Mutations in C2orf37, Encoding a Nucleolar Protein, Cause Hypogonadism, Alopecia, Diabetes Mellitus, Mental Retardation, and Extrapyramidal Syndrome

Hypogonadism, alopecia, diabetes mellitus, mental retardation, and extrapyramidal syndrome (also referenced as Woodhouse-Sakati syndrome) is a rare autosomal recessive multisystemic disorder. We have identified a founder mutation consisting of a single base-pair deletion in C2orf37 in eight families of Saudi origin. Three other loss-of-function mutations were subsequently discovered in patients of different ethnicities. The gene encodes a nucleolar protein of unknown function, and the cellular phenotype observed in patient lymphoblasts implicates a role for the nucleolus in the pathogenesis of this disease. Our findings expand the list of human disorders linked to the nucleolus and further highlight the developmental and/or maintenance functions of this organelle.     

Localization and genetic linkage of the human immunoglobulin heavy chain genes and the creatine kinase brain (CKB) gene: Identification of a hot spot for recombination

The immunoglobulin heavy chain (IGH) gene cluster and the gene coding for the brain form of the enzyme creatine kinase (CKB) have previously been localized to chromosome 14, at 14q32.3 and 14q32, respectively. Here we report more precise regional localization of these genes by dosage studies using DNA from a child hemizygous for the region from 14q32.32 to 14qter. CKB and IGH are present in a single dose in the proband. Dosage studies in a second patient with a similar but smaller deletion due to a ring chromosome 14 show that CKB is proximal to the IGH cluster. An EcoRI restriction site polymorphism was found with probes for the CKB gene. Linkage analysis of family data indicates that CKB is closely linked to IGH. Linkage analysis also revealed unusually high recombination (beta = 3.2%) between the C delta and C gamma 3 genes of the IGH constant region, which are only 60 kb apart. This finding, in combination with a previous observation of linkage equilibrium in the region, suggests that the C delta-C gamma 3 region contains a recombination hot spot.     

Confirmation of linkage of Duane's syndrome and refinement of the disease locus to an 8.8-cM interval on chromosome 2q31

Duane's syndrome is a congenital abnormality of eye movement, which may be inherited as an autosomal dominant trait but usually occurs sporadically. Genetic mapping in a Mexican family has recently identified a locus for Duane's syndrome within a 17.8-cM region of chromosome 2q31. The region was flanked by the microsatellite markers D2S2330 and D2S364. We performed linkage and haplotype analysis in a four-generation UK family with autosomal dominant transmission of Duane's syndrome. Linkage to 2q31 was confirmed with a maximum logarithm of differences (lod) score of 3.3 at theta = 0. The genetic interval was reduced to an 8.8-cM region between markers D2S326 and D2S364 that includes the candidate homeobox D gene cluster.     

Duplication of C7orf58, WNT16 and FAM3C in an Obese Female with a t(7;22)(q32.1;q11.2) Chromosomal Translocation and Clinical Features Resembling Coffin-Siris Syndrome

We characterized the t(7;22)(q32;q11.2) chromosomal translocation in an obese female with coarse features, short stature, developmental delay and a hypoplastic fifth digit. While these clinical features suggest Coffin-Siris Syndrome (CSS), we excluded a CSS diagnosis by exome sequencing based on the absence of deleterious mutations in six chromatin-remodeling genes recently shown to cause CSS. Thus, molecular characterization of her translocation could delineate genes that underlie other syndromes resembling CSS. Comparative genomic hybridization microarrays revealed on chromosome 7 the duplication of a 434,682 bp region that included the tail end of an uncharacterized gene termed C7orf58 (also called CPED1) and spanned the entire WNT16 and FAM3C genes. Because the translocation breakpoint on chromosome 22 did not disrupt any apparent gene, her disorder was deemed to result from the rearrangement on chromosome 7. Mapping of yeast and bacterial artificial chromosome clones by fluorescent in situ hybridization on chromosome spreads from this patient showed that the duplicated region and all three genes within it were located on both derivative chromosomes 7 and 22. Furthermore, DNA sequencing of exons and splice junctional regions from C7orf58, WNT16 and FAM3C revealed the presence of potential splice site and promoter mutations, thereby augmenting the detrimental effect of the duplicated genes. Hence, dysregulation and/or disruptions of C7orf58, WNT16 and FAM3C underlie the phenotype of this patient, serve as candidate genes for other individuals with similar clinical features and could provide insights into the physiological role of the novel gene C7orf58.     

Confirmation of genetic heterogeneity in limb-girdle muscular dystrophy: Linkage of an autosomal dominant form to chromosome 5q

Limb-girdle muscular dystrophy (LGMD) is a clinically and genetically heterogenous group of disorders, with both recessive and dominant forms reported. Recently, a series of recessive LGMD families were linked to chromosome 15q. We report herein the results of our linkage studies in a previously reported large autosomal dominant family. The LGMD gene in this family was localized to chromosome 5q22.3-31.3 by using a series of CA(n) microsatellite repeat markers. Linkage to 15q was excluded. These findings confirm genetic heterogeneity in this clinically diverse syndrome.     

Novel human esophagus-specific gene c1orf10: cDNA cloning, gene structure, and frequent loss of expression in esophageal cancer

We have identified a novel human gene, designated C1orf10, using modified differential display PCR. The C1orf10 gene, which spans 5 kb in length, is composed of three exons. The deduced protein contains 495 amino acids with one transmembrane domain. The amino acid sequence of C1orf10 is characterized by the presence of a calcium-binding motif of about 90 amino acids at its N-terminal and a conserved consecutive repeat sequence of 60 amino acids that was identified previously only in bacterial ice nucleation proteins. In normal adult tissues, C1orf10 is highly expressed only in the esophagus and was undetectable in a total of 15 other tissues examined, suggesting its important role in esophageal cells. The expression of C1orf10 is either dramatically reduced or absent in esophageal cancer cell lines (3/3) as well as primary esophageal cancer tissues (35/37) compared with the corresponding normal esophageal mucosa. Using a radiation hybrid panel, C1orf10 was found to be located on chromosome 1q21. These findings suggest that expression of C1orf10 is unique to esophageal cells and that loss of its expression may play a role in the development of esophageal cancer.     

Splicing mutation of the prostacyclin synthase gene in a family associated with hypertension

Prostacyclin inhibits platelet aggregation, smooth muscle cell proliferation, and vasoconstriction. The prostacyclin synthase (PGIS) gene is a candidate gene for cardiovascular disease. The purpose of this study was to locate possible mutations in the PGIS gene related to hypertension and cerebral infarction. Using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method, we discovered a T to C transition at the +2 position of the splicing donor site of intron 9 in patients with essential hypertension (EH). In vitro expression analysis of an allelic minigene consisting of exons 8-10 revealed that the nucleotide transition causes skipping of exon 9. This in turn alters the translational reading frame of exon 10 and introduces a premature stop codon (TGA). A three-dimensional model shows that the splice site mutation produces a truncated protein with a deletion in the heme-binding region. This splice site mutation was found in only one subject in 200 EH patients and 200 healthy controls. Analysis of the patient's family members revealed the mutation in two of the three siblings. The urinary excretion of prostacyclin metabolites in subjects with the mutation was significantly decreased. All subjects displaying the splice site mutation in the PGIS gene were hypertensive. In this study, we report a novel splicing mutation in the PGIS gene, which is associated with hypertension in a family. It is thought that this mechanism may involve in the pathophysiology of their hypertension.     

Identification and characterization of C6orf37, a novel candidate human retinal disease gene on chromosome 6q14

We have identified a novel human gene, chromosome 6 open reading frame 37 (C6orf37), that is expressed in the retina and maps to human chromosome 6q14, a genomic region that harbors multiple retinal disease loci. The cDNA sequence contains an open reading frame of 1314 bp that encodes a 437-amino acid protein with a predicted molecular mass of 49.2 kDa. Northern blot analysis indicates that this gene is widely expressed, with preferential expression observed in the retina compared to other ocular tissues. The C6orf37 protein shares homology with putative proteins in R. norvegicus, M. musculus, D. melanogaster, and C. elegans, suggesting evolutionary conservation of function. Additional sequence analysis predicts that the C6orf37 gene product is a soluble, globular cytoplasmic protein containing several conserved phosphorylation sites. Furthermore, we have defined the genomic structure of this gene, which will enable its analysis as a candidate gene for chromosome 6q-associated inherited retinal disorders.     

Mutations in C8orf37, encoding a ciliary protein, are associated with autosomal-recessive retinal dystrophies with early macular involvement

Cone-rod dystrophy (CRD) and retinitis pigmentosa (RP) are clinically and genetically overlapping heterogeneous retinal dystrophies. By using homozygosity mapping in an individual with autosomal-recessive (ar) RP from a consanguineous family, we identified three sizeable homozygous regions, together encompassing 46 Mb. Next-generation sequencing of all exons, flanking intron sequences, microRNAs, and other highly conserved genomic elements in these three regions revealed a homozygous nonsense mutation (c.497T>A [p.Leu166^∗]) in C8orf37, located on chromosome 8q22.1. This mutation was not present in 150 ethnically matched control individuals, single-nucleotide polymorphism databases, or the 1000 Genomes database. Immunohistochemical studies revealed C8orf37 localization at the base of the primary cilium of human retinal pigment epithelium cells and at the base of connecting cilia of mouse photoreceptors. C8orf37 sequence analysis of individuals who had retinal dystrophy and carried conspicuously large homozygous regions encompassing C8orf37 revealed a homozygous splice-site mutation (c.156−2A>G) in two siblings of a consanguineous family and homozygous missense mutations (c.529C>T [p.Arg177Trp]; c.545A>G [p.Gln182Arg]) in siblings of two other consanguineous families. The missense mutations affect highly conserved amino acids, and in silico analyses predicted that both variants are probably pathogenic. Clinical assessment revealed CRD in four individuals and RP with early macular involvement in two individuals. The two CRD siblings with the c.156−2A>G mutation also showed unilateral postaxial polydactyly. These results underline the importance of disrupted ciliary processes in the pathogenesis of retinal dystrophies.     

A gene for familial juvenile polyposis maps to chromosome 18q21.1.

Familial juvenile polyposis (FJP) is a hamartomatouspolyposis syndrome in which affected family members develop upper and lower gastrointestinal juvenile polyps and are at increased risk for gastrointestinal cancer. A genetic locus for FJP has not yet been identified by linkage; therefore, the objective of this study was to perform a focused genome screen in a large family segregating FJP. No evidence for linkage was found with markers near MSH2, MLH1, MCC, APC, HMPS, CDKN2A, JP1, PTEN, KRAS2, TP53, or LKB1. Linkage to FJP was established with several markers from chromosome 18q21.1. The maximum LOD score was 5.00, with marker D18S1099 (recombination fraction of .001). Analysis of critical recombinants places the FJP gene in an 11.9-cM interval bounded by D18S1118 and D18S487, a region that also contains the tumor-suppressor genes DCC and DPC4. These data demonstrate localization of a gene for FJP to chromosome 18q21.1 by linkage, and they raise the possibility that either DCC or DPC4 could be responsible for FJP.     

Alternative splicing determines the domain structure of WWP1, a Nedd4 family protein.

Nedd-4-like proteins are E3 ubiquitin-ligase molecules which regulate key trafficking decisions, including targeting of proteins to proteosomes or lysosomes. Here we show that a human Nedd4 family gene, WWP1, is localized on 8q21 and generates at least six isoforms through alternative splicing. We show that alternative splicing affects the domain structure of WWP1, with forms that contain or lack an N-terminal C2 domain. Interestingly, the relative ratio of these forms varies in a tissue-specific manner. Other splice forms were also identified which may disrupt the structure of the C2 domain by removing its predicted C-terminal beta-strands. One splice form generates, through the introduction of a reading frame shift, a C2 domain-only form of WWP1. We discuss the hypothesis that regulation of splice site usage may modulate the activity of WWP1 and possibly other Nedd4 family proteins.