Nucleotide Variants In Genes Research Articles

Analysis of sperm nuclear protein gene polymorphisms and DNA integrity in infertile men

Sperm nuclear proteins, the protamines (PRM) and transition nuclear proteins (TNP) play a crucial role in sperm nuclear condensation. The compact packaging of sperm DNA by protamines maintains sperm genome integrity, which is prerequisite for normal sperm function. However the effect of nucleotide variations in PRM and TNP genes on sperm DNA integrity and male fertility is not clear. This case-control study was planned to analyze PRM and TNP gene nucleotide variations and sperm DNA integrity in 100 oligozoospermic infertile men and 100 fertile controls. Protamine and TNP genes were amplified by polymerase chain reaction and sequenced. Flow cytometry-sperm chromatin structure assay (FC-SCSA) was applied to measure the DNA fragmentation index (DFI) in sperm. Semen analysis was performed as per WHO [1999] guidelines with slight modification. In total, 7 nucleotide variations including two novel changes, a non-synonymous mutation in the exon-2 of PRM2 gene (c.443C > A) and a novel insertion of T (c.396_397InsT) at the 3′ UTR region of TNP1 were detected. None of the nucleotide changes were observed with increased risk frequency in the oligozoospermic infertile men compared to the controls. Though overall DFI was significantly (p < 0.0001) higher in infertile men compared to controls (36.31 ± 7.25 vs. 26.49 ± 2.78) irrespective of nucleotide changes, no such difference was observed between 100 infertile men or pooled population of 200 with and without mutations. However it was observed that two cases with novel nucleotide changes PRM2 c.443C > A and TNP1 c.396_397InsT had higher DFI value of 34.82% and 43.85%, respectively. In conclusion, our pilot study for the first time in the Indian population revealed two rare novel mutations in sperm nuclear protein genes that are perhaps associated with higher sperm DNA fragmentation.

Analysis of Nucleotide Variations in Genes of Iron Management in Patients of Parkinson's Disease and Other Movement Disorders

The capacity to act as an electron donor and acceptor makes iron an essential cofactor of many vital processes. Its balance in the body has to be tightly regulated since its excess can be harmful by favouring oxidative damage, while its deficiency can impair fundamental activities like erythropoiesis. In the brain, an accumulation of iron or an increase in its availability has been associated with the development and/or progression of different degenerative processes, including Parkinson's disease, while iron paucity seems to be associated with cognitive deficits, motor dysfunction, and restless legs syndrome. In the search of DNA sequence variations affecting the individual predisposition to develop movement disorders, we scanned by DHPLC the exons and intronic boundary regions of ceruloplasmin, iron regulatory protein 2, hemopexin, hepcidin and hemojuvelin genes in cohorts of subjects affected by Parkinson's disease and idiopathic neurodegeneration with brain iron accumulation (NBIA). Both novel and known sequence variations were identified in most of the genes, but none of them seemed to be significantly associated to the movement diseases of interest.

Nucleotide variations in genes encoding carbonic anhydrase 8 and 10 associated with femoral bone mineral density in Japanese female with osteoporosis

Osteoporosis is a multi-factorial common disease, which is caused by combination of genetic as well as environmental factors. Among several factors, osteoclast acidification pathways during bone resorption might play an important role. Carbonic anhydrases, consisting of a gene family, are essential for pH regulation by the osteoclast. Clinically, use of carbonic anhydrase inhibitors has been known to be associated with a bone-sparing effect as judged by spine bone mineral density (BMD). Here, we investigated single nucleotide polymorphisms (SNPs) in carbonic anhydrase genes that are expressed in bone tissues, i.e., CA8 and CA10, for possible association with femoral and lumbar BMD among 337 Japanese women with osteoporosis participated in BioBank Japan project. Significant correlation was observed between CA8 SNP, rs6984526, and femoral BMD (P = 0.00029); homozygous carriers of the major (C) allele (n = 166) had the highest BMD (0.754 +/- 0.006 g/cm(2), mean +/- SD), while heterozygous carriers (n = 135) were intermediate (0.741 +/- 0.07 g/cm(2)) and homozygous T-allele carriers (n = 31) had the lowest BMD (0.691 +/- 0.012 g/cm(2)). CA8 SNP as well displayed significant association with lumbar BMD in recessive model (P = 0.00017). In addition, CA10 SNP, rs2106329, also displayed strong association with femoral BMD (P = 0.00002). The results suggest that the variations of CA8 and CA10 loci may be important determinants of osteoporosis in Japanese women.

Increased Single‐Nucleotide Discrimination in Allele‐Specific Polymerase Chain Reactions through Primer Probes Bearing Nucleobase and 2′‐Deoxyribose Modifications

The diagnosis of genetic dissimilarities between individuals is becoming increasingly important due to the discovery that these variations are related to complex phenotypes like the predisposition to certain diseases or compatibility with drugs. The most common among these sequence variations are single-nucleotide polymorphisms (SNPs). The availability of reliable and efficient methods for the interrogation of the respective genotypes is the basis for any progress along these lines. Many methods for the detection of nucleotide variations in genes exist, in which amplification of the target gene is required before analysis can take place. The allele-specific polymerase chain reaction (asPCR) combines target amplification and analysis in a single step. The principle of asPCR is based on the formation of matched or mismatched primer-target complexes. The most important parameter in asPCR is the discrimination of these matched or mismatched pairs. In recent publications we have shown that the reliability of SNP detection through asPCR is increased by employing chemically modified primer probes. In particular, primer probes that bear a polar 4'-C-methoxymethylene residue at the 3' end have superior properties in discriminating single-nucleotide variations by PCR. Here we describe the synthesis of several primer probes that bear nucleobase modifications in addition to the 4'-C-methoxymethylenated 2'-deoxyriboses. We studied the effects of these alterations on single-nucleotide discrimination in allele-specific PCR promoted by a DNA polymerase and completed these results with single-nucleotide-incorporation kinetic studies. Moreover, we investigated thermal denaturing of the primer-probe-template complexes and recorded circular dichroism (CD) spectra for inspecting the thermodynamic and photophysical duplex behaviour of the introduced modifications. In short, we found that primer probes bearing a 4'-C-methoxymethylene residue at the 2'-deoxyribose moiety in combination with a thiolated thymidine moiety have synergistic effects and display significantly increased discrimination properties in asPCR.

Nucleotide variations in genes encoding plasminogen activator inhibitor-2 and serine proteinase inhibitor B10 associated with prostate cancer

Genes encoding the serine proteinase inhibitor B family (SERPINBs) are mainly clustered on human chromosome 18 (18q21). Several serpins are known to affect malignant phenotypes of tumor cells, so aberrant genetic variants in this molecular family are candidates for conferring susceptibility for risk of cancer. We investigated whether eight selected non-synonymous variations within SERPINB loci at 18q21 might be associated with risk of prostate cancer in Japanese men. A case-control study involving 292 prostate-cancer patients and 384 controls revealed significant differences in regard to distribution of four missense variations in genes encoding plasminogen activator inhibitor 2 (PAI2) and SERPINB10. The most significant association was detected for the N120D polymorphism in the PAI2 gene (P = 5.0 x 10(-5)); men carrying the 120-N allele (120-N/N and 120-N/D genotypes) carried a 2.4-fold increased risk of prostate cancer (95% confidence interval 1.45-4.07). Associations were also detected for three other missense polymorphisms in those two genes. Strong linkage disequilibrium in the region encompassing PAI2 and SERPINB10 extended to about 50 kbp. The results suggested that missense variations in one or both of these genes confer important risks for prostate cancer, and may be themselves tumorigenic. Although confirmative replication studies on larger cohorts are awaited, clinical examination of these variations may become useful for identifying individuals at high risk for prostate cancer.

Nucleotide variation in genes involved in wood formation in two pine species

Nucleotide diversity in eight genes related to wood formation was investigated in two pine species, Pinus pinaster and P. radiata. The nucleotide diversity patterns observed and their properties were compared between the two species according to the specific characteristics of the samples analysed. A lower diversity was observed in P. radiata compared with P. pinaster. In particular, for two genes (Pp1, a glycin-rich protein homolog and CesA3, a cellulose synthase) the magnitude of the reduction of diversity potentially indicates the action of nonneutral factors. For both, particular patterns of nucleotide diversity were observed in P. pinaster (high genetic differentiation for Pp1 and close to zero differentiation associated with positive Tajima's D-value for CesA3). In addition, KORRIGAN, a gene involved in cellulose-hemicellulose assembly, demonstrated a negative Tajima's D-value in P. radiata accompanied by a high genetic differentiation in P. pinaster. The consistency of the results obtained at the nucleotide level, together with the physiological roles of the genes analysed, indicate their potential susceptibility to artificial and/or natural selection.

Tuning Single Nucleotide Discrimination in Polymerase Chain Reactions (PCRs): Synthesis of Primer Probes Bearing Polar 4′‐C‐Modifications and Their Application in Allele‐Specific PCR

There are many methods available for the detection of nucleotide variations in genetic material. Most of these methods are applied after amplification of the target genome sequence by the polymerase chain reaction (PCR). Many efforts are currently underway to develop techniques that can detect single nucleotide variations in genes either by means of, or without the need for, PCR. Allele-specific PCR (asPCR), which reports nucleotide variations based on either the presence or absence of a PCR-amplified DNA product, has the potential to combine target amplification and analysis in one single step. The principle of asPCR is based on the formation of matched or mismatched primer-target complexes by using allele-specific primer probes. PCR amplification by a DNA polymerase from matched 3'-primer termini proceeds, whereas a mismatch should obviate amplification. Given the recent advancements in real-time PCR, this technique should, in principle, allow single nucleotide variations to be detected online. However, this method is hampered by low selectivity, which necessitates tedious and costly manipulations. Recently, we reported that the selectivity of asPCR can be significantly increased through the employment of chemically modified primer probes. Here we report further significant advances in this area. We describe the synthesis of various primer probes that bear polar 4'-C-modified nucleotide residues at their 3' termini, and their evaluation in real-time asPCR. We found that primer probes bearing a 4'-C-methoxymethylene modification have superior properties in the discrimination of single nucleotide variations by PCR.

Nucleotide sequence variation does not relate to differences in kinetic properties of neutral trehalase from the insect pathogenic fungus Metarhizium anisopliae.

Genetic variability in a putative virulence factor, the neutral trehalase ( Ntl) gene, was examined in strains of the insect pathogenic fungi Metarhizium anisopliae and Metarhizium flavoviride by restriction fragment length polymorphism (RFLP). The Ntl gene was sequenced from four of these strains that showed dissimilar RFLP patterns. Enzyme kinetic experiments were also performed on the partially purified neutral trehalase in order to assess whether nucleotide changes in these strains related to differences in enzyme catalytic function (i.e., Km, Vmax, and Kcat). Finally, the Metarhizium strains were assessed in bioassays against waxworm larvae in order to relate nucleotide variation with Ntl enzyme kinetics and insect virulence. The greatest RFLP variation was observed with Rsa1. M. flavoviride was found to be most dissimilar in RFLP patterns when compared with the M. anisopliae strains. RFLP patterns for Ntl were diagnostic markers for previously studied genetic groups of M. anisopliae. Comparisons of Ntl sequences showed that the introns were found to be more variable (6.2%) than the exons (3.1%). Comparisons of the translated nucleotide codons showed high levels (91%) of synonymous sequence variation between strains. Another fraction of the remaining mutations was neutral, resulting in amino acid substitutions with similar functions. The neutral trehalase was partially purified by preparative isoelectric focus, revealing a single band of enzyme activity as assessed by analytical isoelectric focusing (pI ca. 5). Kinetic properties of the neutral trehalases revealed no differences between the M. anisopliae strains, while the M. flavovoride had a lower Kcat/Km. However, there was lower virulence in one strain that showed Ntl enzyme kinetic properties that were similar to the other strains, suggesting that factors other than neutral trehalase may be responsible for delimiting virulence in this insect pathogenic fungi. Although there is nucleotide variation in genes involved in pathogenicity, this variation is mostly neutral in nature, and there is strong stabilizing selection to maintain enzyme function.