Ion-induced folding of a kink turn that departs from the conventional sequence

Kink turns (k-turns) are important structural motifs that create a sharp axial bend in RNA. Most conform to a consensus in which a three-nucleotide bulge is followed by consecutive G•A and A•G base pairs, and when these G•A pairs are modified in vitro this generally leads to a failure to adopt the k-turn conformation. Kt-23 in the 30S ribosomal subunit of Thermus thermophilus is a rare exception in which the bulge-distal A•G pair is replaced by a non-Watson–Crick A•U pair. In the context of the ribosome, Kt-23 adopts a completely conventional k-turn geometry. We show here that this sequence is induced to fold into a k-turn structure in an isolated RNA duplex by Mg²⁺ or Na⁺ ions. Therefore, the Kt-23 is intrinsically stable despite lacking the key A•G pair; its formation requires neither tertiary interactions nor protein binding. Moreover, the Kt-23 k-turn is stabilized by the same critical hydrogen-bonding interactions within the core of the structure that are found in more conventional sequences such as the near-consensus Kt-7. T. thermophilus Kt-23 has two further non-Watson–Crick base pairs within the non-canonical helix, three and four nucleotides from the bulge, and we find that the nature of these pairs influences the ability of the RNA to adopt k-turn conformation, although the base pair adjacent to the A•U pair is more important than the other.     

Structure of a rare non-standard sequence k-turn bound by L7Ae protein

Kt-23 from Thelohania solenopsae is a rare RNA kink turn (k-turn) where an adenine replaces the normal guanine at the 2n position. L7Ae is a member of a strongly conserved family of proteins that bind a range of k-turn structures in the ribosome, box C/D and H/ACA small nucleolar RNAs and U4 small nuclear RNA. We have solved the crystal structure of T. solenopsae Kt-23 RNA bound to Archeoglobus fulgidus L7Ae protein at a resolution of 2.95 Å. The protein binds in the major groove displayed on the outer face of the k-turn, in a manner similar to complexes with standard k-turn structures. The k-turn adopts a standard N3 class conformation, with a single hydrogen bond from A2b N6 to A2n N3. This contrasts with the structure of the same sequence located in the SAM-I riboswitch, where it adopts an N1 structure, showing the inherent plasticity of k-turn structure. This potentially can affect any tertiary interactions in which the RNA participates.     

The plasticity of a structural motif in RNA: Structural polymorphism of a kink turn as a function of its environment

The k-turn is a widespread structural motif that introduces a tight kink into the helical axis of double-stranded RNA. The adenine bases of consecutive G•A pairs are directed toward the minor groove of the opposing helix, hydrogen bonding in a typical A-minor interaction. We show here that the available structures of k-turns divide into two classes, depending on whether N3 or N1 of the adenine at the 2b position accepts a hydrogen bond from the O2′ at the −1n position. There is a coordinated structural change involving a number of hydrogen bonds between the two classes. We show here that Kt-7 can adopt either the N3 or N1 structures depending on environment. While it has the N1 structure in the ribosome, on engineering it into the SAM-I riboswitch, it changes to the N3 structure, resulting in a significant alteration in the trajectory of the helical arms.     

Secondary structure models of D2-D3 expansion segments of 28S rRNA for Hoplolaiminae species

The D2-D3 expansion segments of the 28S ribosomal RNA (rRNA) were sequenced and compared to predict secondary structures for Hoplolaiminae species based on free energy minimization and comparative sequence analysis. The free energy based prediction method provides putative stem regions within primary structure and these base pairings in stems were confirmed manually by compensatory base changes among closely and distantly related species. Sequence differences ranged from identical between Hoplolaimus columbus and H. seinhorsti to 20.8% between Scutellonema brachyurum and H. concaudajuvencus. The comparative sequence analysis and energy minimization method yielded 9 stems in the D2 and 6 stems in the D3 which showed complete or partial compensatory base changes. At least 75% of nucleotides in the D2 and 68% of nucleotides in the D3 were related with formation of base pairings to maintain secondary structure. GC contents in stems ranged from 61 to 73% for the D2 and from 64 to 71% for the D3 region. These ranges are higher than G-C contents in loops which ranged from 37 to 48% in the D2 and 33-45% in the D3. In stems, G-C/C-G base pairings were the most common in the D2 and the D3 and also non-canonical base pairs including A•A and U•U, C•U/U•C, and G•A/A•G occurred in stems. The predicted secondary model and new sequence alignment based on predicted secondary structures for the D2 and D3 expansion segments provide useful information to assign positional nucleotide homology and reconstruction of more reliable phylogenetic trees.     

Ficolin-2 levels and FCN2 haplotypes influence hepatitis B infection outcome in Vietnamese patients

Human Ficolin-2 (L-ficolins) encoded by FCN2 gene is a soluble serum protein that plays an important role in innate immunity and is mainly expressed in the liver. Ficolin-2 serum levels and FCN2 single nucleotide polymorphisms were associated to several infectious diseases. We initially screened the complete FCN2 gene in 48 healthy individuals of Vietnamese ethnicity. We genotyped a Vietnamese cohort comprising of 423 clinically classified hepatitis B virus patients and 303 controls for functional single nucleotide polymorphisms in the promoter region (-986G>A, -602G>A, -4A>G) and in exon 8 (+6424G>T) by real-time PCR and investigated the contribution of FCN2 genotypes and haplotypes to serum Ficolin-2 levels, viral load and liver enzyme levels. Haplotypes differed significantly between patients and controls (P = 0.002) and the haplotype AGGG was found frequently in controls in comparison to patients with hepatitis B virus and hepatocellular carcinoma (P = 0.0002 and P<0.0001) conferring a protective effect. Ficolin-2 levels differed significantly between patients and controls (p<0.0001). Patients with acute hepatitis B had higher serum Ficolin-2 levels compared to other patient groups and controls.The viral load was observed to be significantly distributed among the haplotypes (P = 0.04) and the AAAG haplotype contributed to higher Ficolin-2 levels and to viral load. Four novel single nucleotide polymorphisms in introns (-941G>T, -310G>A, +2363G>A, +4882G>A) and one synonymous mutation in exon 8 (+6485G>T) was observed. Strong linkage was found between the variant -986G>A and -4A>G. The very first study on Vietnamese cohort associates both Ficolin-2 serum levels and FCN2 haplotypes to hepatitis B virus infection and subsequent disease progression.     

Association between the C.1161G>A and C.1779C>G Genetic Variants of XRCC1 Gene and Hepatocellular Carcinoma Risk in Chinese Population

The human X-ray repair complementing group 1 gene (XRCC1) is an important candidate gene influencing hepatocellular carcinoma (HCC) susceptibility. The objective of this study was to detect the association between c.1161G>A and c.1779C>G variants of XRCC1 gene and HCC risk. This study was conducted in Chinese population consisting of 623 HCC cases and 639 controls. These two genetic variants could be genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The association of XRCC1 gene variants with the risk of HCC was investigated under different genetic models. Our findings suggested that the genotypes/alleles from c.1161G>A and c.1779C>G genetic variants were statistically associated with HCC risk. As for the c.1161G>A, the AA genotype was statistically associated with the increased risk of HCC compared to GG wild genotype (OR = 2.36, 95% CI 1.63-3.40, P < 0.001). As for the c.1779C>G, the risk of HCC was significantly higher for GG genotype compared to CC wild genotype (OR = 2.17, 95% CI 1.51-3.12, P < 0.001). Furthermore, significant differences in the risk of HCC were also detected in other genetic models for these two variants. The allele-A of c.1161G>A and allele-G of c.1779C>G variants may contribute to the susceptibility of HCC (A versus G: OR = 1.48, 95% CI 1.26-1.75, P < 0.001 and G versus C: OR = 1.51, 95% CI 1.28-1.78, P < 0.001). Our data indicated that these two variants of XRCC1 gene were statistically associated with HCC risk in Chinese population.     

The three faces of riboviral spontaneous mutation: spectrum, mode of genome replication, and mutation rate

Riboviruses (RNA viruses without DNA replication intermediates) are the most abundant pathogens infecting animals and plants. Only a few riboviral infections can be controlled with antiviral drugs, mainly because of the rapid appearance of resistance mutations. Little reliable information is available concerning i) kinds and relative frequencies of mutations (the mutational spectrum), ii) mode of genome replication and mutation accumulation, and iii) rates of spontaneous mutation. To illuminate these issues, we developed a model in vivo system based on phage Qß infecting its natural host, Escherichia coli. The Qß RT gene encoding the Read-Through protein was used as a mutation reporter. To reduce uncertainties in mutation frequencies due to selection, the experimental Qß populations were established after a single cycle of infection and selection against RT ⁻ mutants during phage growth was ameliorated by plasmid-based RT complementation in trans. The dynamics of Qß genome replication were confirmed to reflect the linear process of iterative copying (the stamping-machine mode). A total of 32 RT mutants were detected among 7,517 Qß isolates. Sequencing analysis of 45 RT mutations revealed a spectrum dominated by 39 transitions, plus 4 transversions and 2 indels. A clear template•primer mismatch bias was observed: A•C>C•A>U•G>G•U> transversion mismatches. The average mutation rate per base replication was ≈9.1×10⁻⁶ for base substitutions and ≈2.3×10⁻⁷ for indels. The estimated mutation rate per genome replication, μ_g, was ≈0.04 (or, per phage generation, ≈0.08), although secondary RT mutations arose during the growth of some RT mutants at a rate about 7-fold higher, signaling the possible impact of transitory bouts of hypermutation. These results are contrasted with those previously reported for other riboviruses to depict the current state of the art in riboviral mutagenesis.     

UGT1A6 Polymorphisms Modulated Lung Cancer Risk in a Chinese Population

Uridine diphosphoglucuronosyltransferases (UGTs) 1A6 is the only UGT1A isoform expressed in lung tissue. It is responsible for the detoxification of carcinogens such as benezo[a]pyrene from cigarette smoke. The purpose of this study was to evaluate the association of UGT1A6 polymorphisms and haplotypes with lung cancer risk and to evaluate the functional significance of UGT1A6 polymorphisms. Genomic DNA was isolated from leukocytes. Eight UGT1A6 polymorphisms were sequenced in a test set of 72 Chinese lung cancer patients and 62 healthy controls. Potential risk modifying alleles were validated in a separate set of 95 Chinese lung cancer patients and 100 healthy controls. UGT1A6 19T>G, 541A>G and 552A>C showed significant association with increased lung cancer risk, while UGT1A6 105C>T and IVS1+130G>T were significantly associated with reduced lung cancer risk. Multivariate logistic regression analysis demonstrated a significant association of lung cancer with UGT1A6 541A>G (OR: 3.582, 95% CI: 1.27–10.04, p = 0.015), 552A>C (OR: 5.364, 95% CI: 1.92–14.96, p = 0.001) and IVS1+130G>T (OR: 0.191, 95% CI: 0.09–0.36, p<0.001). Functional test demonstrated that UGT1A6 105C>T increased mRNA stability, providing a plausible explanation of its association with reduced lung cancer risk. Thus UGT1A6 polymorphisms may be used to identify people with increased risk of developing lung cancer.     

The effects of ABCG5/G8 polymorphisms on plasma HDL cholesterol concentrations depend on smoking habit in the Boston Puerto Rican Health Study

Low HDL-cholesterol (HDL-C) is associated with an increased risk for atherosclerosis, and concentrations are modulated by genetic factors and environmental factors such as smoking. Our objective was to assess whether the association of common single-nucleotide polymorphisms (SNPs) at ABCG5/G8 (i18429G>A, i7892T>C, Gln604GluC>G, 5U145A>C, Tyr54CysA>G, Asp19HisG>C, i14222A>G, and Thr400LysC>A) genes with HDL-C differs according to smoking habit. ABCG5/G8 SNPs were genotyped in 845 participants (243 men and 602 women). ABCG5/G8 (i7892T>C, 5U145A>C, Tyr54CysA>G, Thr400LysC>A) SNPs were significantly associated with HDL-C concentrations (P < 0.001–0.013) by which carriers of the minor alleles at the aforementioned polymorphisms and homozygotes for the Thr400 allele displayed lower HDL-C. A significant gene-smoking interaction was found, in which carriers of the minor alleles at ABCG5/G8 (Gln604GluC>G, Asp19HisG>C, i14222A>G) SNPs displayed lower concentrations of HDL-C only if they were smokers (P = 0.001–0.025). Also, for ABCG8_Thr400LysC>A SNP, smokers, but not nonsmokers, homozygous for the Thr400 allele displayed lower HDL-C (P = 0.004). Further analyses supported a significant haplotype global effect on lowering HDL-C (P = 0.002) among smokers. In conclusion, ABCG5/G8 genetic variants modulate HDL-C concentrations, leading to an HDL-C-lowering effect and thereby a potential increased risk for atherosclerosis only in smokers.     

The k-junction motif in RNA structure

The k-junction is a structural motif in RNA comprising a three-way helical junction based upon kink turn (k-turn) architecture. A computer program written to examine relative helical orientation identified the three-way junction of the Arabidopsis TPP riboswitch as an elaborated k-turn. The Escherichia coli TPP riboswitch contains a related k-junction, and analysis of >11 000 sequences shows that the structure is common to these riboswitches. The k-junction exhibits all the key features of an N1-class k-turn, including the standard cross-strand hydrogen bonds. The third helix of the junction is coaxially aligned with the C (canonical) helix, while the k-turn loop forms the turn into the NC (non-canonical) helix. Analysis of ligand binding by ITC and global folding by gel electrophoresis demonstrates the importance of the k-turn nucleotides. Clearly the basic elements of k-turn structure are structurally well suited to generate a three-way helical junction, retaining all the key features and interactions of the k-turn.     

Thermodynamic examination of the pyrophosphate sensor helix in the thiamine pyrophosphate riboswitch

Riboswitches are functional mRNA that control gene expression. Thiamine pyrophosphate (TPP) binds to thi-box riboswitch RNA and allosterically inhibits genes that code for proteins involved in the biosynthesis and transport of thiamine. Thiamine binding to the pyrimidine sensor helix and pyrophosphate binding to the pyrophosphate sensor helix cause changes in RNA conformation that regulate gene expression. Here we examine the thermodynamic properties of the internal loop of the pyrophosphate binding domain by comparing the wild-type construct (RNA WT) with six modified 2 × 2 bulged RNA and one 2 × 2 bulged DNA. The wild-type construct retains five conserved bases of the pyrophosphate sensor domain, two of which are in the 2 × 2 bulge (C65 and G66). The RNA WT construct was among the most stable (ΔG°₃₇ = −7.7 kcal/mol) in 1 M KCl at pH 7.5. Breaking the A•G mismatch of the bulge decreases the stability of the construct ∼0.5–1 kcal/mol, but does not affect magnesium binding to the RNA WT. Guanine at position 48 is important for RNA–Mg²⁺ interactions of the TPP-binding riboswitch at pH 7.5. In the presence of 9.5 mM magnesium at pH 5.5, the bulged RNA constructs gained an average of 1.1 kcal/mol relative to 1 M salt. Formation of a single A+•C mismatch base pair contributes about 0.5 kcal/mol at pH 5.5, whereas two tandem A+•C mismatch base pairs together contribute about 2 kcal/mol.     

Rare Primary Mitochondrial DNA Mutations and Probable Synergistic Variants in Leber's Hereditary Optic Neuropathy

Background

Leber’s hereditary optic neuropathy (LHON) is a maternally inherited blinding disorder, which in over 90% of cases is due to one of three primary mitochondrial DNA (mtDNA) point mutations (m.11778G>A, m.3460G>A and m.14484T>C, respectively in MT-ND4, MT-ND1 and MT-ND6 genes). However, the spectrum of mtDNA mutations causing the remaining 10% of cases is only partially and often poorly defined.

Methodology/Principal Findings

In order to improve such a list of pathological variants, we completely sequenced the mitochondrial genomes of suspected LHON patients from Italy, France and Germany, lacking the three primary common mutations. Phylogenetic and conservation analyses were performed. Sixteen mitochondrial genomes were found to harbor at least one of the following nine rare LHON pathogenic mutations in genes MT-ND1 (m.3700G>A/p.A132T, m.3733G>A-C/p.E143K-Q, m.4171C>A/p.L289M), MT-ND4L (m.10663T>C/p.V65A) and MT-ND6 (m.14459G>A/p.A72V, m.14495A>G/p.M64I, m.14482C>A/p.L60S, and m.14568C>T/p.G36S). Phylogenetic analyses revealed that these substitutions were due to independent events on different haplogroups, whereas interspecies comparisons showed that they affected conserved amino acid residues or domains in the ND subunit genes of complex I.

Conclusions/Significance

Our findings indicate that these nine substitutions are all primary LHON mutations. Therefore, despite their relative low frequency, they should be routinely tested for in all LHON patients lacking the three common mutations. Moreover, our sequence analysis confirms the major role of haplogroups J1c and J2b (over 35% in our probands versus 6% in the general population of Western Europe) and other putative synergistic mtDNA variants in LHON expression.     

Methionine synthase polymorphisms (MTR 2756 A>G and MTR 2758 C>G) frequencies and distribution in the Jordanian population and their correlation with neural tube defects in the population of the northern part of Jordan

BACKGROUND:

The human methionine synthase gene (MTR) is located on chromosome 1q43; it is of 105.24 kb and is made up of 33 exons. Methionine synthase is a cytoplasmic enzyme that requires methylcobalamin for activity and catalyzes the remethylation of homocysteine to methionine. In this reaction, the methyl group of 5-methyltetrahydrofolate is transferred to the enzyme bond cob(I) alamin to generate methylcobalamin, followed by the transfer of the methyl group to homocysteine to reform methionine.

MATERIALS AND METHODS:

The frequencies of the polymorphisms of MTR 2756A>G and MTR 2758C>G have been determined in this study in a sample of 491 individuals collected from all regions of Jordan and representing the Jordanian population. The different alleles and genotypes at the two polymorphic sites were identified using the Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR-RFLP) analysis.

RESULTS:

Showed that the percentages of the polymorphic alleles at the MTR 2756 position in the north, middle and south regions were 90.38, 92.65 and 83.69%, respectively, for the MTR 2756A allele, and were 9.61, 7.34 and 16.30%, respectively, for the MTR 2756G allele, with overall percentages in the whole Jordanian population of 90.73 and 9.27% for the MTR 2756A and MTR 2756G alleles, respectively. The percentages of the genotype MTR 2756AA were 82.90% in the northern region, 86.72% in the middle region and 71.73% in the southern region, and an overall percentage of MTR 2756AA in the whole Jordanian population was 83.50%. The frequencies of MTR 2756AG genotype in the northern, middle and southern regions were 14.95, 11.84 and 23.91%, respectively, with an overall percentage of 14.46% in the whole Jordanian population. The percentages of the genotype MTR 2756GG in the northern, middle and southern regions were 2.13, 1.42 and 4.34%, respectively, with an overall percentage of 2.04% in the whole Jordanian population. Only the wild type allele (C) of the MTR 2758C>G polymorphism was detected in this study. In addition, the association of MTR 2756A>G and MTR 2758C>G polymorphisms with the development of neural tube defects (NTDs) was examined using 17 cases of mothers from the northern part of Jordan, who gave birth to NTD affected children during the period of this study. Results showed no association between these two examined polymorphisms and the increase in maternal risk for giving birth to NTD children.

CONCLUSION:

results of this study recommend that examination should be done on larger populations to arrive at better conclusions. Also, more studies on gene–gene interaction should be done to examine the associations with NTDs.     

Association between genetic variants in DNA and histone methylation and telomere length

Telomere length, a biomarker of aging and age-related diseases, exhibits wide variation between individuals. Common genetic variation may explain some of the individual differences in telomere length. To date, however, only a few genetic variants have been identified in the previous genome-wide association studies. As emerging data suggest epigenetic regulation of telomere length, we investigated 72 single nucleotide polymorphisms (SNPs) in 46 genes that involve DNA and histone methylation as well as telomerase and telomere-binding proteins and DNA damage response. Genotyping and quantification of telomere length were performed in blood samples from 989 non-Hispanic white participants of the Sister Study, a prospective cohort of women aged 35–74 years. The association of each SNP with logarithmically-transformed relative telomere length was estimated using multivariate linear regression. Six SNPs were associated with relative telomere length in blood cells with p-values<0.05 (uncorrected for multiple comparisons). The minor alleles of BHMT rs3733890 G>A (p = 0.041), MTRR rs2966952 C>T (p = 0.002) and EHMT2 rs558702 G>A (p = 0.008) were associated with shorter telomeres, while minor alleles of ATM rs1801516 G>A (p = 0.031), MTR rs1805087 A>G (p = 0.038) and PRMT8 rs12299470 G>A (p = 0.019) were associated with longer telomeres. Five of these SNPs are located in genes coding for proteins involved in DNA and histone methylation. Our results are consistent with recent findings that chromatin structure is epigenetically regulated and may influence the genomic integrity of telomeric region and telomere length maintenance. Larger studies with greater coverage of the genes implicated in DNA methylation and histone modifications are warranted to replicate these findings.     

Survey of genome sequences in a wild sweet potato,Ipomoea trifida (H. B. K.) G. Don

Ipomoea trifida (H. B. K.) G. Don. is the most likely diploid ancestor of the hexaploid sweet potato, I. batatas (L.) Lam. To assist in analysis of the sweet potato genome, de novo whole-genome sequencing was performed with two lines of I. trifida, namely the selfed line Mx23Hm and the highly heterozygous line 0431-1, using the Illumina HiSeq platform. We classified the sequences thus obtained as either ‘core candidates’ (common to the two lines) or ‘line specific’. The total lengths of the assembled sequences of Mx23Hm (ITR_r1.0) was 513 Mb, while that of 0431-1 (ITRk_r1.0) was 712 Mb. Of the assembled sequences, 240 Mb (Mx23Hm) and 353 Mb (0431-1) were classified into core candidate sequences. A total of 62,407 (62.4 Mb) and 109,449 (87.2 Mb) putative genes were identified, respectively, in the genomes of Mx23Hm and 0431-1, of which 11,823 were derived from core sequences of Mx23Hm, while 28,831 were from the core candidate sequence of 0431-1. There were a total of 1,464,173 single-nucleotide polymorphisms and 16,682 copy number variations (CNVs) in the two assembled genomic sequences (under the condition of log₂ ratio of >1 and CNV size >1,000 bases). The results presented here are expected to contribute to the progress of genomic and genetic studies of I. trifida, as well as studies of the sweet potato and the genus Ipomoea in general.     

Multiple elements in the eIF4G1 N-terminus promote assembly of eIF4G1•PABP mRNPs in vivo

eIF4G is the scaffold subunit of the eIF4F complex, whose binding domains for eIF4E and poly(A)-binding protein (PABP) are thought to enhance formation of activated eIF4F•mRNA•PABP complexes competent to recruit 43S pre-initiation complexes. We found that the RNA-binding region (RNA1) in the N-terminal domain (NTD) of yeast eIF4G1 can functionally substitute for the PABP-binding segment to rescue the function of an eIF4G1-459 mutant impaired for eIF4E binding. Assaying RNA-dependent PABP–eIF4G association in cell extracts suggests that RNA1, the PABP-binding domain, and two conserved elements (Box1 and Box2) between these segments have overlapping functions in forming native eIF4G•mRNA•PABP complexes. In vitro experiments confirm the role of RNA1 in stabilizing eIF4G–mRNA association, and further indicate that RNA1 and Box1 promote PABP binding, in addition to RNA binding, by the eIF4G1 NTD. Our findings indicate that PABP–eIF4G association is only one of several interactions that stabilize eIF4F•mRNA complexes, and emphasize that closed-loop mRNP formation via PABP–eIF4G interaction is non-essential in vivo. Interestingly, two other RNA-binding regions in eIF4G1 have critical functions downstream of eIF4F•mRNA assembly.     

Association with litter size of new polymorphisms on ESR1 and ESR2 genes in a Chinese-European pig line

The objective of this study was to search for polymorphisms in the coding region of the estrogen receptors 1 and 2 (ESR1 and ESR2 )and to analyze the effects of these variants and the well known intronic ESR1 Pvu II polymorphism on litter size in a Chinese-European pig line. We identified five silent single nucleotide polymorphisms (SNP) in the ESR1 cDNA: c.669T > C (exon 3), c.1227C > T (exon 5), c.1452C > T (exon 7), c.1665T > C and c.1755A > G (exon 8). One pair of these SNP (c.1665T > C and c.1755A > G) co-segregated in the analyzed line, and the SNP c.669T > C showed the same segregation pattern as the Pvu II polymorphism. These polymorphisms were tested in this study, although the c.1452C > T SNP within exon 7 was not analyzed due to its low informativeness. In the ESR2 cDNA, one missense SNP was found within exon 5, which caused an amino acid substitution in the coded protein: "c.949G > A (p.Val317Met)" and was tested on sow litter size. Information on 1622 litter records from 408 genotyped sows was analyzed to determine whether these SNP influenced the total number of piglets born (TNB) or the number of born alive (NBA). The polymorphisms ESR1: [Pvu II; c.669T > C], ESR1: [c.1665T > C; c.1755A > G] and ESR2: c.949G > A showed no statistically significant association with litter size. However, the ESR1: c.1227T allele was significantly associated with TNB. The additive substitution effect was estimated to be 0.40 piglets born per litter (P < 0.03), and no dominance effects were observed. This SNP could be useful in assisted selection for litter size in some pig lines, as a new genetic marker in linkage disequilibrium with the causative mutation.     

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.     

Interactions of the apolipoprotein A5 gene polymorphisms and alcohol consumption on serum lipid levels

Background

Little is known about the interactions of apolipoprotein (Apo) A5 gene polymorphisms and alcohol consumption on serum lipid profiles. The present study was undertaken to detect the interactions of ApoA5–1131T>C, c.553G>T and c.457G>A polymorphisms and alcohol consumption on serum lipid levels.

Methodology/Principal Findings

A total of 516 nondrinkers and 514 drinkers were randomly selected from our previous stratified randomized cluster samples. Genotyping was performed by polymerase chain reaction and restriction fragment length polymorphism. The levels of serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), ApoA1 and ApoB were higher in drinkers than in nondrinkers (P<0.05–0.001). The genotypic and allelic frequencies of three loci were not different between the two groups. The interactions between –1131T>C genotypes and alcohol consumption on ApoB levels (P<0.05) and the ApoA1/ApoB ratio (P<0.01), between c.553G>T genotypes and alcohol consumption on low-density lipoprotein cholesterol (LDL-C) levels (P<0.05) and the ApoA1/ApoB ratio (P<0.05), and between c.457G>A genotypes and alcohol consumption on TG levels (P<0.001) were detected by factorial regression analysis after controlling for potential confounders. Four haplotypes (T-G-G, C-G-G, T-A-G and C-G-T) had frequencies ranging from 0.06 to 0.87. Three haplotypes (C-G-G, T-A-G, and C-G-T) were significantly associated with serum lipid parameters. The –1131T>C genotypes were correlated with TG, and c.553G>T and c.457G>A genotypes were associated with HDL-C levels in nondrinkers (P<0.05 for all). For drinkers, the –1131T>C genotypes were correlated with TC, TG, LDL-C, ApoB levels and the ApoA1/ApoB ratio (P<0.01 for all); c.553G>T genotypes were correlated with TC, TG, HDL-C and LDL-C levels (P<0.05–0.01); and c.457G>A genotypes were associated with TG, LDL-C, ApoA1 and ApoB levels (P<0.05–0.01).

Conclusions

The differences in some serum lipid parameters between the drinkers and nondrinkers might partly result from different interactions of the ApoA5 gene polymorphisms and alcohol consumption.     

Genetic analysis of OCT1 gene polymorphisms in an Indian population

BACKGROUND:

Genetic variants of the organic cation transporter (OCT1) gene could influence interindividual variation in clinical response to metformin therapy. The genetic basis for the single-nucleotide polymorphism (SNP) of OCT1 gene has been established in other populations, but it remains to be elucidated in the Indian population. This study is focused on OCT1 gene variants rs2282143 (P341L, 1022C>T), rs628031 (M408V, 1222A>G) and rs622342 (1386C>A) frequency distributions in the South Indian Tamilian population.

MATERIALS AND METHODS:

A total of 112 unrelated healthy subjects of South Indian Tamilian origin, aged 18–60 years, of either sex were recruited for the study. Genotyping was determined using the quantitative real time-polymerase chain reaction and polymerase chain reaction followed by restriction fragment length polymorphism methods.

RESULTS:

Allele frequencies of rs2282143, rs628031and rs622342 polymorphisms were 8.9%, 80.3% and 24.5%, respectively. Interethnic differences in the genotype and allele frequencies of OCT1 gene polymorphism were observed when compared with other major populations. The SNPs rs2282143, T allele and rs628031, G allele were more common in Asians (5.5–16.8% and 76.2–81%) and African Americans (8.2% and 73.5%) than in Caucasians (0–2% and 57.4–60%).

CONCLUSION:

This is the first time the frequency of OCT1 gene polymorphism was determined in the Indian population, and is similar to the frequencies observed in African-Americans and other Asian populations but different from those in Caucasians. The data observed in this study would justify further pharmacogenetic studies to potentially evaluate the role of OCT1 gene polymorphism in the therapeutic efficacy of metformin.