Chain Reaction-single Strand Conformation Polymorphism Research Articles

Detection of new single nucleotide polymorphisms in plasma cells carcinoma (myeloma) by polymerase chain reaction–single strand conformation polymorphism and sequencing technical methods

Abstract Background: MicroRNA (miRNA) is a short, single-stranded, non-coding sequence of RNA that does not translate into proteins. Because of their ability to control gene expression, recent studies have shown that these short, non-coding sequences play an important role in cancer. So that, detection the polymorphisms or variants in miRNA genes may help to identify their correlation to cancer susceptibility. Objectives: Study the polymorphisms in miRNA genes or variants miRNA genes and their correlation with myeloma disease. Materials and Methods: Twenty samples were collected from people who visited Marjan Hospital in Province of Babylon, Iraq, from February to July 2022. In addition, 20 samples from healthy people were collected as control. Genotyping of miRNA was accomplished using the polymorphism techniques of polymerase chain reaction and single strand conformation polymorphism (SSCP). DNA sequencing was also applied to categorize all the ranges of SSCP models detected using only gel imaging. Results: The genotypes obtained in this experiment verified that several single nucleotide polymorphisms were obtained between 5-band and 6-band miRNA National center for biotechnology information Primer3 plus reference. Conclusion: The results explain that the miRNA gene polymorphisms may have correlation with the susceptibility to myeloma disease.

The sequence variation of mitochondrial tRNA tyrosine and cysteine among Iranian women with idiopathic recurrent miscarriage: A case-control study.

Recurrent miscarriage is one of the most prevalent reproductive diseases. This phenomenon has several reasons, including maternal, hormonal, immunological, and parental genetic factors. Idiopathic recurrent miscarriage (IRM), with no distinctive etiology, involves about half of the recurrent miscarriage cases. Some mutations in mitochondrial DNA can lead to miscarriage. Mitochondrial tRNA (mt-tRNA) mutations cause nearly half of the mitochondrial disorders. To identify mt- tRNA gene mutations in Iranian women with IRM. In this case-control study, 100 Iranian women with IRM and 100 women as control without any history of miscarriage were investigated by polymerase chain reaction-single strand conformation polymorphism technique followed by gene sequencing. Bioinformatics analysis were done using human mitochondrial genome database, molecular evolutionary genetics analysis, mammalian mitochondrial-tRNA, etc. Results showed 4 mt-tRNA mutations including 1 cysteine mt-tRNA mutation (5824CT) and 3 tyrosine mt-tRNA mutations (5868TA, 5849CT, and 5836TC) in our cases. Amongst the 4 mutations found, one was novel that is still not reported. Our bioinformatics analysis revealed that these mutations can be pathogenic. They occurred in tRNA-conserved regions and their secondary structure was changed, which can result in mitochondrial dysfunction. Mutations of these genes may help in the assessment of IRM. Further study of all 22 mt-tRNAs possible mutations is recommended to describe their etiologic role in IRM.

Polymorphic Variants Analysis in Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-Alpha (PPARGC1A) Gene of Indian and Egyptian Buffaloes

Background-Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) is a candidate gene for milk fat in livestock. The present study was carried out to investigate polymorphism in PPARGC1A and study its potential association. Methods-Two hundred forty animals from Murrah and Bhadawari Indian buffalo breeds and Egyptian buffaloes were selected. The statistical analysis used Tukey’s honest significance test to investigate the association of different genotypes. PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) assays and sequencing technologies were used to confirm the SNPs. Results-Three Novel single nucleotide polymorphisms (SNP1- g.993A greater than T, SNP2- g.1237T greater than A and SNP3-g.1238G greater than C) and one insertion (g.1240_ greater than G bp) were identified in this gene. Due to nucleotide mutations, four non-synonymous mutations were observed amino acid sequences of buffalo breeds. Significant differences in protein and SNF output were discovered in Egyptian buffalo breeds. In both Murrah and Bhadawari buffaloes, fat, protein, and SNF yield were substantially correlated with detected genotypes while there was non-significant association of protein and SNF percent in buffalo breeds. The information provided will be valuable in studying the involvement of PPARGC1A in regulating fat synthesis and enhancing milk quality in buffaloes.

Expression and Variations in EPAS1 Associated with Oxygen Metabolism in Sheep.

Endothelial PAS domain protein 1 gene (EPAS1) is a member of the HIF gene family. This gene encodes a transcription factor subunit that is involved in the induction of oxygen-regulated genes. Several studies have demonstrated that a mutation in EPAS1 could affect oxygen sensing, polycythemia, and hemoglobin level. However, whether EPAS1 mutation affects sheep oxygen metabolism is still unknown. Therefore, we explored the relationship between the variation of EPAS1 and oxygen metabolism in sheep. In this study, variations in ovine EPAS1 exon 15 were investigated in 332 Tibetan sheep and 339 Hu sheep by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. In addition, we studied the effect of these variations on blood gas in 176 Tibetan sheep and 231 Hu sheep. Finally, the mRNA expression of EPAS1 in six tissues of Hu sheep and Tibetan sheep living at different altitudes (2500 m, 3500 m, and 4500 m) was analyzed by real-time quantitative PCR (RT-qPCR). Four alleles (A, B, C, and D) were detected, and their distributions highly differed between Tibetan sheep and Hu sheep. In Tibetan sheep, B was the dominant allele, and C and D alleles were rare, whereas all four alleles were common in Hu sheep. Six single nucleotide polymorphisms (SNPs) were identified between the four alleles and one of them was non-synonymous (p.F606L). While studying the blood gas levels in Tibetan sheep and Hu sheep, one variant region was found to be associated with an elevated pO2 and sO2, which suggested that variations in EPAS1 are associated with oxygen metabolism in sheep. RT-qPCR results showed that EPAS1 was expressed in the six tissues of Hu sheep and Tibetan sheep at different altitudes. In addition, the expression of EPAS1 in four tissues (heart, liver, spleen, and longissimus dorsi muscle) of Hu sheep was lower than that in Tibetan sheep from three different altitudes, and the expression of EPAS1 was positively correlated with the altitude. These results indicate that the variations and expression of EPAS1 is closely related to oxygen metabolism.

Identification of the KAP27-1 gene in sheep and its effect on wool traits

ABSTRACT The keratin-associated proteins (KAPs) are structural components of wool and hair fibres, with over 90 genes that encode these proteins having been identified in mammalian species. A KAP gene called KRTAP27-1 was recently identified in goats, but the gene has not been reported in the closely related ruminant species sheep. In this study, we used a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis to find ovine KRTAP27-1 and investigate nucleotide sequence variation in the gene. Our analysis revealed four sequence variants (named A to D) in 399 New Zealand Merino × Southdown-cross sheep. Within these sequence variants there were five single nucleotide polymorphisms (SNPs) in the coding region, including two non-synonymous SNPs. Six genotypes (AA, AB, AC, BB, BC and BD) of KRTAP27-1 were detected, and for the three genotypes with a frequency of over 5.0%, sheep of genotypes AB and BB produced wool of higher mean staple length (MSL) and higher greasy fleece weight (GFW) than sheep of genotype AA. These results suggest that variation in ovine KRTAP27-1 may be affecting wool growth and thus production.

Major Histocompatibility Complex (MHC) Diversity of the Reintroduction Populations of Endangered Przewalski's Horse.

Major histocompatibility complex (MHC) genes are the most polymorphic in vertebrates and the high variability in many MHC genes is thought to play a crucial role in pathogen recognition. The MHC class II locus DQA polymorphism was analyzed in the endangered Przewalski’s horse, Equus przewalskii, a species that has been extinct in the wild and all the current living individuals descend from 12 founders. We used the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) to detect the polymorphism within the MHC DQA in 31 Przewalski’s horses from two reintroduced populations. Consequently, only seven alleles were identified, with only four presenting in each population. In comparison with other mammals, the Przewalski’s horse demonstrated less MHC variation. The nucleotide genetic distance of the seven ELA-DQA alleles was between 0.012 and 0.161. The Poisson corrected amino acid genetic distance of the founded alleles was 0.01–0.334. The allele and genotype frequencies of both reintroduced populations of Przewalski’s horse deviated from the Hardy–Weinberg equilibrium. Specific MHC DQA alleles may have been lost during the extreme bottleneck event that this species underwent throughout history. We suggest the necessity to detect the genetic background of individuals prior to performing the reintroduction project.

Artificial Neural Network-Based Identification of Associations between UCP2 and UCP3 Gene Polymorphisms and Meat Quantity Traits

In identifying mutations occurring in distinct cow breeds, genetic elements must be taken into consideration. More recently, these hereditary features have gained attention throughout the world. As in many underdeveloped nations, to bridge the deficit in molecular genetics, multiple solutions are required. The inner membrane anion carrier superfamily contains the uncoupling proteins (UCPs), vital to energy regulation. Research on heredity has shown that variations in the UCP2 and UCP3 genes are connected to obesity and metabolic syndrome. This research aimed to investigate if any mutation in the UCP 2 and UCP 3 genes are related to many characteristics in Pakistan’s three indigenous cattle breeds using artificial neural network (ANN). For better analysis, the output of the ANN model is loaded into the Primer Premier 3 software. Using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing, the results of this study indicated 07 variations in the exon 4 region of the UCP2 gene and 03 variants in the exon 3 area of the UCP3 gene among 215 indigenous cow breeds. The association study revealed that the g.C35G mutation in the UCP3 gene is strongly related to meat quantity characteristics such as carcass weight and drip percentage (P0.05) but not with body height or hip width ( P > 0.05 ). Sequence analysis showed five distinct diplotypes: AA, BC, AC, CC, and CD. Cattle with the novel heterozygous diplotype BC perform better in carcass trait and drip percentage than animals with other genotypes. The study’s findings suggest that the UCP3 gene may be utilized for marker-assisted selection (MAS) and breed mixing in Pakistan cattle breeds to aid in the country’s economic growth.

Correlation of lipoprotein lipase gene polymorphism and mRNA expression with intramuscular fat content in Baicheng-Oil chicken.

Lipoprotein lipase (LPL) was often taken as a candidate gene for investigating fat metabolism. However, there are few studies on the effect of LPL on intramuscular fat (IMF) deposition in Baicheng oil chicken (BOC) and Three-yellow Chicken (TYC). In this study, we studied the relationship between polymorphism and messenger RNA (mRNA) expression of LPL with IMF deposition in the chest muscle (CM) and leg muscle (LM) of TYC and BOC. Sixty TYCs and 60 BOCs were raised from 1 d and slaughtered by avascularization at their slaughtering age. IMF contents of the CM and LM in the BOC were markedly higher than those in the TYC. Three genotypes following AA, AB and BB were found by the method of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). The synonymous mutation C12315T was detected. The content of IMF with the AA genotype was significantly higher than the AB genotype in the LM of TYC. The mRNA expression both of CM and LM in BOC was prominently higher than those in TYC, and there was a positive significant correlation between LM and CM in both BOC and TYC. These results suggested that the SNPs polymorphism and mRNA expression of the LPL gene might be helpful for selective breeding in IMF of the chicken.

Variation in caprine KRTAP1-3 and its association with cashmere fibre diameter

Keratin-associated proteins (KAPs) are components of cashmere fibres. The gene encoding the KAP1-3 protein (KRTAP1-3) has been described in goats, but little is known about sequence variation in this gene and if it affects cashmere fibre traits. In this study, we used a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique to screen for nucleotide sequence variation in caprine KRTAP1-3 in 327 Longdong cashmere goats, then analysed association between the genetic variation that was revealed and some cashmere fibre traits. Six PCR-SSCP patterns representing six different variant sequences of KRTAP1-3 (named A to F) were revealed. Among these variant sequences, seven single nucleotide polymorphisms (SNPs) were detected, with two of them being non-synonymous. Goats with genotype AC had higher mean fibre diameter (MFD) than those with genotype AB (P < 0.001), while goats with genotype AB had higher MFD than those with AA (P < 0.001). The presence of C (P < 0.001) and B (P = 0.006) in a genotype was associated with increased MFD, and together this suggests that variation in caprine KRTAP1-3 affects the key fibre trait of MFD.

Comparison of merits of DNA sequencing, PCR-SSCP and MFP assays in the detection of drug resistance in Mycobacterium leprae

Purpose: To compare the merits of conventional Mouse Foot Pad (MFP) assay, Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) analysis, and Direct-DNA sequencing in identification of drug resistance in M. leprae.&#x0D; Methods: A total of 41 leprosy cases were investigated for drug resistance using MFP assay, whereby growth results were obtained in 23 cases. The DNAs extracted from these samples were amplified in polymerase chain reaction (PCR) using specific primers in order to determine the drug resistance-loci. Furthermore, PCRSSCP analysis was carried out using PCR amplicons, while gel electrophoresis was performed to identify any shift in mobility in any one of the DNA strands, as well as the pattern of mutation.&#x0D; Results: A total of 5 isolates exhibited the highest degree of resistance R100, whereas 4 isolates showed intermediate level of resistance R10. In contrast, the least resistance was seen in R1. There were no mutations in 4 out of 10 strains which were dapsone-resistant, i.e., 1 HR, 2 IR and 1 LR. Moreover, there was no mutation in 305 bp sequence of the rpoB gene. DNA sequencing sensitivity was 60 %, whereas the sensitive isolates tested in the experiments exhibited no mutation, resulting in 100 % specificity.&#x0D; Conclusion: These results indicate the advantages of molecular techniques over conventional MFP technique. The study has revealed that PCR-SSCP analysis, a rapid, specific and less expensive method, has greater potential for use in routine testing of the susceptibility of M. leprae to rifampicin and dapsone, than the other tests.

Mitochondrial mutations in protein coding genes of respiratory chain including complexes IV, V, and mt-tRNA genes are associated risk factors for congenital heart disease.

Most studies aiming at unraveling the molecular events associated with cardiac congenital heart disease (CHD) have focused on the effect of mutations occurring in the nuclear genome. In recent years, a significant role has been attributed to mitochondria for correct heart development and maturation of cardiomyocytes. Moreover, numerous heart defects have been associated with nucleotide variations occurring in the mitochondrial genome, affecting mitochondrial functions and cardiac energy metabolism, including genes encoding for subunits of respiratory chain complexes. Therefore, mutations in the mitochondrial genome may be a major cause of heart disease, including CHD, and their identification and characterization can shed light on pathological mechanisms occurring during heart development. Here, we have analyzed mitochondrial genetic variants in previously reported mutational genome hotspots and the flanking regions of mt-ND1, mt-ND2, mt-COXI, mt-COXII, mt-ATPase8, mt-ATPase6, mt-COXIII, and mt-tRNAs (Ile, Gln, Met, Trp, Ala, Asn, Cys, Tyr, Ser, Asp, and Lys) encoding genes by polymerase chain reaction-single stranded conformation polymorphism (PCR-SSCP) in 200 patients with CHD, undergoing cardiac surgery. A total of 23 mitochondrial variations (5 missense mutations, 8 synonymous variations, and 10 nucleotide changes in tRNA encoding genes) were identified and included 16 novel variants. Additionally, we showed that intracellular ATP was significantly reduced (P=0.002) in CHD patients compared with healthy controls, suggesting that the mutations have an impact on mitochondrial energy production. Functional and structural alterations caused by the mitochondrial nucleotide variations in the gene products were studied in-silico and predicted to convey a predisposing risk factor for CHD. Further studies are necessary to better understand the mechanisms by which the alterations identified in the present study contribute to the development of CHD in patients.

Ovine Toll-like Receptor 9 (TLR9) Gene Variation and Its Association with Flystrike Susceptibility.

Simple SummaryFlystrike is a major ectoparasitic disease of sheep and it creates both an economic impact and welfare issue for the sheep industry. Several factors control the responses of sheep to flystrike, and among these, immune response is regarded as an important factor. Toll-like receptors (TLRs) plays a crucial role in the innate immune system by recognizing pathogen-associated molecular patterns derived from various microbes. Of these receptors, TLR9 recognises unmethylated cytosine-phosphate-guanine (CpG) motifs that are known to be prevalent in bacterial genomes and are also reported in Dipteran insects, including Lucilia cuprina, one of the main species associated with flystrike in sheep. In this study, we used a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique to investigate TLR9 variation in sheep with and without flystrike, and found that variation in a gene region containing the coding sequence of the putative CpG-DNA binding domain was associated with the likelihood of flystrike occurrence. This suggests that variation in ovine TLR9 may affect a sheep’s response to flystrike.Toll-like receptors (TLRs) are a family of proteins that play a role in innate immune responses by recognising pathogen-associated molecular patterns derived from various microbes. Of these receptors, TLR9 recognises bacterial and viral DNA containing unmethylated cytosine-phosphate-guanine (CpG) motifs, and variation in TLR9 has been associated with resistance to various infectious diseases. Flystrike is a problem affecting the sheep industry globally and the immune response of the sheep has been suggested as one factor that influences the response to the disease. In this study, variation in ovine TLR9 from 178 sheep with flystrike and 134 sheep without flystrike was investigated using a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) approach. These sheep were collected from both commercial and stud farms throughout New Zealand and they were of 13 different breeds, cross-breds and composites. Four alleles of TLR9 were detected, including three previously identified alleles (*01, *02 and *03) and a new allele (*04). In total six single nucleotide polymorphisms (SNPs) were found. Of the three common alleles in the sheep studied, the presence of *03 was found to be associated with a reduced likelihood of flystrike being present (OR = 0.499, p = 0.024). This suggests that variation in ovine TLR9 may affect a sheep’s response to flystrike, and thus the gene may have value as a genetic marker for improving resistance to the disease.

Identification of sequence variation in the oocyte-derived bone morphogenetic protein 15 (BMP15) gene (BMP15) associated with litter size in New Zealand sheep (Ovis aries) breeds.

Advances in the study of reproductive traits indicate that functional variation in fertility genes may be useful for improving sheep fertility. The aim of this study was to search for variation in the bone morphogenetic protein 15 gene (BMP15) and ascertain any association with litter size in purebred Finnish Landrace sheep (n = 148), Finnish Landrace × Texel-cross sheep (n = 45), and composite sheep (of varying breed background; n = 58) from New Zealand (NZ). A 482bp and 312bp fragment of exon 1 and 2, respectively, of BMP15 were analysed using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). The additive and dominance effect of BMP15 variation on litter size were estimated using animal and sire models. Two variants (A and B) were detected in exon 1; nosequence variation was detected in exon 2. Variant A had the nucleotide sequence CTT between positions c.31 and c.33, while variant B had a deletion (c.31_33del). The observed frequency for variant A in the Finnish Landrace sheep, Finnish Landrace × Texel-cross sheep and the composite sheep, was 0.77, 0.92, and 0.68, respectively while the frequency of variant B (c.31_33del) was 0.23, 0.08, and 0.32, respectively. An association between litter size and c.31_33del (P < 0.001) was observed in composite sheep. Analysis of more sheep will be required to confirm these results. Litter size did not differ significantly between sheep breeds regardless of the presence/absence of c.31_33del. Results suggested that c.31_33del might be a genetic marker for improving fecundity in some NZ sheep.

Variation in a Newly Identified Caprine KRTAP Gene Is Associated with Raw Cashmere Fiber Weight in Longdong Cashmere Goats.

Keratin-associated proteins (KAPs) and keratins determine the physical and chemical properties of cashmere fibers as they are the main components of the fibers. It has been reported that ovine KRTAP1-2 affects clean fleece weight, greasy fleece weight and yield in sheep, but the gene has not been described in goats and its effects on fiber traits are unknown. In this study, we identify the keratin-associated protein 1-2 gene (KRTAP1-2) in the goat genome and describe its effect on cashmere fiber traits in 359 Longdong cashmere goats. Six sequence variants (named CAPHI-KRTAP1-2*A to CAPHI-KRTAP1-2*F) were revealed using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. These sequences have the highest homology with ovine KRTAP1-2 sequences. There were a 60-bp deletion, a 15-bp insertion and five single nucleotide polymorphisms (SNPs) including two non-synonymous SNPs in the coding sequence. The caprine KRTAP1-2 gene was expressed in the skin tissue, but a signal was not observed for the kidneys, liver, lungs, spleen, heart and longissimus dorsi muscle. Variation in caprine KRTAP1-2 was found to be associated with raw cashmere fiber weight, but not with fiber diameter and length.

Polymorphisms in hormone-sensitive lipase and leptin receptor genes and their association with growth traits in Barki lambs.

Background and Aim:Marker-assisted selection has many advantages over conventional selection in animal breeding. The candidate gene approach has been applied to identify genetic markers associated with economically important traits in livestock. This study was established to investigate variation in the hormone-sensitive lipase (HSL) and leptin receptor (LEPR) genes, and their association with growth traits in Barki lambs.Materials and Methods:Records for birth weight (BW), pre-weaning average daily gain (ADG1), weaning weight (WW), post-weaning average daily gain (ADG2), and marketing weight (MW) were obtained from 247 Barki lambs. Polymerase chain reaction–single-stranded conformational polymorphism analyses were used to detect variation in exon 9 of HSL and exon 19 of LEPR. General linear models were used to test for associations between the variation in ovine HSL and LEPR, and growth traits.Results:The SSCP banding patterns for HSL showed three variants (H1, H2, and H3), which contained two nucleotide-sequence differences (c.1865C>T and c.2038T>C). Two SSCP banding patterns (L1 and L2) were observed for LEPR and these contained two nucleotide-sequence differences (c.2800G>A and c.2978C>G). The HSL genotype showed no effect on the studied traits. The LEPR genotype was proven to have significant effects (p<0.05) on ADG2 and MW. The presence of the L1 variant was associated (p<0.01) with decreased ADG2 and MW.Conclusion:The finding of an association between LEPR gene variation and growth rate after weaning in Barki lambs warrants efforts to improve this trait.

Investigation of Polymorphisms in the Upstream Sequence of LIF and LIFR Genes in the Infertile Women

: During pregnancy, the embryo implantation stage is a highly dynamic and molecularly controlled phenomenon. Several genes are involved in the implantation process, among which the leukemia inhibitory factor (LIF) is a marker of implantation. LIF is a multi-functional cytokine located on chromosome 22. The expression of this gene is increased in the middle of the secretion phase from the sexual cycle, and any defect in its expression will cause the implantation failure. LIF receptor or LIFR gene, as the LIF receptor, consists of two membrane proteins called LIFR and GP130. LIFR acts as a signal receptor for LIF in a low-affinity level. In this study, we focused on the screening of polymorphisms in the promoter region of the LIF and LIFR genes in the infertile women using the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) method. In this study, blood samples were collected from 100 women with primary and secondary infertility and 50 healthy women as a control group. Extraction of DNA was done by the phenol-chloroform method, and in the next step, using specific primers for upstream regions of the LIF and LIFR genes, target sequences were amplified and analyzed by the SSCP method. Finally, PCR products with different configurations were selected for sequencing analysis. The results showed two polymorphisms in the upstream region of LIF and LIFR genes of two women, but there were no genetic changes in the control group. The present study was the first in this field, and the results indicated the importance of examining such genes in infertility with an unknown cause.

Evaluation of Genetic Diversity at GDF9 and BMP4 Gene by Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) in Indian Goats

Genetic diversity of three Indian goat breeds namely Barbari, Sirohi and Black Bengal goats (Capra hircus) was evaluated using Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) method to detect polymorphism in two candidate genes (GDF9 and BMP4) in a sample of 90 goats with 30 goats of each breed. Both of the studied loci were polymorphic, having three variants for GDF9 and two variants for BMP4, in the studied population. Polymorphic information content (PIC) criterion revealed an intermediate polymorphism for all analysed phenotypic groups for Barbari, Sirohi and Black Bengal goats. Effective allele number criterion also revealed equally diverse nature of these genes. This preliminary study provides insight into the genetic diversity of Barbari, Sirohi and Black Bengal goats at GDF9 and BMP4 gene locus, which can be utilized for further study polymorphism and association of these polymorphic variants with traits of economic traits in future.

Weak Ligaments and Sloping Joints: A New Hypothesis for Development of Congenital Atlantoaxial Dislocation and Basilar Invagination.

ObjectiveDevelopmental bony craniovertebral junction (CVJ) anomalies seem to have a genetic basis and also abnormal joint morphology causing atlantoaxial dislocation (AAD) and basilar invagination (BI). MethodsDNA extracted polymerase chain reaction single-stranded conformation polymorphism (SSCP) performed for mutation screening of FBN1 gene (n = 50 cases+ 50 age/sex-matched normal; total: 100). Samples with a deviated pattern of bands in SSCP were sequenced to detect the type of variation. Computed tomography (CT) scans of 100 patients (15–45 years old) compared with an equal number of age/sex-matched controls (21.9 ±8.2 years). Joint parameters studied: sagittal joint inclination (SI), craniocervical tilt (CCT), coronal joint inclination (CI). ResultsThirty-nine samples (78%) showed sequence variants. Exon 25, 26, 27, and 28 showed variable patterns of DNA bands in SSCP, which on sequencing gives various types of DNA sequence variations in intronic region of the FBN1 gene in 14%, 14%, 6%, and 44% respectively. CT radiology:SI and CCT correlated with both BI and AAD (p < 0.01). The mean SI value in controls: 83.35° ± 8.65°, and in patients with BI and AAD:129° ± 24.05°. Mean CCT in controls: 60.2° ± 9.2°, and in patients with BI and AAD: 86.0° ± 18.1°. Mean CI in controls:110.3° ± 4.23°, and in cases: 125.15° ± 16.4°. ConclusionThe study showed mutations in FBN1 gene (reported in Marfan syndrome). There is also an alteration of joint morphology, correlating with AAD and BI severity. Hence, we propose a double-hit hypothesis: the presence of weak ligaments (due to FB1 gene alterations) and abnormal joint morphology may contribute to AAD and BI.

Genetic variants of the BMP2 and GDF9 genes and their associations with reproductive performance traits in Barki ewes

• Two BMP2 variants and three GDF9 variants were identified in Barki ewes. • BMP2 variants were associated with TNLW and TWLW; however GDF9 variants were associated with twining rate, TNLB and TWLB. • Variation in ovine BMP2 and GDF9 affects reproductive performance traits of Barki ewes. The aim of this study was to investigate variation in the bone morphogenetic protein 2 ( BMP2 ) and growth differentiation factor 9 ( GDF9 ) genes, and ascertain whether it was associated with reproductive traits in Barki ewes. Records for conception rate of ewe, lambing number of ewe, lambing rate of ewe, twining rate of ewe, total number of lambs born per ewe (TNLB), total weight of lambs born per ewe (TWLB), total number of lambs weaned per ewe (TNLW), total weight of lambs weaned per ewe (TWLW) and ewe rearing ability were obtained for 296 Barki ewes. Polymerase chain reaction–single strand conformational polymorphism (PCR–SSCP) analyses were used to detect variation in exon 2 of BMP2 and exon 1 of GDF9 . The SSCP banding patterns for BMP2 revealed two variants ( A1 and A2 ), which contained one nucleotide sequence difference (c.962A > T). Three SSCP banding patterns ( C1 , C2 and C3 ) were observed for GDF9 and these contained two nucleotide-sequence differences (c.25C > T and c.260 G > A). Association analysis between the variation in BMP2 and GDF9 and reproductive performance traits revealed that BMP2 genotype was associated ( P < 0.05) with TNLW and TWLW, while GDF9 genotype was associated with twining rate of ewe ( P < 0.001), TNLB ( P < 0.001) and TWLB ( P < 0.05). This suggests that selection for the BMP2 and GDF9 genotypes might increase reproductive performance traits in Barki ewes.

Effect of DNMT3A polymorphisms on CpG island hypermethylation in gastric mucosa

BackgroundCpG methylation of tumor suppressor genes occurs in the early stage of carcinogenesis. Detecting risk factors for aberrant CpG methylation is clinically important for predicting cancer development. DNA methyltransferase (DNMT) 3a is considered to play critical roles in the DNA methylation process during pathogenesis. In this study, we evaluated the association between DNMT3A polymorphisms (rs6733868 and rs13428812) and CpG methylation status in non-cancerous gastric mucosa.MethodsWe determined the DNMT3A genotype and CpG methylation status of 4 genes (p14ARF, p16INK4a, DAPK, and CDH1) in 510 subjects without gastric cancer. Helicobacter pylori (HP) infection status was determined by the rapid urease test, urea breath test, speculum examination, or serum antibody test. We determined the DNMT3A genotype using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP). CpG methylation status was determined by methylation-specific polymerase chain reaction (MSP). When the methylated band was stronger than 10 ng/μL according to the DNA marker, we judged CpG island hypermethylation (CIHM) to be present. Associations between genotypes and susceptibilities were assessed by logistic regression analysis.ResultsThe minor allele frequencies of both polymorphisms (rs6733868 and rs13428812) were lower in the CpG methylated groups of each of the 4 genes (p14ARF, p16INK4a, DAPK, and CDH1). Using a dominant genetic model, rs6733868 was significantly associated with the hypermethylation of each gene, whereas rs13428812 was associated with the methylation of 3 genes (all except p14ARF). When low-CIHM was defined as 1 or 2 CpG islands methylated and high-CIHM was defined as 3 or more CpG islands methylated, carrying the minor allele of rs6733868 was associated with both decreased low- and high-CIHM, and that of rs13428812 also was associated with a decrease. Comparing low-CIHM with high-CIHM, carrying the minor alleles of rs6733868 or rs13428812 was related to decreased susceptibility to high-CIHM. In HP-infected subjects, carrying the minor alleles of rs6733868 or rs13428812 had a significantly greater association with decreased susceptibility to high-CIHM.ConclusionsOur study indicates that polymorphisms of DNMT3A are associated with the accumulation of gene methylation in gastric mucosa. Carrying the minor alleles of rs6733868 or rs13428812 inhibits aberrant gene methylations, which are typically enhanced by HP infection.