Introduction of novel splice variants for CASC18 gene and its relation to the neural differentiation.

BACKGROUND: CASC18 along with APPL2, OCC-1 and NUAK1 flanking genes are located in 12q23.3 locus which is known as a potential cancer predisposition locus. Only an uncharacterized EST was initially reported for CASC18 and it was crucial to find its full length sequence and function. METHODS AND RESULTS: In an attempt to search for the CASC18's full-length gene sequence, other related ESTs were bioinformatically collected and four novel splice variants (designated as; CASC18-A, -B, -C and -D) were deduced and some were experimentally validated. Two transcription start sites and two alternative polyadenylation sites were deduced for CASC18 gene, using EST data mining and RACE method. CASC18-A and CASC18-D were exclusively expressed in neural cell lines and CASC18-D expression level was gradually increased during the NT2 differentiation to the neuron-like cells. Consistently, overexpression of CASC18-D variant in NT2 cells resulted in remarkable up-regulation of PAX6 neural differentiation marker, suggesting a crucial role of this variant in neural differentiation. CONCLUSION: Here, we introduced seven novel transcription variants for human CASC18 gene in which CASC18-D has the potential of being used as a neural cell differentiation marker.

Functional characterization of the 12p12.1 renal cancer-susceptibility locus implicates BHLHE41.

Genome-wide association studies have identified multiple renal cell carcinoma (RCC) susceptibility loci. Here, we use regional imputation and bioinformatics analysis of the 12p12.1 locus to identify the single-nucleotide polymorphism (SNP) rs7132434 as a potential functional variant. Luciferase assays demonstrate allele-specific regulatory activity and, together with data from electromobility shift assays, suggest allele-specific differences at rs7132434 for AP-1 transcription factor binding. In an analysis of The Cancer Genome Atlas data, SNPs highly correlated with rs7132434 show allele-specific differences in BHLHE41 expression (trend P value=6.3 × 10(-7)). Cells overexpressing BHLHE41 produce larger mouse xenograft tumours, while RNA-seq analysis reveals that constitutively increased BHLHE41 induces expression of IL-11. We conclude that the RCC risk allele at 12p12.1 maps to rs7132434, a functional variant in an enhancer that upregulates BHLHE41 expression which, in turn, induces IL-11, a member of the IL-6 cytokine family.

Imaging genetics and psychiatric disorders.

Imaging genetics is an integrated research method that uses neuroimaging and genetics to assess the impact of genetic variation on brain function and structure. Imaging genetics is both a tool for the discovery of risk genes for psychiatric disorders and a strategy for characterizing the neural systems affected by risk gene variants to elucidate quantitative and mechanistic aspects of brain function implicated in psychiatric disease. Early studies of imaging genetics included association analyses between brain morphology and single nucleotide polymorphisms whose function is well known, such as catechol-Omethyltransferase (COMT) and brain-derived neurotrophic factor (BDNF). GWAS of psychiatric disorders have identified genes with unknown functions, such as ZNF804A, and imaging genetics has been used to investigate clues of the biological function of these genes. The difficulty in replicating the findings of studies with small sample sizes has motivated the creation of largescale collaborative consortiums, such as ENIGMA, CHARGE and IMAGEN, to collect thousands of images. In a genome-wide association study, the ENIGMA consortium successfully identified common variants in the genome associated with hippocampal volume at 12q24, and the CHARGE consortium replicated this finding. The new era of imaging genetics has just begun, and the next challenge we face is the discovery of small effect size signals from large data sets obtained from genetics and neuroimaging. New methods and technologies for data reduction with appropriate statistical thresholds, such as polygenic analysis and parallel independent component analysis (ICA), are warranted. Future advances in imaging genetics will aid in the discovery of genes and provide mechanistic insight into psychiatric disorders.

Genome-wide analysis associates familial colorectal cancer with increases in copy number variations and a rare structural variation at 12p12.3.

Colorectal cancer (CRC) is one of the most common cancer worldwide. However, a large number of genetic risk factors involved in CRC have not been understood. Copy number variations (CNVs) might partly contribute to the 'missing heritability' of CRC. An increased overall burden of CNV has been identified in several complex diseases, whereas the association between the overall CNV burden and CRC risk is largely unknown. We performed a genome-wide investigation of CNVs on genomic DNA from 384 familial CRC cases and 1285 healthy controls by the Affymetrix 6.0 array. An increase of overall CNV burden was observed in familial CRC patients compared with healthy controls, especially for CNVs larger than 50kb (case/control ratio = 1.66, P = 0.025). In addition, we discovered for the first time a novel structural variation at 12p12.3 and determined the breakpoints by strategic PCR and sequencing. This 12p12.3 structural variation was found in four of 2862 CRC cases but not in 6243 healthy controls (P = 0.0098). RERGL gene (RERG/RAS-like), the only gene influenced by the 12p12.3 structural variation, sharing most of the conserved regions with its close family member RERG tumor suppressor gene (RAS-like, estrogen-regulated, growth inhibitor), might be a novel CRC-related gene. In conclusion, this is the first study to reveal the contribution of the overall burden of CNVs to familial CRC risk and identify a novel rare structural variation at 12p12.3 containing RERGL gene to be associated with CRC.

Novel homozygous mutations in Desert hedgehog gene in patients with 46,XY complete gonadal dysgenesis and prediction of its structural and functional implications by computational methods.

Male to female sex reversal in patients with 46,XY karyotype results from the failure of development of testis which may be due to mutations in the SRY gene. Only 10-15% of cases of 46,XY gonadal dysgenesis are accounted for by different types of mutations in the SRY gene. Hence, majority of such patients may have mutations in other genes involved in the testicular differentiation pathway. Besides SRY, other autosomal and X-linked genes are also involved in sexual development during embryogenesis. We describe here the first report from India wherein, two cases of 46,XY complete gonadal dysgenesis that could be attributable to mutations in the Desert hedgehog (DHH) gene. The mutations found in these two patients were a homozygous deletion (c.271_273delGAG) that resulted in deletion of one amino acid (p.D90del) and a homozygous duplication (c.57-60dupAGCC) that resulted in premature termination resulting in non-functional DHH protein. The structure-function implications of the p.D90del mutation were predicted using computational tools. Structural studies on the p.D90del mutant revealed that the mutation could seriously perturb the interaction of DHH with its binding partners. This is the second report in literature showing homozygous mutation in cases with 46,XY complete gonadal dysgenesis.

Identification of novel suggestive loci for high-grade myopia in Polish families.

PURPOSE: Myopia is the most common human eye disorder with complex genetic and environmental causes. To date, several myopia loci have been identified in families of different geographic origin. However, no causative gene(s) have yet been identified. The aim of this study was the characterization of Polish families with high-grade myopia, including genetic analysis. METHODS: Forty-two multiplex Polish families with non-syndromic high-grade myopia participated in the study. All family members underwent detailed ophthalmic examination and high-grade myopia was defined as ≤-6.0 diopters (D) based on the spherical refractive error. A genome-wide single nucleotide polymorphism (SNP)-based high-density linkage scan was performed using Affymetrix Human SNP Array 6.0 on a selected family (HM-32) with multiple affected individuals. RESULTS: Nonparametric linkage analysis identified three novel loci in family HM-32 at chromosome 7p22.1-7p21.1 ([NPL] 8.26; p=0.006), chromosome 7p12.3-7p11.2 ([NPL] 8.23; p=0.006), and chromosome 12p12.3-12p12.1 ([NPL] 8.02; p=0.006), respectively. The effect of linkage disequilibrium on linkage due to dense SNP map was addressed by systematically pruning SNPs from the linkage panel. CONCLUSIONS: Haplotype analysis with informative crossovers in affected individuals defined a 12.2; 10.9; and 9.5 Mb genomic regions for high-grade myopia spanned between SNP markers rs11977885/rs10950639, rs11770622/rs9719399, and rs4763417/rs10842388 on chromosomes 7p22.1-7p21.1, 7p12.3-7p11.2, and 12p12.3-12p12.1, respectively.

Morphologic Overlap between Infantile Myofibromatosis and Infantile Fibrosarcoma: A Pitfall in Diagnosis.

Infantile myofibromatosis (IM) is a distinctive mesenchymal disorder with different clinical forms, including solitary, multicentric, and generalized with visceral involvement. A wide morphologic spectrum is encountered, with the extremes resembling congenital infantile fibrosarcoma (CIFS) and infantile hemangiopericytoma. We report a series of lesions with mixed features of CIFS and IM and compare them in order to further define their clinicopathologic features and the significance of the so-called composite fibromatosis. Seven lesions with unusual overlapping morphologic "composite" features of both IM and CIFS were selected from a series of 106 myofibroblastic lesions. Three cases classified as composite infantile myofibromatoses (COIM) were highly cellular tumors with a diffuse growth of primitive mesenchymal cells and focal features of IM combined with areas resembling infantile fibrosarcoma (IF). Four cases were classified as IF. Three of these exhibited a biphasic pattern with foci resembling IM, including whorls of primitive and spindle cells and perivascular and intravascular projections of myofibroblastic nodules, and the 4th had a close histologic resemblance to a primitive, immature IM. With reverse transcriptase polymerase chain reaction, the ETV6-NTRK3 transcript was absent in 3 COIM and was detected in 3 CIFS; the other CIFS had typical cytogenetic aberrations. On the basis of currently available information, COIM represents a morphologic variant of IM that can mimic IF. Careful histologic evaluation to detect the typical features of IM is essential to avoid classification as IF. Molecular analysis for the ETV6-NTRK3 gene fusion is an important diagnostic tool in this group of lesions.

The human 2'-5'oligoadenylate synthetase family: unique interferon-inducible enzymes catalyzing 2'-5' instead of 3'-5' phosphodiester bond formation.

The demonstration by Kerr and colleagues that double-stranded (ds) RNA inhibits drastically protein synthesis in cell-free systems prepared from interferon-treated cells, suggested the existence of an interferon-induced enzyme, which is dependent on dsRNA. Consequently, two distinct dsRNA-dependent enzymes were discovered: a serine/threonine protein kinase that nowadays is referred to as PKR and a 2'-5'oligoadenylate synthetase (2'-5'OAS) that polymerizes ATP to 2'-5'-linked oligomers of adenosine with the general formula pppA(2'p5'A)(n), n>or=1. The product is pppG2'p5'G when GTP is used as a substrate. Three distinct forms of 2'-5'OAS exist in human cells, small, medium, and large, which contain one, two, and three OAS units, respectively, and are encoded by distinct genes clustered on the 2'-5'OAS locus on human chromosome 12. OASL is an OAS like IFN-induced protein encoded by a gene located about 8 Mb telomeric from the 2'-5'OAS locus. OASL is composed of one OAS unit fused at its C-terminus with two ubiquitin-like repeats. The human OASL is devoid of the typical 2'-5'OAS catalytic activity. In addition to these structural differences between the various OAS proteins, the three forms of 2'-5'OAS are characterized by different subcellular locations and enzymatic parameters. These findings illustrate the apparent structural and functional complexity of the human 2'-5'OAS family, and suggest that these proteins may have distinct roles in the cell.

Duplication and relocation of the functional DPY19L2 gene within low copy repeats.

BACKGROUND: Low copy repeats (LCRs) are thought to play an important role in recent gene evolution, especially when they facilitate gene duplications. Duplicate genes are fundamental to adaptive evolution, providing substrates for the development of new or shared gene functions. Moreover, silencing of duplicate genes can have an indirect effect on adaptive evolution by causing genomic relocation of functional genes. These changes are theorized to have been a major factor in speciation. RESULTS: Here we present a novel example showing functional gene relocation within a LCR. We characterize the genomic structure and gene content of eight related LCRs on human Chromosomes 7 and 12. Two members of a novel transmembrane gene family, DPY19L, were identified in these regions, along with six transcribed pseudogenes. One of these genes, DPY19L2, is found on Chromosome 12 and is not syntenic with its mouse orthologue. Instead, the human locus syntenic to mouse Dpy19l2 contains a pseudogene, DPY19L2P1. This indicates that the ancestral copy of this gene has been silenced, while the descendant copy has remained active. Thus, the functional copy of this gene has been relocated to a new genomic locus. We then describe the expansion and evolution of the DPY19L gene family from a single gene found in invertebrate animals. Ancient duplications have led to multiple homologues in different lineages, with three in fish, frogs and birds and four in mammals. CONCLUSION: Our results show that the DPY19L family has expanded throughout the vertebrate lineage and has undergone recent primate-specific evolution within LCRs.

Nonmosaic smallest duplication of 12q24.31-qter: the first reported case.

Duplication of the terminal region of the long arm of chromosome 12 is not common. In 13 previous cases, duplication of this region was generally associated with deletions of the derivative chromosomes, larger sized duplications or mosaicism. We have studied a young man with a nonmosaic duplication of 12q24.31-qter translocated to chromosome 5pter. This is the first reported case of pure subtle duplication involving less than two terminal subbands of 12q24.31 to qter. The origin of this genetic material was confirmed by whole chromosome paints and subtelomere specific FISH probes. As both the subtelomere signals for 5p and 12q were present in the der(5) chromosome, it is unlikely that there was any loss of unique DNA sequences from the terminal region of chromosome 5p. This case is compared with 13 other reported cases with a duplication of the 12q terminal segment.

Variable stability of chromosomes containing amplified alpha-satellite sequences in human mesenchymal tumours.

Alpha-satellite sequences are found in the centromeric region of all human chromosomes and have been implicated in centromeric function. We describe the structure and behaviour of chromosomes containing amplified human alphoid DNA from chromosome 12, in an osteosarcoma cell line (OSA) and an atypical lipomatous tumour (ALT). In OSA, the amplified material was detected in one large marker chromosome, whereas in ALT amplified sequences were observed in chromosomes of variable number and appearance. The marker in OSA was mitotically stable, but those in ALT exhibited a high degree of mitotic instability, forming bridges at anaphase and chromatin strings between interphase nuclei. The amplified alpha-satellite arrays reacted positively with human anti-centromeric antiserum and anti-centromere protein B antibodies in both tumours. Centromere protein C, previously shown to be present only in functional kinetochores, was invariably detected at the constriction of the marker in OSA, while one-fifth of markers in ALT appeared to exhibit additional centromere protein C-positive regions outside the primary constriction, indicating that the observed chromosomal instability in ALT might, at least in part, be a consequence of the occasional formation of more than one functional kinetochore. In OSA the alphoid DNA was coamplified with unique sequences from central 12q and the amplified material was C-band negative but in ALT amplified material from central 12q as well as sequences from proximal 12p were detected, resulting in C-band-positive areas. A propensity for additional kinetochore formation might thus be associated with the coamplification of alphoid DNA and pericentromeric sequences from chromosome 12.

Precise localization on chromosome 12 of the ATF-1 gene by fluorescence in situ hybridization.

The ATF-1 gene encodes for a transcription factor normally regulated by cAMP (Hai et al. 1989, Yoshimura et al. 1990). Recently, it has been shown to be involved in the recurrent t(12;22) translocation observed in soft tissue malignant melanoma, in a fusion gene with the EWS gene (Zucman et al. 1993). We report here on its precise localization on chromosome 12 by fluorescence in situ hybridization.

Allelic variations of human keratins K4 and K5 provide polymorphic markers within the type II keratin gene cluster on chromosome 12.

To appreciate point mutations in keratin genes as causes for hereditary epithelial diseases, the normal variation of these gene sequences in the population must be known. Because genetic polymorphism of keratins at the protein level due to allelic variation has been described for the type II keratins 4 and 5, we have analyzed their corresponding genes using single-strand conformation polymorphism gel electrophoresis and sequence analysis of polymerase chain reaction amplified genomic DNA. Although no sequence variations were found in the carboxyl-terminal and rod domains we were able to map the molecular differences among the alleles to their amino-terminal domains. In particular, we have identified three alleles of keratin 4. Two alleles differed by a nucleotide transition causing a neutral amino acid substitution (alanine to valine) and one allele had a 42-bp in-frame deletion corresponding to 14 amino acids within the V1 subdomain. Three alleles were also recognized for the keratin 5 locus, all being elicited by single nucleotide substitutions. Of these, only one altered the amino acid sequence, replacing an uncharged (glycine) with a charged (glutamic acid) amino acid in the H1 subdomain. Pedigree analyses in three families showed the alleles to be inherited as autosomal Mendelian traits. Thus, these normal alleles of keratins 4 and 5 will provide favorable polymorphic markers for linkage analysis directly within the cluster of type II keratin genes located on chromosome 12q to elucidate the potential involvement of these and other keratin genes in disorders of squamous cell differentiation.

Isolation, characterization and chromosomal localization of the human GADD153 gene.

We report the isolation and characterization of the growth arrest and DNA-damage-inducible gene, GADD153, from human cells and show that it is localized in the region 12q13.1-q13.2 on chromosome 12. Comparison of the human gene with the previously described hamster gene revealed a high level of conservation in both the structural and regulatory regions of the genes. Each is composed of four exons with intron/exon junctions maintained at the identical positions. The human Gadd153 protein shares 91% identity with the hamster protein in amino acid sequence, and 78% identity in nucleotide sequence. A 900-bp fragment of 5' flanking sequence from the human gene, when linked to the bacterial cat reporter gene, was found to exhibit promoter activity in HeLa cells which could be further activated by treatment with the DNA alkylating agent, methyl methanesulfonate. Sequence analysis indicated that the human promoter region is relatively G+C-rich and contains putative binding sites for multiple transcription factors, including recognition sites for TATA- and CAAT-binding proteins, six Sp1-binding sites, an activator protein-1 binding site, an E-26-specific sequence-binding protein-1 DNA-binding site, and four interleukin-6 response elements. Many of these sites are also present in an identical position in the hamster gene suggesting they may play an important role in regulating GADD153 expression.

Embryonic simple epithelial keratins 8 and 18: chromosomal location emphasizes difference from other keratin pairs.

The keratins 8 and 18 of simple epithelia differ from stratified epithelial keratins in tissue expression and regulation. To examine the specific properties of human keratin 8, we cloned and sequenced the cDNA from a placental mRNA expression library and defined the optimum state of such clones for expression in bacterial plasmid vectors. Using the polymerase chain reaction we identified and sequenced three introns and located the single active gene for keratin 8, out of a background of 9 to 24 pseudogenes, on chromosome 12. This chromosome contains several genes for type II keratins and also the gene for keratin 18, the type I keratin that is coexpressed with keratin 8. This location of both members of a keratin pair on a single chromosome is thus far unique among the keratin genes; it is consistent with the hypothesis that keratins 8 and 18 may be closer to an ancestral keratin gene than the keratins of more highly differentiated epithelia.