Polymorphism In Exon Research Articles

Novel genetic susceptibility loci identified by family based whole exome sequencing in Han Chinese schizophrenia patients

Schizophrenia (SCZ) is a highly heritable psychiatric disorder that affects approximately 1% of population around the world. However, early relevant studies did not reach clear conclusions of the genetic mechanisms of SCZ, suggesting that additional susceptibility loci that exert significant influence on SCZ are yet to be revealed. So, in order to identify novel susceptibility genes that account for the genetic risk of SCZ, we performed a systematic family-based study using whole exome sequencing (WES) in 65 Han Chinese families. The analysis of 51 SCZ trios with both unaffected parents identified 22 exonic and 1 splice-site de novo mutations (DNMs) on a total of 23 genes, and showed that 12 genes carried rare protein-altering compound heterozygous mutations in more than one trio. In addition, we identified 26 exonic or splice-site single nucleotide polymorphisms (SNPs) on 18 genes with nominal significance (P < 5 × 10−4) using a transmission disequilibrium test (TDT) in all the families. Moreover, TDT result confirmed a SCZ susceptibility locus on 3p21.1, encompassing the multigenetic region NEK4-ITIH1-ITIH3-ITIH4. Through several different strategies to predict the potential pathogenic genes in silico, we revealed 4 previous discovered susceptibility genes (TSNARE1, PBRM1, STAB1 and OLIG2) and 4 novel susceptibility loci (PSEN1, TLR5, MGAT5B and SSPO) in Han Chinese SCZ patients. In summary, we identified a list of putative candidate genes for SCZ using a family-based WES approach, thus improving our understanding of the pathology of SCZ and providing critical clues to future functional validation.

A new allele in the BMP15 gene (FecXRA) that affects prolificacy co-segregates with FecXR and FecXGR in Rasa aragonesa sheep

A FecX-mutated allele called FecXR in the BMP15 gene has been described in Rasa aragonesa sheep. FecXR causes increased prolificacy when heterozygous and sterility when homozygous in ewes. However, highly prolific ewes without the FecXR allele have been found in this breed. Therefore, a genome-wide association study (GWAS) was performed in 158 ewes (tail H: N = 73, mean prolificacy ± standard deviation = 2.14 ± 0.26; tail L: N = 85, mean prolificacy = 1.06 ± 0.08) with the Ovine HD SNP BeadChip. In this analysis, the FecXGR allele was found to have genome-wide significance associated with prolificacy, first described in the Grivette sheep breed. We also identified a novel polymorphism in exon 2 of BMP15 in 9 high prolific ewes by Sanger sequencing. This new mutation, called FecXRA, is a SNP (Oar3.1_X: g. 50970948C > T; NM_001114767.1: c.1172C > T) that produces an amino acid substitution (ENSOART00000010201: p.T400I) that is predicted to be deleterious and to alter the predicted secondary structure of the mature protein. To confirm if this SNP had any the effect on prolificacy, we genotyped sires with known EBVs (Estimated Breeding Values), finding one hemizygous sire for the FecXRA allele with the highest EBV in the breeding program (effect on litter size at + 0.39 lamb per lambing). A very low frequency, ranging from 0.13 to 2%, was found for the FecXGR and FecXRA alleles in 3428 animals belonging to four different flocks. Finally, an association study was performed to validate and quantify the effects of the FecXGR and FecXRA alleles. Significant increased prolificacy of 0.52 ± 0.05, 0.42 ± 0.05 and 0.32 ± 0.01 were found when comparing FecXGR, FecXRA and FecXR heterozygous ewes to wild type homozygous ones. These effects are of the same order of magnitude as the effect of most of other known major genes for prolificacy. Only significant differences between FecXGR and FecXR were found among the three alleles associated with increased prolificacy. However, we cannot confirm the effect of the FecXRA allele at homozygous state because we did not find any homozygous ewes. These results confirm that these three alleles in the BMP15 gene that affect prolificacy co-segregate in Rasa aragonesa sheep.

Single nucleotide polymorphism of the β-lactoglobulin gene in sheep breeds reared in Bulgaria

In the present study, single nucleotide polymorphism in exon II of the β-lactoglobulin gene was investigated in four Bulgarian sheep breeds: Bulgarian Dairy Synthetic Population sheep (BDSP), Copper-red Shumen sheep (CRSH), Stara Zagora sheep (STZG) and Pleven Blackhead sheep (PLBH). Two genetic variants of β-LG gene (A and B) have been identified through PCR-RFLP assay. A 103 bp fragment of the polymorphic β-LG locus were amplified and digested with endonuclease enzyme RsaI. The obtained restriction fragments revealed three genotypes: AA, AB and BB, observed in 31%, 65.5% and 3.5% of the BDSP population and in 48%, 28% and 24% of CRSH sheep population, respectively, with departure from the Hardy-Weinberg equilibrium (P&lt;0.05) in these groups. The allele frequencies demonstrated a prevalence of the A allele (0.638 and 0.620) over the B allele (0.362 and 0.380) in both populations. On the contrary, the distribution of allele frequencies in STZG and PLBH was 0.240 and 0.100 for allele A, respectively and 0.760 and 0.900 for allele B. Therefore, the homozygous BB genotype in these sheep populations was more frequently encountered (0.520 and 0.800) than the heterozygous AB genotype (0.480 and 0.200), with HWE correspondence (P&gt;0.1). The homozygous genotype AA was absent in STZG and PLBH sheep populations. The greatest Nei’s genetic distance calculated by UPGMA method was found between the populations BDSPand PLBH (0.5334), while the closest relationship (0.0006) was established between CRSH and BDSP. The results obtained from the present investigation confirmed the presence of the SNP polymorphism in exon II of the β-lactoglobulin gene. Therefore, the genetic variability established in this polymorphic locus could be applied in further association studies with milk production traits in sheep.

Uncoupling protein gene UCP1-3826A/G, UCP2 Ins/Del and UCP3-55C/T polymorphisms in obese Turkish children.

Mitochondrial uncoupling proteins (UCP) 1, 2 and 3 are members of the anion carrier protein family located in the inner mitochondrial membrane. There are various controversial reports on UCP genotypes and obesity in adults and children. This study aims to investigate the link between mostly studied UCP polymorphisms (UCP1-3826A/G, UCP2 Insertion/Deletion (Ins/Del) polymorphism of exon 8, and UCP3-55C/T Polymorphisms) and obesity in Turkish children. Furthermore, the relationships of UCP polymorphisms are also analyzed within the scope of metabolic parameters of obese children. Molecular screening of the UCP1, UCP2, and UCP3 gene polymorphisms was carried out in 189 children aged 6 to 18 years, 102 of who had exogenous obesity (54 girls) and 87 of whom were healthy controls (48 girls). In the obese group, fasting lipids, glucose and insulin levels were measured. In 60 obese children, an oral glucose tolerance test (OGTT) was performed with 0, 30, 60, 90 and 120 minutes of sampling for plasma glucose and insulin levels. The frequency of UCP polymorphisms was similar in obese and non-obese children. In obese children, fasting lipids, glucose and insulin levels were not different among the UCP 1, 2 and 3 genotypes. While no relationship was found between the UCP 1 and 3 genotypes and glucose/insulin levels during OGTT, carriers of the Insertion allele with UCP2 Ins/Del polymorphism had significantly higher 30-minute insulin levels (p=0.018). Polymorphisms of the UCP1-3826A/G, UCP2 Ins/Del, and UCP3-55C/T are not associated with obesity and related pathologies in Turkish children. However, the presence of the Ins allele of the UCP2 gene has been found to have an unfavorable influence on early insulin excursion after glucose loading.

Determination of the Relationship Between Craving and 4-repeat Allele of DRD4 Gene Polymorphism in the Early Withdrawal Period of Alcohol Use Disorders.

Alcohol use disorder (AUD) is a disease with chronic relapses. Risk factors of craving, which is thought to be one of the predictors of relapse, have been studied for a long time. The DRD4 gene is located on chromosome 11p and has a 48-base pair variable number of tandem repeat (VNTR) polymorphisms in the 3rd exon. This study aimed to investigate if a relationship existed between craving and DRD4 VNTR polymorphism and to determine the predictors of craving. A total of 125 patients with AUD were included in the study. The sociodemographic data form, the Michigan Alcoholism Screening Test (MAST), the Obsessive Compulsive Drinking Scale (OCDS), and the Penn Alcohol Craving Scale (PACS) were applied to the patients. Polymerase chain reaction (PCR) was used to determine the DRD4 VNTR variant of all participants in the peripheral blood sample. In the 4R/4R homozygous group, it was found that the age at first alcohol use was higher and the scores of the OCDS on the seventh day were lower, but this relationship could not be demonstrated in further statistical analyses. In the stepwise linear regression model, the age at first alcohol use, MAST score, duration of AUD, and delirium tremens history were found to be the predictors of craving. DRD4 VNTR polymorphism does not play a role as a predictor of craving. A decrease in age at first alcohol use, an increase in the MAST score, and the presence of delirium tremens were found to be the predictors of craving among the participants of this study.

Association between the MUC1 rs4072037 Polymorphism and Risk of Gastric Cancer and Clinical Outcomes.

PurposeMucin 1 (MUC1) was identified as a gastric cancer (GC) susceptibility gene by genome-wide association studies in Asians and candidate gene studies in Europeans. This study aimed to investigate the association between the MUC1 rs4072037 polymorphism and GC in terms of the Lauren classification and long-term clinical outcomes.Materials and MethodsA total of 803 patients with GC and 816 unrelated healthy controls were enrolled in the study. The association between the MUC1 rs4072037 variant and GC histological types and clinical outcomes, including tumor recurrence and prognosis was investigated.ResultsThe major A allele of rs4072037 was associated with increased GC risk (P<0.05). In subtype analysis, the association was most significant for diffuse-type GC (P<0.05) and in a dominant model (P<0.05), whereas there was no association with intestinal-type GC (P>0.05). Cox proportional hazards analysis revealed the heterozygote AG rs4072037 allele as an independent risk factor influencing tumor recurrence and disease-related death in diffuse-type GC (P<0.05). but not in intestinal-type GC (P>0.05).ConclusionsThe exonic single nucleotide polymorphism rs4072037 in MUC1 was associated with diffuse-type GC and was an independent risk factor influencing tumor recurrence and disease-related death in diffuse-type GC.

The VNTR 48 bp Polymorphism in the DRD4 Gene Is Associated with Higher Tobacco Smoking in Male Mexican Mestizo Smokers with and without COPD.

Cigarette smoking is influenced by nicotine’s effects on dopaminergic activity, which appear to be moderated by genetic variation, particularly a variable number tandem repeat (VNTR, 48 bp) polymorphism in the third exon of the dopamine receptor gene (DRD4). Smokers with the VNTR ≥7 repeats (long, L allele) report markedly increased participation in some smoking behaviors; hence, our aim was to evaluate the association of the L allele in Mexican Mestizo smokers with and without COPD. The DRD4 VNTR 48 bp was genotyped in 492 Mexican Mestizo smokers: 164 COPD patients (≥20 cigarettes per day, cpd), 164 heavy smokers without COPD (HS, ≥20 cpd) and 164 light smokers without COPD (LS, 1–10 cpd). In the dominant model analysis (SL + LL vs. SS), men in the COPD and HS groups showed a statistical difference compared to LS (p = 0.01, OR = 2.06, CI 95% 1.17–3.64 and p = 0.05, OR = 1.88, CI 95% 1.03–3.45, respectively). In addition, by clustering smokers >20 cpd (COPD + HS) and comparing with the LS group, we found an association with increased risk of higher tobacco smoking p = 0.01, OR = 1.99, CI 95% 1.18–3.34. In conclusion, the long allele (L) in the VNTR of the DRD4 gene is associated with the risk of presenting higher tobacco smoking in male Mexican Mestizo smokers.

Association between Kidney Injury Molecule-1 Gene Polymorphism and Acute Kidney Injury among the Lebanese Patients

Acute kidney injury is a common condition associated with longer hospital stay and increased mortality. Kidney injury molecule-1 (KIM-1) is one of the early and sensitive biomarkers for acute kidney injury diagnosis. Therefore we examined the relationship between kidney injury molecule-1 gene polymorphism and acute kidney injury in Lebanese hospitalized patients. Genomic DNA was isolated from blood samples collected from 50 patients and 40 controls. Kidney injury molecule-1 exon 4 was amplified by polymerase chain reaction and the amplified products were sequenced at Macrogen. Serum creatinine and urea levels were measured and compared between controls and patients. Three out of the five known single nucleotide polymorphisms showed significant association with susceptibility to the disease (P ≤ 0.05). Data analysis implied that carriers of the risk allele of these 3 single nucleotide polymorphisms were more predisposed to acute kidney injury. No association was found between the studied nucleotides variations and creatinine/urea levels. Haplotype analysis showed high association of the block CTA with acute kidney injury incidence and high creatinine and urea levels. Our results suggest that polymorphisms in exon 4 of kidney injury molecule-1 in the Lebanese population may be associated with acute kidney injury.

Polymorphism of the Myostatin (MSTN) Gene in Landes and Kielecka Geese Breeds.

Simple SummaryIdentification of mutations in the myostatin gene, affecting the occurrence of the double muscling phenotype in some breeds of beef cattle, was an impetus for further analysis and identification of mutations within this gene in other animal breeds, characterized by increased meat performance parameters. The number of geese in poultry livestock production in Poland is small. The native geese breeds can be successfully used to produce high-quality poultry meat and can be a very good source of goose meat production for regional and organic products. The aim of the study was to identify a mutation in the MSTN (Myostatin) gene and investigate whether this polymorphism can affect body weight in different periods of life in Landes and Kielecka breeds. Measurements of the examined trait were taken with time intervals to demonstrate the putative effect of the identified SNP (Single Nucleotide Polymorphism) on body weight over the course of bird growth. In conclusion, the identified c.1231C>T polymorphism suggests a possible link between the polymorphism and the BW (body weight) of Kielecka geese in the 12th week of life. The most significant factors affecting the BW values in geese are breed and sex.Myostatin, also known as growth differentiation factor 8 (GDF8), belongs to the TGF- superfamily of proteins. MSTN is a highly conserved protein that acts as a negative regulator of skeletal muscle growth. Loss of myostatin functionality causes the phenotype to appear in the form of ‘double musculature’, among others in cattle, sheep, and house mice. The presented results of the research were carried out on two geese breeds—Landes and Kielecka. The aim of the study was to identify mutations in the MSTN gene and study their impact on body weight in both geese breeds in different periods of life. Analysis of the obtained results showed the existence of polymorphism in exon 3 (c.1231C>T) and suggested a possible association (p < 0.05) between BW and genotype in 12 weeks of life in male Kielecka geese breed. The identified polymorphism may be one of the factors important for improving body weight in the studied Kielecka breed, therefore, it is necessary to conduct further research on a larger population of geese breeds in order to more accurately estimate the effect of the identified SNP c.1231C>T on BW in geese.

Analysis of Cerebrospinal Fluid Soluble TREM2 and Polymorphisms in Sporadic Parkinson's Disease in a Chinese Population.

Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglial surface receptor that mediates the degradation disorder of amyloid β (Aβ) in Alzheimer's disease. However, the role of TREM2 in Parkinson's disease (PD) and α-Synclein (α-Syn) degradation is largely unknown. In this case-control study on Chinese population, we sequenced for polymorphisms in exon 2 of the TREM2 gene in 1,292 individuals, PD cases (n = 612), healthy controls (n = 680) by Sanger sequence, and compared the distribution of allelic frequencies between the two groups by the Fisher's exact test. Additionally, we developed and used the enzyme-linked immunosorbent assay to evaluated soluble TREM2 (sTREM2) levels in the cerebrospinal fluid (CSF), and plasma in partial of sequenced groups (55 PD and 40 healthy controls) analyzed their relationship with total a-syn (t-a-Syn). Two novel variants were detected in exon 2 of the TREM2 gene, namely, p.S81 N, p.G58D; however, these were not significantly associated with PD (612 PD and 680 healthy controls). sTREM2 in CSF was significantly upregulated in PD patients compared to healthy controls (433.1 ± 24.7 pg/mL vs. 275.2 ± 17.9 pg/mL, p < 0.0001), but not in plasma (281.7 ± 29.3 pg/mL vs. 257.8 ± 16.5 pg/mL, p = 0.805). In PD patients, sTREM2 was positively correlated with t-α-syn (r = 0.62, p = 0.0001) in CSF, but not in plasma (r = 0.02, p = 0.89). Although it may not indicate that exon 2 polymorphisms of TREM2 play a role in the pathogenesis of PD in the Chinese population, our findings described above highlight the relevance of CSF sTREM2 as a promising biomarker and are extremely possible to the therapeutic target for PD in the future.

Cloning, characterization of TaGS3 and identification of allelic variation associated with kernel traits in wheat (Triticum aestivum L.)

BackgroundGrain weight is an important yield component. Selection of advanced lines with heavy grains show high grain sink potentials and strong sink activity, which is an increasingly important objective in wheat breeding programs. Rice OsGS3 has been identified as a major quantitative trait locus for both grain weight and grain size. However, allelic variation of GS3 has not been characterized previously in hexaploid wheat.ResultsWe cloned 2445, 2393, and 2409 bp sequences of the homologs TaGS3-4A, TaGS3-7A, and TaGS3-7D in wheat ‘Changzhi 6406’, a cultivar that shows high grain weight. The TaGS3 genes each contained five exons and four introns, and encoded a deduced protein of 170, 169, and 169 amino acids, respectively. Phylogenetic analysis of plant GS3 protein sequences revealed GS3 to be a monocotyledon-specific gene and the GS3 proteins were resolved into three classes. The length of the atypical Gγ domain and the cysteine-rich region was conserved within each class and not conserved between classes. A single-nucleotide polymorphism in the fifth exon (at position 1907) of TaGS3-7A leads to an amino acid change (ALA/THR) and showed different frequencies in two pools of Chinese wheat accessions representing extremes in grain weight. Association analysis indicated that the TaGS3-7A-A allele was associated with higher grain weight in the natural population. The TaGS3-7A-A allele was favoured in global modern wheat cultivars but the allelic frequency varied among different wheat-production regions of China, which indicated that this allele is of potential utility to improve wheat grain weight in certain wheat-production areas of China.ConclusionsThe novel molecular information on wheat GS3 homologs and the KASP functional marker designed in this study may be useful in marker-assisted breeding for genetic improvement of wheat.

Somatic Mutations and Loss of Heterozygosity of HLA Genes Are Frequently Occurred and Tightly Associated with Poor Prognosis in Adult T Cell Leukemia-Lymphoma

Introduction: Adult T cell leukemia-lymphoma (ATL) is clinically divided into aggressive (acute and lymphoma) and indolent (chronic and smoldering) subtypes. Recent studies revealed that ATL has a wide variety of gene alternations (Nat Genet. 2015; 47:1304, Nature. 2016; 534:402). Consistent with the notion that human leukocyte antigen (HLA) play a critical role for immune response, reduced expression of HLA has been shown to associate with poor prognosis in various types of malignancies (Oncogene. 2008; 27:5869). However, it is extremely difficult to assess the impact of somatic mutations of HLA genes in cancer cells because of its highly polymorphic nature. Taking advantage of computational tools using whole-exome sequencing data, somatic mutations of HLA class I genes were previously reported in about 3% of various types of cancers (Nat Biotechnol. 2015; 33:1152). To date, HLA typing has been performed through partial sequencing data of polymorphic exons, resulting in the loss of critical information on the entire region of HLA genes. In this context, using a next generation sequence (NGS)-based HLA DNA typing method, we analyzed the entire region of HLA genes in ATL cells and its impact on clinical outcomes. Methods: The present study was approved by the Investigational Review Board of University of the Ryukyus and Tokai University. Peripheral blood mononuclear cells collected from 25 ATL patients (Pts) (20 with acute type and 5 with chronic type) were analyzed. Cell adhesion molecular 1 (CADM1)-positive ATL cells and CADM1-negative non-ATL cells were separated by a magnetic-activated cell-sorting protocol, and genomic DNAs in each fraction were distinctively extracted. The PCR products of the eight HLA loci (HLA-A, -B, -C, -DRB1, -DQA1, -DQB1, -DPA1 and -DPB1) were obtained using the previously designed long ranged PCR primers (Tissue Antigens. 2012; 80:305) that cover the entire HLA gene sequences from the enhancer-promoter region to the 3'UTR region. The full-length level HLA genotypes were determined by mapping of sequence reads obtained by the Ion Torrent system to HLA allele reference sequences in the international ImMunoGeneTics project (IMGT)-HLA database. Somatic mutations and loss of heterozygosity (LOH) generated in ATL cells were identified by detail comparison of sequence data between the ATL and non-ATL cells. Survival curves were estimated using the Kaplan-Meier method and compared by the log-rank test. Results: We found a total of 20 somatic mutations in ATL cells among 9 Pts, including 15 single nucleotide variations (SNVs) and 5 insertions/deletions (indels) (Table 1). Of the 15 SNVs, 3 were nonsense mutations in exon 2/3 of HLA-A/B, 7 were nonsynonymous mutations in exon 1/4/5 of HLA-A/B/C, and 2 were mutations at splice sites in intron 2 of HLA-B/C. The mutations of splice sites in intron 2 and all the 5 indels caused frameshift generating premature stop codons. In contrast to the exons, there were only 3 mutations found in introns. Of 8 Pts harboring LOH, two Pts showed LOH in HLA-A gene, 4 Pts in HLA-A, -C, and -B genes, one Pt in HLA-DRB1, -DQA1, -DQB1, -DPA1 and -DPB1 genes, and one Pt in all the HLA-genes from HLA-A to HLA-DPB1. All the somatic mutations altering the structure of the HLA proteins (St-mt) and/or LOH were found in Pts with acute type ATL, while no St-mt or LOH were detected in Pts with chronic type ATL. Pts with St-mt/LOH (n = 9) significantly worsened in 3 years overall survival rate as compared to that in Pts without St-mt/LOH (n = 16) (11 % vs. 32 %, P = 0.016) (Fig. 1A). Among Pts with acute type ATL, Pts with St-mutations/LOH (n = 9) also showed a trend to worsen in the survival rate as compared to those in Pts without St-mutation /LOH (n = 11) (11 % vs. 30 %, P=0.064) (Fig. 1B). Conclusion: NGS-based HLA gene analyses at full-length level revealed that St-mt/LOH were frequently occurred predominantly in HLA class I loci in tumor cells from Pts with aggressive ATL. Importantly, St-mt/LOH in HLA genes were associated with aggressive phenotypes and poor prognosis, suggesting the possibility that ATL cells would develop immune escape mechanisms through a variety of acquired abnormalities in HLA gene. Since allogeneic stem cell transplantation is an essential therapeutic option for aggressive ATL, comprehensive knowledges on HLA gene abnormalities provide considerable advantage to optimize therapeutic modalities. Disclosures Fukushima: Daiichi-Sankyo: Research Funding.

Sirtuin3 gene tissue expression profiling, SNP detection and its association with body conformation traits in goats

Abstract Sirtuin3 (SIRT3), a NAD+ dependent deacetylase, play a predominant role to control various metabolic processes involved in mammalian energy homeostasis. A study was conducted on two native goat breeds of Kerala, Malabari and Attappady Black, to analyze the tissue expression profile of caprine SIRT3, detect potential polymorphisms in exon 6 of SIRT3 and to study their association with production traits in goats. The mRNA isolated from ovary, uterus, liver and muscle of Malabari and Attappady Black goats were subjected to quantitative PCR (qPCR). Relative expression of caprine SIRT3 was significantly (p ≤ 0.05) higher in muscle, followed by uterus, liver and ovary. DNA was isolated from a total of 282 goats (176 Malabari and 106 Attappady Black) which exhibited three distinct banding patterns (PP, PQ and PR) for exon 6 fragment of SIRT3, on PCR-SSCP analysis. Sequencing the representative PCR products revealed the presence of two novel non-synonymous SNPs (c.802C > T and c.835 G > A) in the amplicon. The effect of the amino acid substitution on protein function was predicted by PolyPhen-2 tool which indicated that the amino acid change at position 802 was probably damaging and that at position 835 was benign. Further association analysis revealed that the diplotypes of exon 6 fragment had a significant (p ≤ 0.01) association with body weight. PR diplotpye was observed to be superior to PP and PQ diplotypes. All these results highlight the role of SIRT3 in growth traits and thus SIRT3 may be considered as a potential candidate gene for the production traits of goats.

Polymorphisms of pro-opiomelanocortin gene and the association with reproduction traits in chickens

Pro-opiomelanocortin (POMC) is a member of prohormone family and has important functions in stress response, skin pigmentation, thermoregulation and reproduction. In this study, the single nucleotide polymorphisms (SNPs) of POMC gene exons were detected by direct sequencing in 317 Zhenning yellow chickens. The sequencing results indicated there were seven mutation sites (g.1140C > T, g.1185 T > C, g.2085 T > C, g.3566A > C, g.3572 G > A, g.3594 G > A and g.3628 G > A) and all of these were synonymous. Furthermore, seven haplotypes were formed and sixteen diplotypes were obtained. The associations between the POMC gene polymorphisms or diplotypes and reproduction traits were also analyzed. The association analysis results indicated that the SNP of g.1140C > T was associated with egg production at 300 d of age (E300), fertilization rate (FR), hatching rate of hatching eggs (HEHR) and hatching rate of fertilized eggs (FEHR; P < 0.05). The SNP of g.3566A>C was associated with FR (P < 0.05), SNP of g.3594G>A was associated with egg weight at 300d of age (EW300; P < 0.05), and SNP of g.3628G>A was associated with HEHR and FEHR (P < 0.01), respectively. Furthermore, chickens with H2H3 diplotype had greater EW300 and FR than those with H1H7 and H3H4 diplotypes (P < 0.05). These results indicate the expression of the POMC gene had significant genotype effects on the reproduction traits of Zhenning yellow chickens, and that the H2H3 diplotype could be used as a potential genetic marker to improve the reproduction traits in chicken breeding.

Molecular Analysis of Leptin Gene Polymorphism in Achai, Sahiwal Cattle and Nili-ravi Buffalo Breeds of Pakistan

Pakistan is blessed with rich sheep genetic resources and being a source of milk, meat and wool sheep breeds are reared across the country. Despite its great economic importance very limited work has been done in Pakistan on sheep genetic exploration. Leptin hormone plays important role in milk yield, body weight, energy balance, feed intake, immune function and fertility performance in animals. The current study was carried out to investigate genetic polymorphism in exon III of Leptin gene (LEP) in indigenous animal breeds (Nilli-ravi buffalo, Sahiwal and Achai cattle) of Pakistan using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). PCR-RFLP analysis revealed that all the three studied animals breeds were polymorphic for i>LEP gene (having all the three genotypes AA, AB and BB) with frequency of 60%, 33% and 7% in Nili-ravi buffalo, 55%, 41% and 4% in Achai cattle and 52%, 45% and 3% in Sahiwal cattle breed. The genotype BB is highly desirable in cattle and buffalo breeds but its frequency is very low in our indigenous population. Studying genetic polymorphism of milk protein is important due to its application in dairy industry and these in formations could further be utilized for future breeding programs particularly for the enrichment of the rare genotype.

Analysis of testosterone pathway genes in dogs (78,XY; SRY-positive) with ambiguous external genitalia revealed a homozygous animal for 2-bp deletion causing premature stop codon in HSD17B3.

The genetic background of disorders of sex development (DSD) in dogs with a normal male sex chromosome set (78,XY) is poorly described. In this study, we present for the first time, an analysis of six genes of the testosterone pathway, encoding enzymes (CYP17A1, HSD3B2, HSD17B3, SRD5A2) and transcription factors (NR5A1, AR). The entire coding sequence and flanking regions of the introns, 5'-UTR and 3'-UTR were analyzed in five DSD dogs (78,XY, SRY-positive) with ambiguous external genitalia and in 15 control dogs. A homozygous deletion of 2bp in exon 2 of HSD17B3 (hydroxysteroid 17-beta dehydrogenase 3) was found in a Dachshund dog with enlarged clitoris, vulva and abdominal gonads and decreased serum testosterone level. In silico analysis revealed that this deleterious variant causes truncation of the encoded polypeptide (from 306 to 65 amino acids) and deprivation of the active site of the encoded enzyme. Genotyping of 23 control Dachshund dogs showed a normal homozygous genotype. Thus, we assumed that the 2-bp deletion is the causative variant. Moreover, 24 SNPs (four in CYP17A1, three in HSD3B2, six in HSD17B3, five in SRD5A2, one in AR and five in NR5A1), two intronic indels (one in HSD3B2 and one in SRD5A2) and two microsatellite polymorphisms in exon 1 of AR were found. Six SNPs appeared to be novel. No association with DSD phenotype was observed. Identification of the first case of DSD in domestic animals caused by a deleterious variant of a gene involved in testosterone synthesis showed that these genes are important candidates in such studies.

SRSF1 and PTBP1 Are trans-Acting Factors That Suppress the Formation of a CD33 Splicing Isoform Linked to Alzheimer's Disease Risk.

A single nucleotide polymorphism (SNP) in exon 2 of the CD33 gene is associated with reduced susceptibility to late-onset Alzheimer's disease (AD) and causal for elevated mRNA lacking exon 2. In contrast to full-length CD33, transcripts lacking exon 2 result in CD33 protein unable to suppress activation responses in myeloid cells, including microglia. Currently, little is known about the regulation of CD33 exon 2 splicing. Using functional genomics and proteomic approaches, we found that SRSF1 and PTBP1 act as splicing enhancers to increase CD33 exon 2 inclusion in mRNA. Binding of PTBP1 to RNA sequences proximal to the intron 1-exon 2 splice junction is altered by the SNP and represents a potential mechanism behind the SNP-genotype dependent alternative splicing. Our studies also reveal that binding of SRSF1 to the CD33 RNA is not altered by the SNP genotype. Instead, a putative SRSF1 binding sequence at the 3' end of exon 2 directs CD33 exon 2 inclusion into the mRNA, indicating that PTBP1 and SRSF1 promote full-length isoform expression through different mechanisms. Our findings shed light on molecular interactions that regulate CD33 exon 2 splicing, ultimately impacting receptor expression on the cell surface. These data aid in the understanding of CD33's regulation of microglial signaling underpinning the AD genetic associations.

The potential of VKORC1 polymorphisms in Mustelidae for evolving anticoagulant resistance through selection along the food chain.

In response to strong selection, new mutations can arise quickly and sweep through populations, particularly, if survival and reproduction depend on certain allele copies for adaptation to rapidly changing environments, like resistance against deadly diseases or strong toxins. Since the 1950s, resistance to anticoagulant rodenticides in several rodents has emerged through single nucleotide mutations in the vitamin-K-epoxid-reductase-complex-subunit-1 (VKORC1) gene, often located in its exon 3. Detection of high prevalence and concentrations of anticoagulant rodenticides in non-target vertebrates, including carnivorous Mustelidae, let us assume that secondary exposure by feeding on poisoned prey may also cause selection along the food chain and we hypothesized that VKORC1-based resistance might also have evolved in rodents’ predators. Using newly-developed mustelid-specific primers for direct sequencing of genomic DNA, we studied VKORC1-DNA-polymorphisms in 115 mustelids of five species (Martes martes, M. foina, Mustela nivalis, M. erminea, M. putorius), obtained from northern Denmark, yielding six sites with nonsynonymous and several synonymous amino acid polymorphisms in exon 3. Comparison of these VKORC1-genotypes with hepatic rodenticide residues (obtained by HPLC combined with fluorescence or mass spectrometry) in 83 individuals (except M. martes), using generalized linear models, suggested that anticoagulant levels depended on species and specific polymorphisms. Although most VKORC-1 polymorphisms may present standing genetic variation, some are situated in resistance-mediating membrane parts of the VKORC1-encoded protein, and might be a result of selection due to exposure to anticoagulant poisons. Our new molecular markers might allow detecting indirect effects of anticoagulant rodenticides on rodent predator populations in the future.

Determination of association between the polymorphism in exon 3 of dopamine receptor gene type 4 with attention deficit-hyperactivity disorder

IntroductionEvidences suggest that attention deficit-hyperactivity disorder (ADHD) is a hereditary disorder and at least 20 potential genes associated with ADHD have been identified. Dopamine receptor gene type 4 (DRD4) has been more considered due to a stronger relationship with ADHD. However, no study has yet been conducted on the Iranian population to assess the association. ObjectiveIn this study, the association between polymorphism of DRD4 gene with ADHD has been studied among capital of Iran population. Materials and methodsThis study is a case-control study conducted on children aged 6–12 years with ADHD referred to child and adolescent psychiatric clinic Imam Hussein (AS) and normal subjects in 2011. Diagnosis was done based on the DSM-IV-TR criteria and interviewing by two child and adolescent psychiatrists. If parental were consent, then saliva samples of subjects were prepared and DRD4 gene and related allele were evaluated using PCR method. The K-SADS questionnaire was also used to assess comorbid disorders. ResultsIn this study, 114 patients in ADHD group and 109 patients in the control group were studied. The most frequency was obtained for allele 4 allele that has been observed in about 90% of both case and control groups. However, frequency of allele 6 in the case group was 8.8% where the frequency was 5% in the control group (p = 0.02). The presence of repeat of allele 6 increased chance of suffering from ADHD to 1.809 (95% equal to 3.871–0.845). ConclusionFor the first time this study showed that in Iranian population repeat of DRD4 gene allele 6 unlike the other geographic areas is relatively common and it will increase the chances of suffering from ADHD. However, additional studies are required.

Polymorphisms of the melatonin receptor 1A gene that affects the reproductive seasonality and litter size in Small Tail Han sheep.

Previous researches have shown that MTNR1A plays an essential role in sheep reproduction. However, most researches focused more on the reproductive seasonality of sheep, and few scientists had studied the association of polymorphisms of the MTNR1A gene with ovine litter size and reproductive seasonality. Therefore, we chose MTNR1A gene to detect its novel sequence polymorphisms and population genetics and analyse their association with seasonal reproduction and litter size in ewes. The mRNA expression level in hypothalamus, pituitary and ovary was also detected. In this study, five polymorphisms (g.15118664G > T, g.15118683C > T, g.15118756C > T, g.15118774C > T and g.15118951G > A) were identified in exon 2. Most importantly, the g.15118683C > T and g.15118951G > A were significant difference between year‐round oestrous sheep and seasonal oestrous sheep (p < .01), and g.15118756C > T had a great effect on litter size of Small Tail Han sheep (p < .05). In addition, the mRNA expression level of MTNR1A in the hypothalamus of polytocous Small Tail Han sheep was significantly higher than that in monotocous Small Tail Han sheep (p < .01) and the expression of MTNR1A in the hypothalamus of year‐round oestrous sheep was significantly higher than that in seasonal oestrous sheep (p < .01). Polymorphisms in exon 2 may regulate the reproductive seasonality and litter size of ewes by influencing gene expression to regulate the reproductive seasonality and litter size of ewes. Our studies provided useful guidance in marker‐assisted selection of the litter size in Small Tail Han sheep.