Polymorphism detection of <i>DGAT1</i> and <i>Lep</i> genes in Anatolian water buffalo (<i>Bubalus bubalis</i>) populations in Turkey

Acyl-CoA: diacylglycerol–acyltransferase 1 (DGAT1) enzyme plays a key role in controlling the synthesis rate triglyceride from diacylglycerol. Leptin (LP, OB, obese) is an important hormone that synthesizes mostly from adipose tissue and regulates glucose metabolism and homeostasis. DGAT1 and Lep genes are closely related to reproduction, growth, milk yield and composition in water buffalo breeds. This study aimed to identify genetic variation in the DGAT1 and Lep gene regions in 150 water buffalo individuals from five different provinces of Turkey using DNA sequencing. A total of 38 nucleotide variations and indels have identified 761 bp long partial intron 2 and exon 3 and 5′ UTR regions of the Lep gene in Anatolian water buffalo populations; 422 bp long partial exon 7–9 and exon 8 regions of DGAT1 gene were amplified and two mutations were defined in the point of 155 and 275 nucleotide that is three genotypes for S allele and Y allele of DGAT1 gene in intron 7 in Anatolian buffalo populations, respectively. These SNPs may have an effect on reproduction, growth, milk yield and composition in water buffalo populations and may prove to be useful for water buffalo breeding.

GHR/HindIII Locus Polymorphisms in Intron-2 GHR Gene of Papua Local Chicken

This study aimed to detect single nucleotide polymorphisms (SNPs) in intron-2 on growth hormone receptor (GHR) gene in Papua local chickens using the PCR-RFLP method to study its relationship with growth characteristics. Data on the bodyweight of 49 chickens aged 1, 2, 3, and 4 months (22 males, 27 females) and DNA samples were used for this study. The DNA fragment of size 718 bp in intron-2 of the GHR gene from the study chicken was successfully amplified using a pair of specific primers. The PCR-RFLP/HindIII analysis results found this locus's two genotypes (HindIII++ and HindIII--). HindIII+ and HindIII- alleles were 0.02 and 0.98, respectively.

RHD Genotypes in a Chinese Cohort of Pregnant Women

RHD variants in D¯ Chinese pregnant women arose difficulties in management during pregnancy. Therefore, this study aims to precisely manage D¯ pregnant women by evaluating the spectrum of RHD mutations in D¯ pregnant women and getting insight into the possible rare alleles of RHD. A total of 76 D¯ pregnant women were analyzed by performing polymerase chain reactions with sequence-specific primers (PCR-SSP), the 10 RHD exons Sanger sequencing, RHD zygosity detection, and mRNA sequencing (mRNA-seq). About 40% of alleles are variations of RHD, including RHD 1227A homozygous, RHD-CE(2-9)-D, et al. Therefore, we developed a molecular diagnostic strategy for Chinese women, and most D¯ pregnant women can be diagnosed with this simple decision tree. After RHD genotyping for D¯ pregnancy women, we eliminated at least 15% unnecessary ante- and postpartum injections of Rh immunoglobulin (RhIG). As the first pedigree study and the first functional analysis under physiological conditions, mRNA-seq revealed that c.336-1G&gt;A mutation mainly led to the inclusion of the intron 2, which indirectly explained the D¯ phenotype in this family. We also developed a robust protocol for determining fetal RhD status from maternal plasma. All 31 fetuses were predicted as RhD positive and confirmed the RhD status after birth.

Sudden infant death syndrome revisited: serotonin transporter gene, polymorphisms and promoter methylation

Background Based on findings in the brain stems of SIDS victims, the serotonin transporter (5-HTT) gene has been discussed to be associated with SIDS. Methods In the largest study to date, we investigated the promoter length (5-HTTLPR) and intron 2 VNTR polymorphisms in 274 cases and 264 controls and the Ile425Val polymorphism in 65 cases and 64 controls. Moreover, the methylation of the internal promoter region was investigated in 35 cases and 14 controls. Results For 5-HTTLPR, we observed a trend towards an association of allele L (58.8% vs. 53.4%) with SIDS and significant results were observed after stratifying for age, season at death, and prone position. Nevertheless, when pooling all published data, a significant association of allele L with SIDS is confirmed (p: 0.001). For the intron 2 VNTR polymorphism, no significant differences were observed. After pooling, a significant accumulation of the rare allele 9 was observed in SIDS (2.1% vs. 0.6%; p: 0.018). For the Ile425Val polymorphism, no differences were observed. Conclusion We conclude that genetic variation at this gene might be of some importance in SIDS. Epigenetic analysis of the internal promoter, however, revealed no influence on the relative risk to succumb to SIDS. Impact This is the largest study published up to now on 5-HTT gene polymorphisms and SIDS. Polymorphisms in the 5-HTT gene appear to contribute (although to a small degree) to the risk to die from SIDS. There is no evidence that a methylation of the promoter region is of impact for the etiology of SIDS.

Pklr Is a Genetic Modifier of Sickle Cell Disease

Background: Acute pain, the most prominent complication of sickle cell disease (SCD), results from vasoocclusion triggered by sickling of deoxygenated red blood cells (RBCs). A key factor influencing HbS oxygenation is the intracellular concentration of 2,3- diphosphoglycerate (2,3-DPG). 2,3-DPG, an intermediate substrate in the glycolytic pathway, decreases oxygen binding and stabilizes the deoxygenated hemoglobin. Pyruvate kinase (gene PKLR, protein PKR) is a rate-limiting enzyme in glycolysis; variants in PKLR may affect PKR activity, 2,3-DPG levels in RBCs, subsequent frequency of sickling and acute pain episodes (APE). There is thus a strong biological basis for exploring PKLR as a candidate gene affecting acute pain in SCD. Methods: The study population for genetic association consists of 2 cohorts: 1) 242 adults with HbSS from King's College Hospital (KCH), London, UK, with complete hospitalisation records over 10 years (2004-2013 inclusive) as the "discovery" cohort; 2) 977 children with HbSS or HbSb 0 thalassemia from the Silent Infarct Transfusion (SIT) trial, with a 3-year history of severe vasoocclusive pain based on hospitalization, as the "validation" cohort. Both studies were approved by the local Institutional Review Boards at KCH and Vanderbilt University Medical Center, respectively. An independent cohort comprises 52 adults with SCD enrolled under 3 protocols - NCT00011648, NCT00081523, and NCT03685721 - approved by the NHLBI Review Board (NIH), for evaluation of imbalance in allele expression. Genome scan for the KCH cohort was performed using llumina's Infinium "MEGA" chip (1.7m markers). The SIT DNA samples were genotyped using Illumina HumanHap650Y array 5 (661K markers) or Illumina Infinium HumanOmni1-Quad array (1.1m markers). The results were quality controlled followed by genotype imputation based on the 1000 Genomes Project phase 3 data. An annualised "hospitalisation rate" as a measure of pain incidence rate, was calculated by dividing the number of hospital admissions for severe acute pain by the number of years of observation for KCH and SIT cohorts (Fig A). We performed association analysis with common SNPs at PKLR locus using data from our genome-wide SNP set and a linear mixed modelling approach incorporating a genetic relatedness matrix to take account of relatedness, plus sex and age as fixed covariates. We corrected for multiple testing after quantifying the linkage disequilibrium (LD) within PKLR and used this to calculate appropriate significance levels. For the PKLR region and hospitalisation rate, the modified significance level was p&lt;0.001268 for the discovery (KCH) cohort. For the allele expression assays, a synonymous variant, rs1052176 (R596R), in exon 11 of PKLR acted as a marker of relative expression levels of the 2 alleles of the gene. Allele specific expression was carried using the Bio-Rad digital droplet PCR system. Results: 7 of 47 variants evaluated in PKLR were associated with hospitalization rate (LnLnHospRate) in the discovery cohort: intron 4 - rs071053, and intron 2 - rs8177970, rs116244351, rs114455416, rs12741350, rs3020781, and rs8177964). All 7 were validated in Fisher's meta-analyses of the KCH and the SIT cohorts using p&lt;0.0071 as threshold to correct for multiple testing (Fig B). We examined the pairwise LD between PKLR variants, and found all the intron 2 variants in tight LD, while R596R belongs to another LD block (Fig C). 52 SCD individuals had the R596R variant, of which 29 were heterozygous and 23 homozygous for the intron 2 haplotype associated with APE in SCD. We performed a Wilcoxon rank sum test and compared the variation in PKLR expression between the 2 alleles in subjects homozygous and heterozygous for the wildtype intron 2 haplotype, using genomic DNA as internal control for each subject. The results reveal a significant deviation from the expected expression ratio in those heterozygous for the intron 2 haplotype (mean 0.2073, +/- SD 0.0135) when compared with to those without the variant (mean 0.1239, +/- SD 0.0682), p=0.0297 (Fig D). Conclusion: Intronic variants of PKLR are associated with hospitalization rate for acute pain episodes in adults and children with SCD. We show that the intronic variants are likely to influence acute pain by affecting expression of the PKLR gene using allele-specific expression analyses, although the causal variant is unclear. These results support PKLR as a genetic modifier of SCD. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Converging evidence for differential regulatory control of APOEε4 on African versus European haplotypes

INTRODUCTION. The difference in APOEϵ4 risk for Alzheimer disease (AD) between different populations is associated with APOEϵ4 local ancestry (LA). We examined LA SNPs with significant frequency differences between African and European/Japanese APOEϵ4 haplotypes for areas of differential regulation. METHODS. We performed two enhancer Massively Parallel Reporter Assay (MPRA) approaches, supplemented with single fragment reporter assays. We utilized Capture C analyses to support interactions with the APOE promoter. RESULTS. The TOMM40 intron 2 and 3 region showed increased enhancer activity in the European/Japanese versus African LA haplotypes in astrocytes and microglia. This region overlaps with APOE promoter interactions as assessed by Capture C analysis. Single variant analyses pinpoints rs2075650/rs157581, and rs59007384 as functionally different on these haplotypes. DISCUSSION. Both differential regulatory function and Capture C data support an intronic region in TOMM40 as contributing to the differential APOE expression between African and European/Japanese LA.

Molecular Analysis of STin2 (Intron 2) Variant of The SLC6A4 Gene in Children and Adolescents With Attention Deficit Hyperactivity Disorder

Background Attention deficit hyperactivity disorder (ADHD) is recognized as one of the most familiar childhood psychiatric disorders. Many molecular genetic reviews suggest that genes play a crucial role in susceptibility to ADHD. The serotonin transporter gene (SLC6A4) has polymorphisms that seem to correlate with ADHD development. The association between ADHD and the SLC6A4 gene variants in the Iranian population has not been investigated yet. This study analyzes the STin2 (intron 2) variant of the SLC6A4 gene in Iranian children and adolescents with ADHD . Materials and Methods In this retrospective case-control study, 86 ADHD patients and 99 healthy volunteers aged 5 to 14 years old were enrolled as the case group and the control group, respectively. The STin2 (intron2) fragment of the SLC6A4 gene was amplified using specific primers by conventional PCR, and three STin2 alleles of the SLC6A4 gene (STin2.9, STin2.10, and STin2.12) were examined using the acrylamide gel method. Results We found no significant difference between the ADHD and the control groups in STin2.9(34.9% vs 39.4%, p-value = 0.824), STin2.10(29.1% vs 23.2%, p-value = 1.354), and STin2.12(36% vs 36.4%, p-value = 0.986) variants. Conclusion It is concluded that there was no association between the frequency of STin2 variant alleles of the SLC6A4 gene andADHD, but in the study of risk estimation, it was found that allele 10 of this variant is a risk allele in ADHD patients.

Avian Beta Defensin 2 (AvBD2) Gene Polymorphism Identification in IPB-D1 Chicken

<p class="abstrak3">Avian Beta Defensin 2 (AvBD2) gene, which is located in chromosome 3, plays an important role in the immune system of the chicken by inhibiting the development of microorganisms such as bacteria that infect body tissues. Defensins are produced through epithelial cells immediately after tissue injury or infection, which then processes the maturation of dendritic cells to initiate an immune response in the lymph nodes. The purpose of this study was to discover the polymorphism of the AvBD2 gene in IPB-D1 chickens. PCR and direct-DNA sequencing methods were used to identify the diversity of intron 1, exon 2, and intron 2 AvDB2 genes in 47 chickens. Genotype and allele frequency, and heterozygosity calculations were carried out to obtain information of the AvBD2 gene polymorphism. A total of 10 single nucleotide polymorphisms were found in the AvBD2 gene located in intron 1 (g.4843T&gt;A, g.4853G&gt;A, and g.4859T&gt;C), exon 2 (g.4881A&gt;G, g.4889G&gt;A, and g.5002C&gt;T), and intron 2 (g.5075C&gt;T, g.5111T&gt;G, g.5116G&gt;T, and g.5177G&gt;T). All SNPs are polymorphic. The g.5002C&gt;T mutation causes changes in the amino acid Ala to Val which has the potential to be a candidate for characterizing disease resistance in IPB-D1 chickens.</p>