The murine orthologue of the Golgi-localized TPTE protein provides clues to the evolutionary history of the human TPTE gene family

Abstract.: The human TPTE gene encodes a testis-specific protein that contains four potential transmembrane domains and a protein tyrosine phosphatase motif, and shows homology to the tumor suppressor PTEN/MMAC1. Chromosomal mapping revealed multiple copies of the TPTE gene present on the acrocentric chromosomes 13, 15, 21 and 22, and the Y chromosome. Zooblot analysis suggests that mice may possess only one copy of TPTE. In the present study, we report the isolation and initial characterization of the full-length cDNA of the mouse homologue Tpte. At least three different mRNA transcripts (Tpte.a, b, c) are produced via alternative splicing, encoding predicted proteins that would contain four potential transmembrane domains and a protein tyrosine phosphatase motif. Transfection of a 5′EGFP-TPTE fusion protein in Hela cells revealed an intracellular localization within the Golgi apparatus. Tpte was mapped by radiation hybrid to a region of mouse chromosome 8 that shows conserved synteny with human 13q14.2-q21 between NEK3 and SGT1. This region of the human genome was found to contain a partial, highly diverged copy of TPTE that is likely to represent the ancestral copy from which the other copies of TPTE arose through duplication events. The Y chromosome copy of TPTE is a pseudogene and is not therefore involved in the testis expression of this gene famil

The transmembrane serine protease (TMPRSS3) mutated in deafness DFNB8/10 activates the epithelial sodium channel (ENaC) in vitro

TMPRSS3 encodes a transmembrane serine protease that contains both LDLRA and SRCR domains and is mutated in non-syndromic autosomal recessive deafness (DFNB8/10). To study its function, we cloned the mouse ortholog which maps to Mmu17, which is structurally similar to the human gene and encodes a polypeptide with 88% identity to the human protein. RT-PCR and RNA in situ hybridization on rat and mouse cochlea revealed that Tmprss3 is expressed in the spiral ganglion, the cells supporting the organ of Corti and the stria vascularis. RT-PCR on mouse tissues showed expression in the thymus, stomach, testis and E19 embryos. Transient expression of wild-type or tagged TMPRSS3 protein showed a primary localization in the endoplasmic reticulum. The epithelial amiloride-sensitive sodium channel (ENaC), which is expressed in many sodium-reabsorbing tissues including the inner ear and is regulated by membrane-bound channel activating serine proteases (CAPs), is a potential substrate of TMPRSS3. In the Xenopus oocyte expression system, proteolytic processing of TMPRSS3 was associated with increased ENaC mediated currents. In contrast, 6 TMPRSS3 mutants (D103G, R109W, C194F, W251C, P404L, C407R) causing deafness and a mutant in the catalytic triad of TMPRSS3 (S401A), failed to undergo proteolytic cleavage and activate ENaC. These data indicate that important signaling pathways in the inner ear are controlled by proteolytic cleavage and suggest: (i) the existence of an auto-catalytic processing by which TMPRSS3 would become active, and (ii) that ENaC could be a substrate of TMPRSS3 in the inner ea

Molecular and Clinical Characteristics in 46 Families Affected with Peutz-Jeghers Syndrome

Germline mutations of the tumor suppressor gene LKB1/STK11 are responsible for the Peutz-Jeghers syndrome (PJS), an autosomal-dominant disorder characterized by mucocutaneous pigmentation, hamartomatous polyps, and an increased risk of associated malignancies. In this study, we assessed the presence of pathogenic mutations in the LKB1/STK11 gene in 46 unrelated PJS families, and also carried genotype-phenotype correlation in regard of the development of cancer in 170 PJS patients belonging to these families. All LKB1/STK11 variants detected with single-strand conformational polymorphism were confirmed by direct sequencing, and those without LKB1/STK11 mutation were further submitted to Southern blot analysis for detection of deletions/rearrangements. Statistical analysis for genotype-phenotype correlation was performed. In 59% (27/46) of unrelated PJS cases, pathogenic mutations in the LKB1/STK11 gene, including 9 novel mutations, were identified. The new mutations were 2 splice site deletion-insertions, 2 missenses, 1 nonsense, and 4 abnormal splice sites. Genotype-phenotype analysis did not yield any significant differences between patients carrying mutations in LKB1/STK11 versus those without mutations, even with respect to primary biliary adenocarcinoma. This study presents the molecular characterization and cancer occurrence of a large cohort of PJS patients, increases the mutational spectrum of LKB1/STK11 allelic variants worldwide, and provides a new insight useful for clinical diagnosis and genetic counseling of PJS familie

GENCODE: producing a reference annotation for ENCODE

BACKGROUND: The GENCODE consortium was formed to identify and map all protein-coding genes within the ENCODE regions. This was achieved by a combination of initial manual annotation by the HAVANA team, experimental validation by the GENCODE consortium and a refinement of the annotation based on these experimental results. RESULTS: The GENCODE gene features are divided into eight different categories of which only the first two (known and novel coding sequence) are confidently predicted to be protein-coding genes. 5' rapid amplification of cDNA ends (RACE) and RT-PCR were used to experimentally verify the initial annotation. Of the 420 coding loci tested, 229 RACE products have been sequenced. They supported 5' extensions of 30 loci and new splice variants in 50 loci. In addition, 46 loci without evidence for a coding sequence were validated, consisting of 31 novel and 15 putative transcripts. We assessed the comprehensiveness of the GENCODE annotation by attempting to validate all the predicted exon boundaries outside the GENCODE annotation. Out of 1,215 tested in a subset of the ENCODE regions, 14 novel exon pairs were validated, only two of them in intergenic regions. CONCLUSION: In total, 487 loci, of which 434 are coding, have been annotated as part of the GENCODE reference set available from the UCSC browser. Comparison of GENCODE annotation with RefSeq and ENSEMBL show only 40% of GENCODE exons are contained within the two sets, which is a reflection of the high number of alternative splice forms with unique exons annotated. Over 50% of coding loci have been experimentally verified by 5' RACE for EGASP and the GENCODE collaboration is continuing to refine its annotation of 1% human genome with the aid of experimental validation

Disease-Causing 7.4 kb Cis-Regulatory Deletion Disrupting Conserved Non-Coding Sequences and Their Interaction with the FOXL2 Promotor: Implications for Mutation Screening

To date, the contribution of disrupted potentially cis-regulatory conserved non-coding sequences (CNCs) to human disease is most likely underestimated, as no systematic screens for putative deleterious variations in CNCs have been conducted. As a model for monogenic disease we studied the involvement of genetic changes of CNCs in the cis-regulatory domain of FOXL2 in blepharophimosis syndrome (BPES). Fifty-seven molecularly unsolved BPES patients underwent high-resolution copy number screening and targeted sequencing of CNCs. Apart from three larger distant deletions, a de novo deletion as small as 7.4 kb was found at 283 kb 5′ to FOXL2. The deletion appeared to be triggered by an H-DNA-induced double-stranded break (DSB). In addition, it disrupts a novel long non-coding RNA (ncRNA) PISRT1 and 8 CNCs. The regulatory potential of the deleted CNCs was substantiated by in vitro luciferase assays. Interestingly, Chromosome Conformation Capture (3C) of a 625 kb region surrounding FOXL2 in expressing cellular systems revealed physical interactions of three upstream fragments and the FOXL2 core promoter. Importantly, one of these contains the 7.4 kb deleted fragment. Overall, this study revealed the smallest distant deletion causing monogenic disease and impacts upon the concept of mutation screening in human disease and developmental disorders in particular

Cloning of a human homolog of the Drosophila enhancer of zeste gene (EZH2) that maps to chromosome 21q22.2

To identify genes that map on human chromosome 21 (HC21) and that may contribute to the phenotype of Down syndrome (DS), exon trapping was applied to cosmid DNA from an HC21-specific library LL21NCO2-Q. More than 550 potential exons were cloned and partially characterized. One of these, hmc23b04 (GenBank X88270) showed strong homology to the Drosophila Enhancer of zeste protein (GenBank U00180) from amino acid 665 to 694 (p = 7.6 x 10(-11). We have cloned the full-length cDNA for this human homolog of the Drosophila E(z) gene (termed EZH2) and mapped it to within YACs 64f11 and 809b11 between markers D21S65 and ERG on human chromosome 21q22.2. Sequence analysis indicates that EZH2 encodes a 746-amino-acid polypeptide that shows 60.5% identity to the Drosophila E(z) protein and contains a trithorax-like domain and a DNA-binding motif. Northern blot analysis revealed that EZH2 is expressed in several tissues. Alternatively spliced mRNA species have been observed. The Drosophila E(z) protein is a member of the polycomb group genes that maintain homeotic gene repression and are thought to control gene expression by regulating chromatin. The strong sequence conservation suggests a possible function of EZH2 in regulation of gene transcription and chromatin structure; it may therefore contribute to certain phenotypes of Down syndrome by altered regulation of its target genes

Frequency of replication/transcription errors in (A)/(T) runs of human genes

To estimate the error rate of the gene expression machinery and its possible age-related increase, we compared the occurrence of polymerase errors during replication and transcription in (A)/(T) runs, in DNA and RNA of young and old individuals and of early- and late-passage cultured fibroblasts. We analyzed three human genes: TPRD, TGFBR2, and ATRX containing stretches of (A)8, (A)10, and (T)13, respectively. The error rate was determined by sequencing 100 cloned PCR or RT-PCR fragments from each DNA and RNA sample. The error rates in replication and transcription increased with the stretch length. The pooled error rates for genomic DNA were: TPRD (A)8, TGFBR2 (A)10, and ATRX (T)13: 1%+/-0.41, 15.8%+/-1.3, and 31.3%+/-2.9, while those for RNA were: 3.8%+/-0.5, 19.3%+/-2.1, and 54.3%+/-1.8, respectively. The deletions of one nucleotide were the most frequent errors. In the replication analysis, a significant difference was found in old versus young individuals for the ATRX (T)13. In the transcription analysis, significantly higher error rates were obtained in old versus young individuals for the TPRD (A)8 and TGFBR2 (A)10. For these genes, the error rate in RNA isolated from fibroblasts was significantly higher than that in blood. The data show a trend of age-related increase in replication/transcription errors; however further studies are necessary to confirm this hypothesis, since the sample size is small. This imperfect fidelity of the gene expression process may explain the evolutionary disadvantage of nucleotide repeats within coding sequences, and that these repeats are targets for mutations in human diseases

Cloning of the TMPRSS2 gene, which encodes a novel serine protease with transmembrane, LDLRA, and SRCR domains and maps to 21q22.3

To contribute to the development of the transcription map of human chromosome 21 (HC21), we have used exon trapping from pools of HC21-specific cosmids. Using selected trapped exons, we have identified a novel gene (named TMPRSS2) that encodes a multimeric protein with a serine protease domain. The TMPRSS2 3.8-kb mRNA is expressed strongly in small intestine and weakly in several other tissues. The full-length cDNA encodes a predicted protein of 492 amino acids that contains the following domains: (i) A serine protease domain (aa 255-492) of the S1 family that probably cleaves at Arg or Lys residues. (ii) An SRCR (scavenger receptor cysteine-rich) domain (aa 149-242) of group A (6 conserved Cys). This type of domain is involved in the binding to other cell surface or extracellular molecules. (iii) An LDLRA (LDL receptor class A) domain (aa 113-148). This type of domain forms a binding site for calcium. (iv) A predicted transmembrane domain (aa 84-106). No typical signal peptide was recognized. The gene was mapped to 21q22.3 between markers ERG and D21S56 in the same P1 as MX1. The physiological role of TMPRSS2 and its involvement in trisomy 21 phenotypes or monogenic disorders that map to HC21 are unknown

Cloning of the cDNA for the human ATP synthase OSCP subunit (ATP5O) by exon trapping and mapping to chromosome 21q22.1-q22.2

Exon trapping was used to clone portions of potential genes from human chromosome 21. One trapped sequence showed striking homology with the bovine and rat ATP synthase OSCP (oligomycin sensitivity conferring protein) subunit. We subsequently cloned the full-length human ATP synthase OSCP cDNA (GDB/HGMW approved name ATP50) from infant brain and muscle libraries and determined its nucleotide and deduced amino acid sequence (EMBL/GenBank Accession No. X83218). The encoded polypeptide contains 213 amino acids, with more than 80% identity to bovine and murine ATPase OSCP subunits and over 35% identity to Saccharomyces cerevisiae and sweet potato sequences. The human ATP5O gene is located at 21q22.1-q22.2, just proximal to D21S17, in YACs 860G11 and 838C7 of the Chumakov et al. (Nature 359:380, 1992) YAC contig. The gene is expressed in all human tissues examined, most strongly in muscle and heart. This ATP5O subunit is a key structural component of the stalk of the mitochondrial respiratory chain F1F0-ATP synthase and as such may contribute in a gene dosage-dependent manner to the phenotype of Down syndrome (trisomy 21)

Cloning of a novel homeobox-containing gene, PKNOX1, and mapping to human chromosome 21q22.3

To contribute to the development of the transcript map of human chromosome 21 and to the understanding of the pathogenesis of Down syndrome, we have used exon trapping to identify portions of genes from pools of HC21-specific cosmids. More than 550 potential exons have been isolated to date. One such trapped exon, hmc37a09 (GenBank Accession No. X88106), was identical to a region of a human EST, L12425 (GenBank Accession No. D31072). Its predicted amino acid sequence was homologous to the homeodomain region of homeobox-containing genes. Using the trapped sequence and the EST as probes to screen human fetal brain and kidney cDNA libraries, we have cloned the corresponding full-length cDNA. This novel gene encodes a homeodomain-containing polypeptide of 436 amino acids. The most closely related sequence is that of the mouse Meis1, a PBX-like homeobox gene. The homeodomain of the novel gene is closely related to those of the mammalian PBX family and the plant Knotted1 family (involved in plant development). This gene is named PKNOX1 by the Human Nomenclature Committee. By PCR amplification, hybridization, and genetic linkage analysis using a (GT)n polymorphism in the 3'UTR, we have precisely localized PKNOX1 to chromosome 21q22.3 between markers D21S212 and D21S25 on YAC350F7. PKNOX1 is expressed in many human tissues tested by Northern blot analysis. The involvement of the PKNOX1 gene in Down syndrome and/or monogenic disorders associated with dysfunction of this gene is presently unknown. Targeted disruption of the PKNOX1 homolog in mice will enhance our understanding of its biological function in normal mammalian development