The gene mutated in juvenile nephronophthisis type 4 encodes a novel protein that interacts with nephrocystin

Nephronophthisis, the most common genetic cause of chronic renal failure in children, is a progressive tubulo-interstitial kidney disorder that is inherited as an autosomal recessive trait. The disease is characterized by polyuria, growth retardation and deterioration of renal function during childhood or adolescence. The most prominent histological features are modifications of the tubules with thickening of the basement membrane, interstitial fibrosis and, in the advanced stages, medullary cysts. Nephronophthisis can also be associated with conditions affecting extrarenal organs, such as retinitis pigmentosa (Senior-L?ken syndrome) and ocular motor apraxia (Cogan syndrome). Three loci are associated with the juvenile, infantile and adolescent forms, on chromosomes 2q13 (NPHP1; refs 5,6), 9q22 (NPHP2; ref. 7) and 3q21 (NPHP3; ref. 8), respectively. NPHP1, the only gene identified so far, encodes nephrocystin, which contains a Src homology 3 (SH3) domain and interacts with intracytoplasmic proteins involved in cell adhesion. Recently, a second locus associated with the juvenile form of the disease, NPHP4, was mapped to chromosome 1p36 (ref. 14). We carried out haplotype analysis of families affected with nephronophthisis that were not linked to the NPHP1, NPHP2 or NPHP3 loci, using markers covering this region. This allowed us to reduce the NPHP4 interval to a one centimorgan interval between D1S2795 and D1S2870, which contains six genes. We identified five different mutations in one of these genes, designated NPHP4, in unrelated individuals with nephronophthisis. The NPHP4 gene encodes a 1,250-amino acid protein of unknown function that we named nephrocystin-4. We demonstrated the interaction of nephrocystin-4 with nephrocystin suggesting that these two proteins participate in a common signaling pathway.     

The Pendred syndrome gene encodes a chloride-iodide transport protein

Pendred syndrome is the most common form of syndromic deafness and characterized by congenital sensorineural hearing loss and goitre. This disorder was mapped to chromosome 7 and the gene causing Pendred syndrome (PDS) was subsequently identified by positional cloning. PDS encodes a putative transmembrane protein designated pendrin. Pendrin is closely related to a family of sulfate transport proteins that includes the rat sulfate-anion transporter (encoded by Sat-1; 29% amino acid sequence identity), the human diastrophic dysplasia sulfate transporter (encoded by DTD; 32%) and the human sulfate transporter 'downregulated in adenoma' (encoded by DRA; 45%). On the basis of this homology and the presence of a slightly modified sulfate-transporter signature sequence comprising its putative second transmembrane domain, pendrin has been proposed to function as a sulfate transporter. We were unable to detect evidence of sulfate transport following the expression of pendrin in Xenopus laevis oocytes by microinjection of PDS cRNA or in Sf9 cells following infection with PDS-recombinant baculovirus. The rates of transport for iodide and chloride were significantly increased following the expression of pendrin in both cell systems. Our results demonstrate that pendrin functions as a transporter of chloride and iodide, but not sulfate, and may provide insight into thyroid physiology and the pathophysiology of Pendred syndrome.     

The pallid gene encodes a novel, syntaxin 13-interacting protein involved in platelet storage pool deficiency

Pallid (pa) is 1 of 13 platelet storage pool deficiency (SPD) mouse mutants. pa animals suffer from prolonged bleeding time, pigment dilution, kidney lysosomal enzyme elevation, serum alpha1-antitrypsin activity deficiency and abnormal otolith formation. As with other mouse mutants of this class, characterization of pa mice suggests a defect in organelle biosynthesis. Here we describe the physical mapping, positional cloning, and mutational and functional analysis of the gene that is defective in pa mice. It encodes a ubiquitously expressed, highly charged 172-amino-acid protein (termed pallidin) with no homology to known proteins. We detected a nonsense mutation at codon 69 of this gene in the pallid mutant. In a yeast two-hybrid screen, we discovered that pallidin interacts with syntaxin 13, a t-SNARE protein that mediates vesicle-docking and fusion. We confirmed this interaction by co-immunoprecipitation assay. Immunofluorescence studies corroborate that the cellular distribution of pallidin overlaps that of syntaxin 13. Whereas the mocha and pearl SPD mutants have defects in Ap-3, our findings suggest that pa SPD mutants are defective in a more downstream event of vesicle-trafficking: namely, vesicle-docking and fusion.     

The amnionless gene, essential for mouse gastrulation, encodes a visceral-endoderm-specific protein with an extracellular cysteine-rich domain

Fate-mapping experiments in the mouse have revealed that the primitive streak can be divided into three functional regions: the proximal region gives rise to germ cells and the extra-embryonic mesoderm of the yolk sac; the distal region generates cardiac mesoderm and node-derived axial mesendoderm; and the middle streak region produces the paraxial, intermediate and lateral plate mesoderm of the trunk. To gain insight into the mechanisms that mediate the assembly of the primitive streak into these functional regions, we have cloned and functionally identified the gene disrupted in the amnionless (amn) mouse, which has a recessive, embryonic lethal mutation that interferes specifically with the formation and/or specification of the middle primitive streak region during gastrulation. Here we report that the gene Amn encodes a novel type I transmembrane protein that is expressed exclusively in the extra-embryonic visceral endoderm layer during gastrulation. The extracellular region of the Amn protein contains a cysteine-rich domain with similarity to bone morphogenetic protein (BMP)-binding cysteine-rich domains in chordin, its Drosophila melanogaster homolog (Short gastrulation) and procollagen IIA (ref. 3). Our findings indicate that Amn may direct the production of trunk mesoderm derived from the middle streak by acting in the underlying visceral endoderm to modulate a BMP signaling pathway.     

The gene mutated in bare patches and striated mice encodes a novel 3beta-hydroxysteroid dehydrogenase.

X-linked dominant disorders that are exclusively lethal prenatally in hemizygous males have been described in human and mouse. None of the genes responsible has been isolated in either species. The bare patches (Bpa) and striated (Str) mouse mutations were originally identified in female offspring of X-irradiated males. Subsequently, additional independent alleles were described. We have previously mapped these X-linked dominant, male-lethal mutations to an overlapping region of 600 kb that is homologous to human Xq28 (ref. 4) and identified several candidate genes in this interval. Here we report mutations in one of these genes, Nsdhl, encoding an NAD(P)H steroid dehydrogenase-like protein, in two independent Bpa and three independent Str alleles. Quantitative analysis of sterols from tissues of affected Bpa mice support a role for Nsdhl in cholesterol biosynthesis. Our results demonstrate that Bpa and Str are allelic mutations and identify the first mammalian locus associated with an X-linked dominant, male-lethal phenotype. They also expand the spectrum of phenotypes associated with abnormalities of cholesterol metabolism.     

The gene mutated in ataxia-ocular apraxia 1 encodes the new HIT/Zn-finger protein aprataxin

The newly recognized ataxia-ocular apraxia 1 (AOA1; MIM 208920) is the most frequent cause of autosomal recessive ataxia in Japan and is second only to Friedreich ataxia in Portugal. It shares several neurological features with ataxia-telangiectasia, including early onset ataxia, oculomotor apraxia and cerebellar atrophy, but does not share its extraneurological features (immune deficiency, chromosomal instability and hypersensitivity to X-rays). AOA1 is also characterized by axonal motor neuropathy and the later decrease of serum albumin levels and elevation of total cholesterol. We have identified the gene causing AOA1 and the major Portuguese and Japanese mutations. This gene encodes a new, ubiquitously expressed protein that we named aprataxin. This protein is composed of three domains that share distant homology with the amino-terminal domain of polynucleotide kinase 3'- phosphatase (PNKP), with histidine-triad (HIT) proteins and with DNA-binding C2H2 zinc-finger proteins, respectively. PNKP is involved in DNA single-strand break repair (SSBR) following exposure to ionizing radiation and reactive oxygen species. Fragile-HIT proteins (FHIT) cleave diadenosine tetraphosphate, which is potentially produced during activation of the SSBR complex. The results suggest that aprataxin is a nuclear protein with a role in DNA repair reminiscent of the function of the protein defective in ataxia-telangiectasia, but that would cause a phenotype restricted to neurological signs when mutant.     

PALB2, which encodes a BRCA2-interacting protein, is a breast cancer susceptibility gene

PALB2 interacts with BRCA2, and biallelic mutations in PALB2 (also known as FANCN), similar to biallelic BRCA2 mutations, cause Fanconi anemia. We identified monoallelic truncating PALB2 mutations in 10/923 individuals with familial breast cancer compared with 0/1,084 controls (P = 0.0004) and show that such mutations confer a 2.3-fold higher risk of breast cancer (95% confidence interval (c.i.) = 1.4-3.9, P = 0.0025). The results show that PALB2 is a breast cancer susceptibility gene and further demonstrate the close relationship of the Fanconi anemia-DNA repair pathway and breast cancer predisposition.     

The gene defective in leukocyte adhesion deficiency II encodes a putative GDP-fucose transporter

Leukocyte adhesion deficiency II (LAD II) is characterized by the lack of fucosylated glycoconjugates, including selectin ligands, causing immunodeficiency and severe mental and growth retardation. No deficiency in fucosyltransferase activities or in the activities of enzymes involved in GDP-fucose biosynthesis has been found. Instead, the transport of GDP-fucose into isolated Golgi vesicles of LAD II cells appeared to be reduced. To identify the gene mutated in LAD II, we cloned 12 cDNAs from Caenorhabditis elegans, encoding multi-spanning transmembrane proteins with homology to known nucleotide sugar transporters, and transfected them into fibroblasts from an LAD II patient. One of these clones re-established expression of fucosylated glycoconjugates with high efficiency and allowed us to identify a human homolog with 55% identity, which also directed re-expression of fucosylated glycoconjugates. Both proteins were localized to the Golgi. The corresponding endogenous protein in LAD II cells had an R147C amino acid change in the conserved fourth transmembrane region. Overexpression of this mutant protein in cells from a patient with LAD II did not rescue fucosylation, demonstrating that the point mutation affected the activity of the protein. Thus, we have identified the first putative GDP-fucose transporter, which has been highly conserved throughout evolution. A point mutation in its gene is responsible for the disease in this patient with LAD II.     

BBS10 encodes a vertebrate-specific chaperonin-like protein and is a major BBS locus

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous ciliopathy. Although nine BBS genes have been cloned, they explain only 40-50% of the total mutational load. Here we report a major new BBS locus, BBS10, that encodes a previously unknown, rapidly evolving vertebrate-specific chaperonin-like protein. We found BBS10 to be mutated in about 20% of an unselected cohort of families of various ethnic origins, including some families with mutations in other BBS genes, consistent with oligogenic inheritance. In zebrafish, mild suppression of bbs10 exacerbated the phenotypes of other bbs morphants.     

Mutations in a new gene encoding a thiamine transporter cause thiamine-responsive megaloblastic anaemia syndrome.

Thiamine-responsive megaloblastic anaemia syndrome (TRMA; MIM 249270) is an autosomal recessive disorder with features that include megaloblastic anaemia, mild thrombocytopenia and leucopenia, sensorineural deafness and diabetes mellitus. Treatment with pharmacologic doses of thiamine ameliorates the megaloblastic anaemia and diabetes mellitus. A defect in the plasma membrane transport of thiamine has been demonstrated in erythrocytes and cultured skin fibroblasts from TRMA patients. The gene causing TRMA was assigned to 1q23.2-q23.3 by linkage analysis. Here we report the cloning of a new gene, SLC19A2, identified from high-through-put genomic sequences due to homology with SLC19A1, encoding reduced folate carrier 1 (refs 8-10). We cloned the entire coding region by screening a human fetal brain cDNA library. SLC19A2 encodes a protein (of 497 aa) predicted to have 12 transmembrane domains. We identified 2 frameshift mutations in exon 2. a 1-bp insertion and a 2-bp deletion, among four Iranian families with TRMA. The sequence homology and predicted structure of SLC19A2, as well as its role in TRMA, suggest that its gene product is a thiamine carrier, the first to be identified in complex eukaryotes.     

Mutations in the gene encoding B, a novel transporter protein, reduce melanin content in medaka

Pigmentation of the skin is of great social, clinical and cosmetic significance. Several genes that, when mutated, give rise to altered coat color in mice have been identified; their analysis has provided some insight into melanogenesis and human pigmentation diseases. Such analyses do not, however, fully inform on the pigmentation of lower vertebrates because mammals have only one kind of chromatophore, the melanocyte. In contrast, the medaka (a small, freshwater teleost) is a suitable model of the lower vertebrates because it has all kinds of chromatophores. The basic molecular genetics of fish are known and approximately 70 spontaneous pigmentation mutants have been isolated. One of these, an orange-red variant, is a homozygote of a well-known and common allele, b, and has been bred for hundreds of years by the Japanese. Here, we report the first successful positional cloning of a medaka gene (AIM1): one that encodes a transporter that mediates melanin synthesis. The protein is predicted to consist of 12 transmembrane domains and is 55% identical to a human EST of unknown function isolated from melanocytes and melanoma cells. We also isolated a highly homologous gene from the mouse, indicating a conserved function of vertebrate melanogenesis. Intriguingly, these proteins have sequence and structural similarities to plant sucrose transporters, suggesting a relevance of sucrose in melanin synthesis. Analysis of AIM1 orthologs should provide new insights into the regulation of melanogenesis in both teleosts and mammals.     

Fanconi anemia is associated with a defect in the BRCA2 partner PALB2

The Fanconi anemia and BRCA networks are considered interconnected, as BRCA2 gene defects have been discovered in individuals with Fanconi anemia subtype D1. Here we show that a defect in the BRCA2-interacting protein PALB2 is associated with Fanconi anemia in an individual with a new subtype. PALB2-deficient cells showed hypersensitivity to cross-linking agents and lacked chromatin-bound BRCA2; these defects were corrected upon ectopic expression of PALB2 or by spontaneous reversion.     

Mutations in CEP290, which encodes a centrosomal protein, cause pleiotropic forms of Joubert syndrome

Joubert syndrome-related disorders (JSRD) are a group of syndromes sharing the neuroradiological features of cerebellar vermis hypoplasia and a peculiar brainstem malformation known as the 'molar tooth sign'. We identified mutations in the CEP290 gene in five families with variable neurological, retinal and renal manifestations. CEP290 expression was detected mostly in proliferating cerebellar granule neuron populations and showed centrosome and ciliary localization, linking JSRDs to other human ciliopathies.     

IFT80, which encodes a conserved intraflagellar transport protein, is mutated in Jeune asphyxiating thoracic dystrophy

Jeune asphyxiating thoracic dystrophy, an autosomal recessive chondrodysplasia, often leads to death in infancy because of a severely constricted thoracic cage and respiratory insufficiency; retinal degeneration, cystic renal disease and polydactyly may be complicating features. We show that IFT80 mutations underlie a subset of Jeune asphyxiating thoracic dystrophy cases, establishing the first association of a defective intraflagellar transport (IFT) protein with human disease. Knockdown of ift80 in zebrafish resulted in cystic kidneys, and knockdown in Tetrahymena thermophila produced shortened or absent cilia.     

The gene encoding a newly discovered protein, chorein, is mutated in chorea-acanthocytosis

Chorea-acanthocytosis is a neurodegenerative disorder with peripheral red cell acanthocytosis. Linkage of chorea-acanthocytosis to chromosome 9q21 has been found. We refined the locus region and identified a previously unknown, full-length cDNA encoding a presumably structural protein, which we called chorein. We found a deletion in the coding region of the cDNA leading to a frame shift resulting in the production of a truncated protein in both alleles of patients and in single alleles of obligate carriers.     

Maternal imprinting of the mouse Snrpn gene and conserved linkage homology with the human Prader-Willi syndrome region.

Prader-Willi syndrome (PWS) is associated with paternal gene deficiencies in human chromosome 15q11-13, suggesting that PWS is caused by a deficiency in one or more maternally imprinted genes. We have now mapped a gene, Snrpn, encoding a brain-enriched small nuclear ribonucleoprotein (snRNP)-associated polypeptide SmN, to mouse chromosome 7 in a region of homology with human chromosome 15q11-13 and demonstrated that Snrpn is a maternally imprinted gene in mouse. These studies, in combination with the accompanying human mapping studies showing that SNRPN maps in the Prader-Willi critical region, identify SNRPN as a candidate gene involved in PWS and suggest that PWS may be caused, in part, by defects in mRNA processing.