Novelty of Axin 2 and lack of Axin 1 gene mutation in colorectal cancer: a study in Kashmiri population

Colorectal cancer is (CRC) one of the leading causes of mortality and morbidity. Various genetic factors have been reported to be involved in the development of colorectal cancers including Axin gene. Axin, a major scaffold protein, plays an important role in various bio signaling pathways. We aim to study mutational pattern of Axin gene in colorectal cancer patients of Kashmiri population. The paired tumor and adjacent normal tissue specimens of 50 consecutive patients with CRC were used in our study. The DNA preparations were evaluated for the occurrence of Axin 1 and Axin 2 gene mutations by direct DNA sequencing. We analyzed exon 1a, 1b, 1c, 2, 4, 6, and 10 of Axin 1 and exon 7 of Axin 2. In this study, we found a novel mutation of G>T (GCT>TCT) transversion in exon 7 of Axin 2 gene at codon G695T (p.alanine >?serine) at a frequency of 6% (3/50). In the same exon of Axin 2 gene a single nucleotide polymorphism (SNP) was detected in codon L688L (CCT>CTT) at a frequency of 36% (18/50). In exon 1c of Axin 1 a SNP was detected at codon D726D (GAT>GAC) at a frequency of 62.5% (31/50). Both the SNPs were synonymous hence do not lead to change of amino acid. Although Axin 1 and Axin 2 gene mutations have been found to be involved in the development of colorectal cancers, it seems to be a relatively rare event in Kashmiri population. However, an interesting finding of this study is the novelty of Axin 2 gene mutations which may be a predisposing factor in ethnic Kashmiri population to CRC.     

Functional analysis of ISC1 by site-directed mutagenesis

We previously reported that the yeast Saccharomyces cerevisiae ISC1 gene (Yer019w), which has homology to the bacterial sphingomyelinase gene, encodes inositol phosphosphingolipids-phospholipase C, Isc1p [Sawai, H., Okamoto, Y., Luberto, C., Mao, C., Bielawska, A., Domae, M., and Hannun, Y. A. (2000) J. Biol. Chem. 275, 39793-39798]. The present study was conducted to determine specific domains in Isc1p required for catalysis. Several amino acid residues are conserved from bacterial sphingomyelinase to mammalian sphingomyelinase and are also found in ISC1. Individual mutation of the conserved E100, N233, and H334 resulted in complete loss of Isc1p activity, suggesting an essential role in catalysis for these amino acid residues. Isc1p also contains a domain (from G162 to S169) with homology to P-loop domains, found in nucleotide-binding proteins. In addition, two amino acid residues from this domain, D163 and K168, are conserved from bacterial to mammalian sphingomyelinases in this "P-loop-like domain". G162, D163, G167, K168, and S169 were replaced individually with alanine using site-directed mutagenesis. D163A and K168A lost activity completely. Mutations in the other three positions rendered enzyme versions with much reduced but detectable activity. The V(max) values for G162A, G167A, and S169A were reduced, compared with wild type, but the K(m) values for G162A, G167A, and S169A were similar to that of wild type, indicating that the substrate binding efficiency was not greatly altered in these mutants and that the P-loop-like domain of ISC1 might be essential in catalysis of Isc1p. Furthermore, the Mg(2+) K(a) constants for G162A, G167, and S169A were higher than that for wild type, suggesting that this P-loop-like domain may be involved in Mg(2+) binding. Although cell lysates from yeast cells overexpressing all mutants similarly bound to phosphatidylserine (PS), an anionic lipid activator of Isc1p, G162A and G167A required 13.3 mol % PS to achieve maximum activity compared to 6.7 mol % for the wild-type enzyme, suggesting that PS might play a role in optimal catalytic efficiency of Isc1p via this P-loop-like domain. This study provides novel insight into a new domain found in Isc1p and related enzymes.     

A SNP in GmBADH2 gene associates with fragrance in vegetable soybean variety ��Kaori�� and SNAP marker development for the fragrance

Fragrance in soybean is due to the presence of 2-acetyl-1-pyrroline (2AP). BADH2 gene coding for betaine aldehyde dehydrogenase has been identified as the candidate gene responsible for fragrance in rice (Oryza sativa L.). In this study, using the RIL population derived from fragrant soybean cultivar "Kaori" and non-fragrant soybean cultivar "Chiang Mai 60" (CM60), STS markers designed from BADH2 homolog were found associating with 2AP production. Genetic mapping demonstrated that QTL position of fragrance and 2AP production coincides with the position of GmBADH2 (Glycine max betaine aldehyde dehydrogenase 2). Sequence comparison of GmBADH2 between Kaori and non-fragrant soybeans revealed non-synonymous single-nucleotide polymorphism (SNP) in exon 10. Nucleotide substitution of G to A in the exon results in an amino acid change of glycine (GGC; G) to aspartic acid (GAC; D) in Kaori. The amino acid substitution changes the conserved EGCRLGPIVS motif of GmBADH2, which is essential for functional activity of GmBADH2 protein, to EGCRLDPIVS motif, suggesting that the SNP in GmBADH2 is responsible for the fragrance in Kaori. Five single nucleotide-amplified polymorphism (SNAP) markers which are PCR-based allele specific SNP markers were developed for fragrance based on the SNP in GmBADH2. Two markers specific to A allele produced a band in only Kaori, while three markers specific to G alleles produced a band in only CM60. The simple PCR-based allele specific SNAP markers developed in the present study are useful in marker-assisted breeding of fragrant soybean.     

Variation in the yak dectin-1 gene (CLEC7A)

C-type lectin domain family 7, member A (CLEC7A, dectin-1) plays an important role in antifungal immunity and belongs to the family of C-type lectin receptors. Variation in the extended exon 4-6 region of the yak dectin-1 gene (CLEC7A), which encodes the β-glucan recognition domain, was investigated using polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP). Three and two variant sequences were identified in exons 4 and 5, respectively, and no variation was found in exon 6. The variation included two single-nucleotide substitutions in exon 4 and one single-nucleotide polymorphism in exon 5, and two of these substitutions would nominally cause amino acid substitutions (p.I158T and p.S197G) in yak dectin-1. This is the first report identifying the yak dectin-1 gene, revealing that it is polymorphic and this polymorphism might affect the structure and function of this gene in yak.     

Congenital afibrinogenemia: intracellular retention of fibrinogen due to a novel W437G mutation in the fibrinogen Bbeta-chain gene

Congenital afibrinogenemia is a rare autosomal recessive coagulation disorder characterised by hemorrhagic manifestations of variable entity and by severe plasma fibrinogen deficiency. Among the 31 afibrinogenemia-causing mutations so far reported, only 2 are missense mutations and both are located in the fibrinogen Bbeta-chain gene. Direct sequencing of the fibrinogen gene cluster in two afibrinogenemic Iranian siblings revealed a novel homozygous T>G transversion in exon 8 (nucleotide position 8025) of the fibrinogen Bbeta-chain gene. The resulting W437G missense mutation involves a highly conserved amino acid residue, located in the C-terminal globular D domain. The role of the W437G amino acid substitution on fibrinogen synthesis, folding, and secretion was assessed by in vitro expression experiments in COS-1 cells, followed by qualitative and quantitative analyses of intracellular and secreted mutant fibrinogen. Results of both pulse-chase experiments and enzyme-linked immunosorbent assays demonstrated intracellular retention of the mutant W437G fibrinogen and marked reduction of its secretion. These data, besides elucidating the pathogenetic role of the W437G mutation in afibrinogenemia, underline the importance of the Bbeta-chain D domain in fibrinogen folding and secretion.     

Single nucleotide polymorphism of CACNA2D1 gene and its association with milk somatic cell score in cattle

The objective of the present study was to identify polymorphisms of the CACNA2D1 gene, and to analyze associations between these polymorphisms and mastitis in several cattle breeds. Through PCR-RFLP methods and DNA sequencing, an allelic variant corresponding to the A→G mutations and Aspartic (Asp) to Glycine (Gly) amino acid replacement at positions 526745 in the exon 25 of bovine CACNA2D1 gene could be detected. Two alleles, A and G, and three genotypes, AA, AG and GG were defined. Genetic character in the studied populations indicated that the A526745G loci of CACNA2D1 gene was moderate polymorphism and fitted with Hardy-Weinberg equilibrium (P > 0.05). The effects of CACNA2D1 polymorphisms on somatic cell score (SCS) were analyzed and significant association was found between A526745G and SCS. The mean of genotype GG was significantly lower than those of genotype AG and AA (P = 0.0469). Information provided in this research could be useful in further studies to determine the role of CACNA2D1 gene in the mastitis resistance.     

Nucleotide sequence analysis of NIPBL gene in Indian Cornelia de Lange syndrome cases

BACKGROUND:

Cornelia de Lange syndrome (CdLS) is a multisystem developmental disorder in children. The disorder is caused mainly due to mutations in Nipped-B-like protein. The molecular data for CdLS is available from developed countries, but not available in developing countries like India. In the present study, the hotspot region of NIPBL gene was screened by Polymerase Chain Reaction which includes exon 2, 22, 42, and a biggest exon 10, in six CdLS patients and ten controls.

MATERIALS AND METHODS:

The method adopted in present study was amplification of the target exon by using polymerase chain reaction, qualitative confirmation of amplicons by Agarose Gel Electrophoresis and use of amplicons for Conformation Sensitive Gel Electrophoresis to find heteroduplex formation followed by sequencing.

RESULTS:

We report two polymorphisms in the studied region of gene NIPBL. The polymorphisms are in the region of intron 1 and in exon 10. The polymorphism C/A is present in intron 1 region and polymorphism T/G in exon 10.

CONCLUSION:

The intronic region polymorphism may have a role in intron splicing whereas the polymorphism in exon 10 results in amino acid change (Val to Gly). These polymorphisms are disease associated as these are found in CdLS patients only and not in controls.     

A de novo nonsense mutation in exon 28 of the neurofibromatosis type l (NF1) gene

We have screened a total of 105 unrelated patients with neurofibromatosis type l (NF1) for mutations in exon 28 of the NF1 gene using heteroduplex analysis and single strand conformation polymorphism analysis. One novel mutation has been identified and characterised. This mutation involves a 13-bp deletion (AAACTGGCTGAGC or AACTGGCTGAGCA) from base position 5077 (or 5078) to 5089 (or 5090) of the cDNA coding sequence. This alteration leads to a reading frame shift with a premature amber termination signal (TAG) at codon 1694. In addition, there is a change from lysine to threonine at codon 1693. The truncated gene product is estimated to be 1125 amino acid residues shorter than the predicted normal protein (2818 amino acids).     

Production of a monoclonal antibody directed against the recombinant SEL1L protein

SEL1L, highly similar to the C elegans sel-1 gene, is a recently cloned human gene whose function is under investigation. SEL1L is differentially expressed in tumors and normal tissues and seems to play a role in tumor growth and aggressiveness. We used the recombinant N-terminus of the SEL1L protein to immunize a Balb/c mouse and produce a monoclonal antibody. A hybridoma secreting an antibody specifically reacting on the SEL1L recombinant fragment was selected. This monoclonal antibody, named MSel1, recognizes the SEL1L protein by Western blotting, immunofluorescence and immunohistochemistry on normal and tumor cells. MSel1 is able to recognize SEL1L even on archival tumor specimens and is therefore particularly appropriate to study SEL1L involvement in tumor progression.     

Extensive amino acid polymorphism at the pgm locus is consistent with adaptive protein evolution in Drosophila melanogaster

PGM plays a central role in the glycolytic pathway at the branch point leading to glycogen metabolism and is highly polymorphic in allozyme studies of many species. We have characterized the nucleotide diversity across the Pgm gene in Drosophila melanogaster and D. simulans to investigate the role that protein polymorphism plays at this crucial metabolic branch point shared with several other enzymes. Although D. melanogaster and D. simulans share common allozyme mobility alleles, we find these allozymes are the result of many different amino acid changes at the nucleotide level. In addition, specific allozyme classes within species contain several amino acid changes, which may explain the absence of latitudinal clines for PGM allozyme alleles, the lack of association of PGM allozymes with the cosmopolitan In(3L)P inversion, and the failure to detect differences between PGM allozymes in functional studies. We find a significant excess of amino acid polymorphisms within D. melanogaster when compared to the complete absence of fixed replacements with D. simulans. There is also strong linkage disequilibrium across the 2354 bp of the Pgm locus, which may be explained by a specific amino acid haplotype that is high in frequency yet contains an excess of singleton polymorphisms. Like G6pd, Pgm shows strong evidence for a branch point enzyme that exhibits adaptive protein evolution.     

SEL1L, the human homolog of C. elegans sel-1: refined physical mapping, gene structure and identification of polymorphic markers

We have cloned the human full-length cDNA SEL1L, which is highly similar to the C. elegans sel-1 gene, an important negative regulator of the "notch" pathway which acts as a key regulator of the cellular proliferation and specification processes in both vertebrates and invertebrates. The SEL1L gene maps to 14q24.3-31 and here we report its fine localization by HAPPY mapping, which determines its molecular distance to microsatellite markers isolated in the region. We have found two new polymorphic (CA)n microsatellites located in the gene, and have identified the exon-intron boundaries. The gene is composed of 21 exons spanning 70 kb of genomic DNA. Human SEL1L protein exhibits a high degree of similarity compared to the mouse and nematode homologs.     

Tyrosine Residue in Exon 14 of the Cytoplasmic Domain of Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1/CD31) Regulates Ligand Binding Specificity

Platelet/endothelial cell adhesion molecule (PECAM-1) is a cell adhesion molecule of the immunoglobulin superfamily that plays a role in a number of vascular processes including leukocyte transmigration through endothelium. The presence of a specific 19– amino acid exon within the cytoplasmic domain of PECAM-1 regulates the binding specificity of the molecule; specifically, isoforms containing exon 14 mediate heterophilic cell–cell aggregation while those variants missing exon 14 mediate homophilic cell–cell aggregation. To more precisely identify the region of exon 14 responsible for ligand specificity, a series of deletion mutants were created in which smaller regions of exon 14 were removed. After transfection into L cells, they were tested for their ability to mediate aggregation. For heterophilic aggregation to occur, a conserved 5–amino acid region (VYSEI in the murine sequence or VYSEV in the human sequence) in the mid-portion of the exon was required. A final construct, in which this tyrosine was mutated into a phenylalanine, aggregated in a homophilic manner when transfected into L cells. Inhibition of phosphatase activity by exposure of cells expressing wild type or mutant forms of PECAM-1 to sodium orthovanadate resulted in high levels of cytoplasmic tyrosine phosphorylation and led to a switch from heterophilic to homophilic aggregation. Our data thus indicate either loss of this tyrosine from exon 14 or its phosphorylation results in a change in ligand specificity from heterophilic to homophilic binding. Vascular cells could thus determine whether PECAM-1 functions as a heterophilic or homophilic adhesion molecule by processes such as alternative splicing or by regulation of the balance between tyrosine phosphorylation or dephosphorylation. Defining the conditions under which these changes occur will be important in understanding the biology of PECAM-1 in transmigration, angiogenesis, development, and other processes in which this molecule plays a role.     

The importance of arginine mutation for the evolutionary structure and function of phenylalanine hydroxylase gene

Phenylalanine hydroxylase (PAH) gene mutations were investigated in 23 (46 alleles) unrelated phenylketonuria (PKU) patients in Cukurova region. First, all exons of PAH gene were screened by denaturing high performance liquid chromatography (DHPLC), and then, the suspicious samples were analyzed by direct sequencing technique. Consequently, the following results were obtained: IVS10-11g-->a splicing mutation in 27/46 (58.7%), R261Q mutation in 7/46 (15.2%) and E178G, R243X, R243Q, P281L, Y386C, R408W mutations, each found in the frequency of 2/46 (4.3%). In many countries, Arginine mutations have the highest frequency among PAH gene mutations in PKU patients. Although, CpG dinucleotids are effective in mutations resulting in arginine changes, this finding originated from the studies on the causes of mutations rather than the studies on the importance of arginine amino acid. In our analyses, we have detected that a majority of mutations causing a change in arginine and other amino acids concentrated in exon 7 comprising the catalytic domain (residues 143-410) of PAH gene. Several studies has emphasized the role of arginine amino acid; with the following outcomes; arginine repetition is significant for RNA binding proteins, and for histon proteins in eukaryotic gene expression, and also arginine repetition occurring in the structure of signal recognition particle's (SRPs) as a consequence of post-translational processes is very important in terms of gene expression. Therefore, the role of arginine amino acid in PAH gene is rather remarkable in that it shows the role of amino acids in the protein/RNA interaction that has started in the evolutionary process and is still preserved and maintained in the motif formation of active domain structure due to its strong binding properties. Thus, such properties imply that both arginine amino acid and exon 7 is of great significance with regards to the structure and function of the PheOH enzyme.     

An extensive search for RFLP in the human glucose-6-phosphate dehydrogenase locus has revealed a silent mutation in the coding sequence.

The genetic polymorphism of an approximately 100-kb DNA region comprising and flanking the glucose-6-phosphate dehydrogenase (G6PD) gene on human chromosome Xq28 has been analyzed in detail. By using 14 unique sequence probes and 18 restriction enzymes, we have characterized 257 restriction fragments or 370 restriction sites. On testing 12-57 individual X chromosomes, all sites but one were nonpolymorphic. However, a PstI site that maps to exon 10 of the G6PD gene, which is still monomorphic in all British and Italian subjects tested, is polymorphic in west-African people. Specifically, it is absent from 22% of Nigerian X chromosomes. By sequence analysis we have shown that the absence of this PstI site results from a G----A replacement at position 1116, corresponding to the third base of a glutamine codon; no amino acid change is produced in the protein. Thus, a polymorphic silent mutation is demonstrated in a human gene.     

Molecular analysis in Brazilian cystic fibrosis patients reveals five novel mutations

We have performed molecular genetic analyses on 160 Brazilian patients diagnosed with cystic fibrosis (CF). Screening of mutations in 320 CF chromosomes was performed through single strand conformation polymorphism (SSCP) and heteroduplex analyses assay followed by DNA sequencing of the 27 exons and exon/intron boundaries of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The frequency of CFTR variants of T-tract length of intron 8 (IVS8 Tn) was also investigated. This analysis enabled the detection of 232/320 CF mutations (72.2%) and complete genotyping of 61% of the patients. The deltaF508 mutation was found in 48.4% of the alleles. Another fifteen mutations (previously reported) were detected: G542X, R1162X, N1303K, R334W, W1282X, G58E, L206W, R553X, 621+1G-->T, V232D, 1717-1G-->A, 2347 delG, R851L, 2789+5G-->A, and W1089X. Five novel mutations were identified, V201M (exon 6a), Y275X (exon 6b), 2686 insT (exon 14a), 3171 delC (exon 17a), and 3617 delGA (exon 19). These results contribute to the molecular characterization of CF in the Brazilian population. In addition, the identification of the novel mutation Y275X allowed prenatal diagnosis in a high-risk fetus.     

The impact of factor XIIIa V34L polymorphism on plasma factor XIII activity in the Chinese and Asian Indians from Singapore

Factor XIII (FXIII) is a plasma transglutaminase that is essential for normal haemostasis and fibrinolysis. A few polymorphic sites have been identified in the gene, one of them being a point mutation (V34L) in exon 2 of the FXIIIa subunit gene leading to an amino acid change of valine to leucine. We have examined the role of this polymorphism in relation to plasma FXIII activity in a total of 532 healthy individuals belonging to two ethnic groups in Singapore. The frequency of the L34 allele was significantly higher (P<0.001) among the Asian Indians (0.08) when compared with the Chinese (0.005). No significant difference in frequency of the L34 allele was observed between Asian Indian CAD patients and controls. The mean FXIII levels were significantly higher (P<0.0005) among the Asian Indians (148.4%±35.5) when compared with Chinese (111.2%±26.7). The L34 variant was associated with increased FXIII activity among Asian Indian females. This study shows that both racial and genetic components play a significant role in determining plasma FXIII activity. The effect of V34L polymorphism on FXIII activity in the Indian females is independent of the effects of the P564L and E651Q polymorphic sites in the FXIIIa gene.C.-K. Heng and S. Lal contributed equally to this work     

The spectrum of mutations in the low-density lipoprotein receptor gene in the Russian population

The spectrum of mutations in the low-density lipoprotein (LDL) receptor gene was studied in a sample of hypercholesterolemia patients of Caucasoid origin from the population of Russia. The examined patients were 45 to 49 years old and had the highest level of total serum cholesterol in this age group. Seven previously nondescribed mutations have been revealed in exon 9 (R410G; M412V) and in exon 12 (Y/Y576; N/N591; L605V; L605R; A612G). Twelve previously described mutations have been identified in exons 2 (C/C27), 5 (C261F; E240X), 6 (E288K), 8 (A391T), 9 (E418G; L432R; D433E), 11 (G/G549; E558K; L/L568), and 12 (G592E). Only one of these mutations was previously described in Russia in a clinical sample of patients with familial hypercholesterolemia. The spectrum of LDL receptor gene mutations in the population sample of patients with hypercholesterolemia significantly differs from the mutation spectrum in patients with familial hypercholesterolemia (clinical samples). Sequencing of the LDL receptor gene is a highly efficient method for identifying the markers of hypercholesterolemia predisposition in a population.     

静原鸡ELOVL2基因多态性及其生物信息学分析

极长链脂肪酸延长酶(elongation of very-long-chain fatty acids, ELOVLs)是脂肪酸合成过程中的关键限速酶,在脂类的生物合成及脂肪酸代谢等调控方面作用显著。为研究宁夏地方优良品种静原鸡ELOVL2基因多态性及其编码蛋白质的结构与功能,以原鸡ELOVL2基因序列作为参考序列,针对ELOVL2基因的8个外显子序列设计特异引物并扩增,经DNA混合池测序筛选出存在突变位点的外显子序列,运用PCR-SSCP技术检测其多态性,经序列拼接后对ELOVL2 CDS区序列进行功能生物信息学分析。研究结果表明,静原鸡ELOVL2基因仅exon7和exon8发生单碱基突变(exon7为c.708 G>A, exon8为c.888 T>C),均未引起氨基酸的改变,属同义突变。在exon7和exon8扩增片段中分别检测出3种(AA, AG和GG)、2种(CC和TC)基因型。遗传特性分析显示,E2e7呈中度多态(0.250.05)。生物信息学分析表明,ELOVL2 CDS序列全长为894 bp,共编码297个氨基酸。其原子组成为C1638H2444N394O406S15,分子量为34.63 kD,等电点(pI)为9.40;存在7个跨膜结构,21个磷酸化位点,无信号肽序列,为稳定的水溶性蛋白;ELOVL2基因在进化中高度保守。该研究结果将为进一步研究ELOVL2基因在静原鸡肉质方面的作用及功能提供参考。