1E19 and D1S2870 (double asterisks) in individuals F32 II-1 and F60 II-1, respectively. This refines the critical genetic region to a suggestive interval <700 kb. Because of the presence of only two affected individuals in each family, this refinement is only suggestive. p-ter = Telomeric; cen = centromeric; nd = not done.