Single-strand Conformation Polymorphism Research Articles

Ovine KRT81 Variants and Their Influence on Selected Wool Traits of Commercial Value

Keratins are the main structural protein components of wool fibres, and variation in them and their genes (KRTs) is thought to influence wool structure and characteristics. The PCR–single strand conformation polymorphism technique has been used previously to investigate genetic variation in selected coding and intron regions of the type II sheep keratin gene KRT81, but no variation was identified. In this study, we used the same technique to explore the 5′ untranslated region of KRT81 and detected three sequence variants (A, B and C) that contain four single nucleotide polymorphisms. Among the 389 Merino × Southdown cross sheep investigated, variant B was linked to a reduction in clean fleece weight, while C was associated with an increase in both greasy fleece weight and clean fleece weight. No discernible effects on staple length or mean-fibre-diameter-related traits were observed. These findings suggest that variation in ovine KRT81 might influence wool growth by changing the density of wool follicles in the skin, the density of individual fibres, or the area of the skin producing fibre, as opposed to changing the rate of extrusion of fibres or their diameter.

Four microRNA gene polymorphisms are associated with Iraqi patients with colorectal cancer

BackgroundColorectal cancer (CRC) is a major cause to global cancer-related mortality. The development of colorectal cancer is linked to hereditary variables that exhibit variability.ObjectivesThe objective of this investigation was to assess the potential correlation between microRNA gene polymorphisms and colorectal cancer (CRC) throughout the Iraqi population.MethodsDNA samples were obtained from a cohort of 100 individuals diagnosed with the (CRC) disease, as well as 100 samples as control group. Four primers were designed to amplify four specific high-frequency variants found within microRNA molecules. These variants include Mir146a G\\C, Mir423 A\\C, Mir196a2, and Mir370. The genotyping of the PCR fragments was performed using the single-strand conformation polymorphism (SSCP) method, followed by direct sequencing of each genotype.ResultsGenotyping experiments confirmed the variability of four targeted variants, namely Mir146a G\\C, Mir 423 A\\C, Mir196a2, and Mir370 tend to exhibit a significant association with (CRC). Individuals with Mir146a: GC and Mir 423 A\\C genotype showed a possible association with the increased risk of (CRC), respectively (P = 0.001; OD 0.50; CI 95% 0.33–0.76; P = 0.002; OD 0.53; CI 95% 0.36–0.80). Individuals with Mir196a2: TT and Mir370 GG genotype exhibited a potential association with (CRC) (P = 0.017; OD 0.44; CI 95% 0.22–0.86; P ≤ 0.001; OD 0.24; CI 95% 0.11–0.50).ConclusionsThe study reveals that single nucleotide polymorphisms (SNPs) in microRNA have a notable and distinct correlation with the heightened susceptibility to colorectal cancer (CRC).

FBXW7 polymorphism asserts susceptibility to colorectal cancer

FBXW7, belonging to the F-Box protein family, is considered a candidate cancer susceptibility gene. Our findings indicate that single nucleotide polymorphisms (SNPs) in the FBXW7 gene are linked to cancer risk, strengthening FBXW7′s role in the pathogenesis of colorectal cancer. Our case-control study comprised of 450 patients diagnosed with colorectal cancer (CRC) and an equal number of 450 healthy subjects. FBXW7 SNPs rs2255137C>T and rs6842544C>T were genotyped using PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) and Single-Stranded Conformation Polymorphism (SSCP) techniques and further cross-checked by direct sequencing. Linkage disequilibrium and haplotype analyses of these SNPs were also assessed. The in-silico approach was used to reveal the functional analysis between the nonsynonymous variation (rs6842544) and CRC followed by its validation at the protein level by western blotting and reverse transcription-PCR. A significant association of colorectal cancer was detected with rs6842544 SNP. However, there was no association between FBXW7 rs2255137 polymorphism and CRC. The homozygous individuals carrying the C variant in FBXW7 rs6842544 showed a slightly higher risk for colorectal cancer (OR = 1.590, 95%CI = 0.39 ∼ 2.89, p = 0.011). The haplotype CC identified in this study seemed to be associated with good prognosis (OR = 1.22, 95% CI = 1.00 ∼ 1.47, p = 0.0013) whereas the TT haplotype was found to reduce the CRC risk (OR = 0.642, 95%CI = 0.48 ∼ 0.84, p = 0.039). In-silico prediction proposed that the variant R133G is responsible for the lower expression of FBXW7. Additionally, the expression profiling of FBXW7 nonsynonymous SNP was significantly lower in primary CRC tissues than in the paired non-cancerous tissues at protein and mRNA levels. The study indicates that the FBXW7 rs6842544 is associated with the risk of development of CRC and could serve as a molecular biological marker to screen high-risk groups for CRC.

Genetic variants in the TAC1 transcriptional regulatory region affect on trainability and excitability levels in Belgian Malinois dogs.

Trainability in dogs is affected by learning aptitude and memory capacity. While this trait has a heritable basis in canines, the specific genetic loci responsible for it remain unknown. Our previous results suggested that the BDNF, CCK and TAC1 genes are associated with learning and memory in canines. Experimental validation is crucial to confirm the effects of these candidate genes on trainability. Understanding the genetic foundation of this trait would offer insight into the inheritance pattern of complex behavioural characteristics. This study aimed to assess the genetic variations within candidate genes and explore their potential associations with behavioural phenotypes in dogs. The behavioural characteristics of 123 male Belgian Malinois dogs were assessed using a customised questionnaire. Target regions of candidate genes were screened for genetic variation by single-strand conformational polymorphism (SSCP). Following that, SSCP banding patterns were sequenced, and putative transcription factor binding sites were predicted using bioinformatics tools. Quantitative association analysis between identified genetic variants and behavioural trait scores was performed using the general linear model (GLM). Sequencing the coding and flanking regions revealed three mutations (c.-89C>T, c.-162G>C and c.*33T>A) in the dog TAC1 gene. Bioinformatics analysis predicted two single nucleotide polymorphisms (SNPs) were located within the putative TAC1 promoter and could disrupt transcription factor binding sites. Statistical tests revealed that the c.-89C>T was significantly associated with excitability (p < 0.01), while the c.-162G>C was significantly associated with trainability level (p < 0.05). In summary, we identified two regulatory SNPs in the 5'-UTR promoter region of the TAC1 gene that are associated with excitability and trainability in Belgian Malinois dogs. These genetic variations have the potential to alter the binding sites of transcription factors NRF1 and OTX1, consequently influencing TAC1 expression and related behavioural characteristics. Our findings implicate TAC1 polymorphisms as candidates influencing breed-specific behavioural characteristics in canines. Further studies on diverse breeds of dogs are necessary to validate these SNPs' effects.

Correlation analysis between polymorphism of leptin and IGFI genes and body measurements in Barki and Farafra sheep

BackgroundGrowth performance, which can be evaluated using various body measurements, is crucial for providing red meat for human consumption. This study aims to improve the growth performance of sheep by identifying the different genotypes of leptin and IGFI genes associated with higher body measurements.MethodsIn this study, we collected blood samples from Barki (n = 30) and Farafra (n = 30) sheep, and measured their body height, width, length, and weight, and tail width and length to assess their growth performance. We used single strand conformation polymorphism polymerase chain reaction (SSCP-PCR) and sequencing analysis to identify single nucleotide polymorphisms (SNPs) and different genotypes of the leptin and insulin-like growth factor receptor (IGFI) genes associated with sheep body measurements.ResultsThe leptin gene (209 bp) and IGFR gene (345 bp) were found to have three (TT, TG, and GG) and (TT, TC, and CC) genotypes, respectively, by SSCP-PCR. Sequence analysis revealed a SNP in the leptin gene (200G > T) with two alleles (G allele: OR058867 and T allele: OR058868). This SNP resulted in the substitution of the amino acid valine with leucine, which increased the width, length, and weight of the body and tail width in the heterozygous form (TG). The IGFI gene had three SNPs: 305 G > T in TT genotype (OR260670), 184C > T, 265T > C, and 305G > T in TC genotype (OR260669), and 265T > C in CC genotype (OR260671). The TC genotype of the IGFR gene was correlated with higher values of body measurements. Barki sheep were found to have the highest values for body weight, width, length, and tail width compared to Farafra sheep.ConclusionWe recommend the use of entire Barki sheep in inbreeding programs to improve growth performance in Egyptian sheep.

In Silico Analysis of Functional SNPs in Genes of Complete Androgen Insensitivity Syndrome (CAIS): A Retrospective, Case-Control Study.

Complete androgen insensitivity syndrome (CAIS) is one of the categories of androgen insensitivity syndrome (AIS) described as complete failure of the cell to react to androgens with external genitalia of a normal female. People with AIS condition are genetically male, with XY karyotype in each cell, but their bodies are unable to respond to male sex hormones (called androgens). It is associated with infertility as well as developing cancerous conditions. The genetic association of CAIS involves polymorphism of genes such as NR5A1, SOX9, SRD5A2, CBX2, GATA4, and SRY. Their mutation and participation in genetics of CAIS can be studied by Single Nucleotide polymorphism (SNP) analysis which is a way to detect genetic variations. SNP in coding region leads to synonymous and non-synonymous mutations. Hence, this study highlights analysis of SNPs associated with CAIS. Our aim is to study SNP analysis of NR5A1, SOX9, SRD5A2, CBX2, GATA4, SRY genes in Complete Androgen Insensitivity Syndrome. SIFT and Polyphen analysis was performed for all the genes and samples were subjected for PCR-SSCP technique. SNPs were analyzed for the genes associated with CAIS. Benign and damaging SNPs were identified. DNA Samples were amplified using PCR technique and they will be analyzed using Single-strand conformation polymorphism (SSCP). As SNPs have decreased stability, damaging and benign character, they can be used as candidate hallmarks in study of Complete Androgen Insensitivity Syndrome.

Regional bias of tumor suppressor gene mutations of STARD8 and WNK2 in colon cancers

StAR-related lipid transfer domain protein 8 (STARD8), encoding a Rho-GTPase-activating protein, and WNK2, encoding a serine/threonine kinase are candidate tumor suppressor genes (TSGs) in human cancers. Inactivation of these genes that would promote cancer pathogenesis is largely unknown in colon cancer (CC). Our study addressed to address whether STARD8 and WNK2 genes are mutated in CC. STARD8 and WNK2 genes possess mononucleotide repeats in their exons, which could be the targets for frameshift mutations in cancers with high microsatellite instability (MSI-H). By single-strand conformation polymorphism (SSCP) analysis, we analyzed the repeated sequences in 140 CCs (95 CCs with MSI-H and 45 CCs with stable MSI (MSS)). By DNA sequencing, we found that five MSI-H CCs (5/95: 5.3%) harbored the frameshift mutations, whereas MSS CCs (0/45) did not. In addition, we detected regional heterogeneous frameshift mutations of these genes in four (25%) of 16 MSI-H CCs. In immunohistochemistry for WNK2, WNK2 expression in the MSI-H CCs was significantly lower than that in the MSS CCs. Our results for the mutation and expression indicate that STARD8 and WNK2 genes are altered at various levels (frameshift mutation, expression, and regional heterogeneity) in MSI-H CCs, which might play a role in the pathogenesis by inactivating their TSG functions.

Genetic variations of α-casein and K-casein genes associated with milk composition in goats

The quality of animal milk varies between individuals of the same breed. The present study aimed to detect the favorable SNPs for α-casein (CSN1S1) and k-casein (CSN3) genes associated with high-quality milk traits. Blood and milk samples of Zaraibi and Damascus goats were collected from Sakha farm in Kafr El-Shiekh. The solid non-fat, lactose, protein, and fat contents, were determined in milk using the biochemical method, and amino acids were determined using HPLC. To identify genotypes and nucleotide variations (SNPs), single-strand conformation polymorphism (SSCP) and sequencing methods were used. One-way ANOVA test was used to determine the effect of different genotypes on milk traits. The SSCP-PCR results in Zaraibi and Damascus goats showed that the CSN1S1 gene has 3 genotypes (AA, AB, and BB), and also the CSN3 gene has 3 genotypes (AA, AG, and GG). The results of sequencing showed three SNPs at positions 41G˂T, 69A˂G, and 80C˂T in the CSN1S1 gene, and three SNPs at positions 49C˂T, 111A˂G, and 340C˂T in the CSN3 gene. The milk results showed that the high protein, fat, and lactose content were associated with the AB genotype in the CSN1S1 gene while they were associated with the AG genotype in the CSN3 gene. Likewise, essential and branched-chain amino acids were increased in goat milk with the AB and AG genotypes of CSN1S1 and CSN3 genes, respectively. The essential amino acids for protein synthase were higher in Zaraibi than in Damascus goats. Therefore, the Zaraibi goats may more possess genotypes in the CSN1S1 and CSN3 genes, which affect the fat and protein level in milk.

Ovine KRTAP36-2: A New Keratin-Associated Protein Gene Related to Variation in Wool Yield.

Keratin-associated proteins (KAPs) are structural components of wool fibres. High-glycine/tyrosine (HGT)-KAPs are a subset of the KAP family, and their abundance in fibres varies. In this study, we report the discovery of an ovine HGT-KAP gene to which we assigned the name KRTAP36-2. Polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analyses revealed four variants of this gene in a screening population of 170 sheep from a variety of breeds. The DNA sequencing of the variants revealed four single-nucleotide polymorphisms (SNPs) and a dinucleotide deletion. Three of these SNPs were in the coding region, and one of these was non-synonymous and potentially led to the amino acid substitution p.Cys27Gly near the middle of the protein. The remaining SNP was located near the putative TATA box, and the di-nucleotide deletion was near the putative transcription initiation site. The effect of this variation in KRTAP36-2 was investigated in 274 Southdown × Merino lambs that were the progeny of five sires. Variation was only found to be associated with wool yield, that is, the proportion of the greasy fleece that remained as clean fleece upon scouring (expressed as a percentage). This may have some value in increasing wool production.

Transmission of isolates of Citrus tristeza virus by single aphids (Toxoptera citricida)

Citrus tristeza virus (CTV) can be transmitted by several aphid species in a semi-persistent mode, with Toxoptera citricida being the most efficient vector. In Brazil, mild CTV isolates are used for pre-immunization of citrus trees against severe isolates. We aimed to determine the capacity and efficiency of T. citricida in separating the viral complex into haplotypes from three well-characterized CTV isolates (PIAC, CS1, and Forte Rolândia). Single-aphid transmission assays were conducted to determine CTV transmission efficiency. The results showed that T. citricida transmitted only haplotypes from the PIAC and CS1 isolates, with efficiencies of 8 and 4%, respectively. Both isolates caused mild CTV symptoms in Brazil. However, isolate Forte Rolândia, which causes severe symptoms in citrus trees, was not transmitted by T. citricida. The detection of CTV haplotypes from PIAC and CS1 isolates in sweet orange (Citrus sinensis) plants after a single aphid transmission occurred at different time points. The first haplotype observed was from PIAC 150 days after citrus was challenged by a single aphid, followed by CS1 at day 210 after transmission. In addition, differences in the single-strand conformation polymorphism patterns between the CTV isolates and CTV haplotypes were determined, suggesting that an aphid can acquire and transmit only one CTV haplotype to citrus plants during phloem feeding. The study of the mechanism of transmission by the vector can increase our knowledge of the interactions among hosts, vectors, and pathogens, which are often neglected.

Analysis of genetic polymorphism in genes encoding cathelicidins from Vietnam indigenous yellow cattle breeds

Cathelicidins include antimicrobial peptides (AMPs) and are involved in the innate immune defense against infections in mammals. Polymorphisms in the DNA sequence of cathelicidin genes could be relevant to inherited variations of host innate immunity, adaptation, and pathogen resistance. This study aims to investigate the sequence polymorphism of cathelicidin genes including CATHL2 and CATHL4 from local indigenous yellow cattle breeds of Vietnam. Genomic DNA samples were extracted from 52 individuals collected from different cattle populations in Vietnam including Ha Giang, Thanh Hoa, Nghe An and Phu Yen. The CATHL2 and CATHL4 genes were amplified by PCR, following sequencing for identification of the single nucleotide polymorphisms (SNPs) and/or insertion-deletion (indel) DNA sequence variations. The copy number variations (CNVs) of the CATHL4 gene were determined by cloning, single strand conformation polymorphism (SSCP) and real-time PCR. The comparative analysis results showed that there are 13 SNPs detected in all sequences of CATHL2 gene including 9 SNPs in intron 2 and intron 3, and 4 missense substitutions in exon 1, 2, and 3. These SNPs do not affect the predicted tertiary structure of Bac5 encoded by this gene. For CATHL4, the results revealed 15 SNPs, 3 indels and 1 repeat variation as motif (TG)n(G)n/(AC)n(C)n. Among these variations, the deletion 46delC in exon 1 of CATHL4 was detected in a sample of the Ha Giang breed causing a truncated polypeptide as predicted. The average copy number of CATHL4 of Vietnam indigenous cattle breeds using GADPH as a reference gene showed in range from 2.53 to 2.89 copies.

Monitoring Drought Tolerance in Oil Palm: Choline Monooxygenase as a Novel Molecular Marker.

Water scarcity negatively impacts oil palm production, necessitating the development of drought-tolerant varieties. This study aimed to develop molecular markers for oil palm breeding programs focused on drought tolerance. Genes associated with drought tolerance were selected, and single nucleotide polymorphism (SNP)-based markers were developed. Genomic DNA was successfully extracted from 17 oil palm varieties, and 20 primers out of 44 were effectively amplified. Screening with single-strand conformation polymorphism (SSCP) revealed an informative SNP marker from the choline monooxygenase (CMO) gene, exhibiting CC, CT, and TT genotypes. Notably, the oil palm variety La Mé showed the CT genotype, while Surat Thani 2 (Deli × La Mé) exhibited the CT and CC genotypes in a 1:1 ratio. Gene expression analysis confirmed the association of the CMO gene with drought tolerance in commercial oil palm varieties. The full-length CMO gene was 1308 bp long and shared sequence similarities with other plant species. However, amino acid sequence variations were observed compared with existing databases. These findings highlight the potential utility of the CMO marker for drought tolerance selection, specifically within the La Mé parent of oil palm Surat Thani 2 varieties, and strongly confirm the La Mé S5 population and Surat Thani 2 as drought-tolerant varieties.

Association Between of the Growth Hormone Gene GH-1 with Growth Indicators of Common Carp Cyprinus carpio L. in Three Different Ecosystems

This study was conducted in the central of health laboratory/Animal and Fish Resource Research Center, Agricultural Research Department, Ministry of Science and Technology, for the period from 15/8/2021 to 15/11/2021 in order to investigate the polymorphism of the growth hormone gene GH-1 and its relationship to the productive performance (body weight, total length, age and case factor) of common carp Cyprinus carpio, in 90 fish representing three different ecosystems (30 fish for each site) including floating cages, ponds, and the environment of the Tigris River, south of Baghdad, with an average weight of (975-1517±150) g. Some physical and chemical measurements of water were measured, including temperature, dissolved oxygen concentration, pH, and salinity. These fish were transferred to the laboratory, and the weight, total length, and age of each fish were measured, as well as DNA extraction for the purpose of amplifying the size of a segment (459pb) of the growth hormone gene that includes exon 4, intron 4, and exon 5 by polymerase chain reaction PCR using primers designed for this gene. The genotypes of the growth hormone gene were investigated using the Single-Stranded Conformation Polymorphism (SSCP) technique, and the produced bands were compared to distinguish different genotypes of the gene by the electrophoresis method. The results showed that the water temperature was close to the environmental systems under study and ranged between (25.4-27 ± 0.50), the rates of dissolved oxygen concentration were (6.5 ± 0.09) mg/l, pH (7.0 ± 0.02) and salinity 0.8 g/l higher in ponds compared to the lowest value of them in the river. The analysis of the SSCP technique revealed the presence of six different genotypes of this hormone, including A, B, C, E, F, and H with a frequency of 24.4, 5.6, 15.6, 15.6, 16.4, and 22.4, respectively, for the fish community in the studied sites. The H genotype was significantly (P< 0.05) superior in the floating cages in the average body weight (1600 ± 0 g) and the relative case coefficient (2.5 ± 0). significant differences were observed in the average total body length and age, according to the multiplicity of genotypes. We conclude from this study an improvement in the growth and health status of common carp in cages and ponds compared to the river at the probability level (P<0.05) and the possibility of focusing on the polygenic genotypes of the growth hormone gene and adopting the H genotype as a guide for selecting fish mothers in genetic improvement programs for artificial propagation and achieving the largest economic return from fish breeding projects.

Screening for causative mutations in ovine BMPR1B and BMP15 genes and their homologous fragments in human.

The BMPR1B and BMP15 genes are well known for their considerable associations with prolificacy in sheep. These genes may also affect fertility or prolificacy in other species, including human. This study was conducted to investigate possible causative mutations in BMPR1B and BMP15 genes in human and an indigenous breed of sheep. Blood samples were collected from 83 singleton- and prolific Mehraban ewes and 81 infertile, singleton- and twin-bearing women. A 190-bp fragment, containing the FecB mutation in ovine BMPR1B, a 380-bp fragment in ovine BMP15 gene and their homologous fragments in human were amplified and then investigated by single-stranded conformation polymorphism and DNA sequencing methods. The FecB mutation of BMPR1B (g.159A>G) was detected in the sheep population, but no polymorphic loci were found in the homologous fragment in studied human samples. The studied fragments of BMP15 were monomorphic in both sheep and human samples. A total of nine and 69 point-differences in the studied fragments of BMPR1B and BMP15 genes were detected between the species, respectively. In sheep, the G allele of BMPR1B had a positive effect on litter size (p<0.05), whereby all AG or GG ewes were prolific. The FecB mutation for the first time was detected in Mehraban sheep and therefore could be considered for marker-assisted selection in this breed. The studied fragments of BMPR1B and BMP15 genes are not responsible for reproduction variation in human. More studies on other genes, associated with fertility in human, are necessary in the future.

Effect of genetic polymorphisms in LALBA and Prolactin genes on milk traits in Barki ewes

This study investigated the genetic polymorphisms in Alpha-lactalbumin (LALBA) and Prolactin (PRL) genes in the Egyptian Barki sheep. The investigated genes were previously identified to be associated with milk and reproductive traits in several livestock species. Nevertheless, little is known about their contribution to milk traits in Egyptian sheep. Milk traits of daily milk yield (DMY), protein percentage (PP), fat percentage (FP), lactose percentage, and total solid percentage (TSP) were available for 256 and 233 Barki ewes for LALBA and PRL, respectively. The association analysis between the phenotypes of milk traits was carried out using the general linear model implemented in SAS software. The single strand conformation polymorphism identified 3 (A, B and C) and 2 (G and C) distinctive alleles in LALBA and PRL, respectively. Allele frequencies for LALBA variants were 0.73 (A), 0.16 (B) and 0.11 (C), and 0.63 (G), and 0.37 (C) for PRL variants. Results showed that age and parity of the ewe significantly (P < 0.05) influenced the studied milk traits. Additionally, a significant (P < 0.05) effect of LALBA variants was reported on milk composition but not DMY. Likewise, a significant (P < 0.05) effect of PRL variants was observed on DMY and TSP. Interestingly, Animals with BB genotypes of LALBA gene produced milk with high FP (5.65 ± 1.25 %), PP (5.77 ± 1.17 %), LP (7.4 ± 1.22 %) and lower TSP (13.06 ± 5.1 %) compared with animals that carry other genotypes. Regarding the PRL polymorphisms, the heterozygote ewes (GC) tended to significantly produce more milk (368.57 ± 20.39 g/day) with higher TSP (19.81 ± 1.57) compared with the homozygotes. This study reported the LALBA and PRL as candidate genes with significant effects on milk yield and composition, which may be appropriate for a wide range of selection programs with breeding objectives that focus on milk yield and composition in sheep. However, further investigations with larger sample size are recommended to confirm these findings with higher accuracy.

Association of ovine follistatin gene polymorphisms with body measurements, fat-tail traits and morphometric of head in Iranian Mehraban sheep

The mature body size of sheep is a polygenic trait that is used as a main selection criterion for increasing meat production through genetic selection. The purpose of this study was to characterize polymorphisms of the follistatin gene (FST locus), which is known to regulate muscle hypertrophy by inhibiting both myostatin and activin. Using single-strand conformation polymorphism analysis (SSCP) and Sanger sequencing, we examined polymorphisms of the ovine FST gene (OAR_v.3.1; Chr 16, NC_019473.1) in Iranian Mehraban sheep (n = 100, seven-month-old male lambs) with records of body measurements, fat-tails, and head morphometry. Among the nine SSCP patterns A to I, we identified five single nucleotide polymorphisms (SNPs), including g.25632659 G>C, g.25632701 G>A, g.25633069 A>G, g.25633069 A>T, and g.25634085 G>C. The only amino acid-altering variant was g.25632659 G>C in exon 3 (p.216Thr > Ser). According to a subsequent statistical analysis, SSCP patterns A (g.25632659 G/C, g.25632701 G/G), B (g.25632659 C/C, g.25632701 G/A), C (g.25632659 C/C, g.25632701 A/A), D (homozygous reference; g.25633069 A/A), E (heterozygous; g.25633069 A/G) and F (homozygous variant; g.25633069 T/T) were significantly (P < 0.05) connected with body measurements. Furthermore, patterns A and B had significantly (P ≤ 0.05) longer forehead lengths than the lambs with the pattern C. Pattern I (homozygous variant; g.25634085 C/C) was significantly associated with an increased mouth width, and decreased eye length compared to patterns H (heterozygous; g.25634085 G/C) and G (homozygous reference; g.25634085 G/G). The findings of this study provide primary evidence of linking between the polymorphisms of the FST gene and body size in a small panel of Iranian Mehraban sheep population.

Clinical significance of SPOP and APC gene alterations in colorectal cancer in Indian population.

Speckle-Type Poz Protein (SPOP) involved in the regulation of proteasome-mediated degradation of several oncoproteins, resulting in cancer initiation and progression. Mutations in Adenomatous Polyposis Coli (APC) gene is reported in most sporadic and hereditary colorectal cancer (CRC). Identifying the cellular changes involved in carcinogenesis when APC is mutated is an important issue that needs attention. The tumor suppressive function of SPOP and APC has long been a major focus in the research field of colorectal cancer. However, the clinical significance of SPOP and APC gene alteration in CRC has not been established to date. Mutational analysis was performed by single-strand conformational polymorphism followed by Sanger sequencing, methylation status by methylation-specific PCR, and protein expression by immunohistochemistry on 142 tumor tissues along with their adjacent non-cancerous specimens. The overall survival (OS) and recurrence free survival (RFS) were estimated by Kaplan-Meier Curve. Mutation rates of APC and SPOP gene were 2.8% and 11.9% while that of promoter hypermethylation were 37% and 47%, respectively. The grade of differentiation and Lymph node metastasis were significantly correlated with APC methylation pattern (p ≤ 0.05). The down regulation of APC was more often seen in colonic cancer compared to rectal cancer (p = 0.07) and more commonly in T3-4 depth of invasion (p = 0.07) and in patients without lymphovascular and perineural invasion (p = 0.007, p = 0.08 respectively). The median overall survival and recurrence free survival (RFS) was 67 & 36months while 3-yr and 5-yr OS and RFS were 61.1% & 56.4% and 49.2% & 44.8%, respectively. APC promoter methylation had a better overall survival (p = 0.035) while loss of SPOP expression had a worse survival (p = 0.09). Our findings reveal high percentage of SPOP gene mutations in CRC. A significant link is found between promoter hyper methylationand protein expression in all mutant cases of APC and SPOP, suggesting that both genes may be associated in the development of colorectal cancer in people of Indian decent. Hypermethylation of APC gene and loss of SPOP expression have shown an association with disease prognosis and could be further studied looking at its potential role in planning adjuvant treatment in CRC patients.

High‐throughput genotyping in estimating genetic resources and detecting pathogens in aquaculture

High‐throughput genotyping in estimating genetic resources and detecting pathogens in aquaculture

One intron variant in the oocyte expressed protein (OOEP) gene is associated with twinning rate, prolificacy, and several body measurement traits in Awassi ewes

Awassi breed is an important sheep in the Middle East, but its lower twinning potential is a limitation against improvement. Due to the dynamic role of oocyte-expressed protein (OOEP) gene in controlling folliculogenesis, this study was conducted to evaluate the possible association between OOEP and twinning rate, as well as body measurement traits, in Awassi ewes. A total of 232 Awassi ewes (123 ewes-producing singletons and 109 ewes-producing twins) were included in this study. Three PCR fragments (OOEP-1 to OOEP-3) were designed to cover the majority of OOEP sequences and genotyped by single-strand conformation polymorphism (SSCP) method. One nucleic acid variant was identified in the intron-2 of OOEP-3, or rs430263839:A>G. Ewes with rs430263839:AG genotype showed variable significant values of larger head length, chest and abdominal circumferences, and higher prolificacy and twinning rates than ewes with the rs430263839:AA genotype. The conducted computations predicted that the detected SNP had caused altered splicing mechanisms in ewes with rs430263839:AG genotype. The increased abdominal and chest diameters in ewes with the rs430263839:AG genotype were thought to help the uterus accommodate more lambs. The predicted splicing alteration caused by this SNP is the putative mechanism by which these ewes may improve reproductive and productive traits. This is the first study that demonstrated the importance of OOEP as a potential biomarker for the twinning rate, prolificacy, and some body measurement traits in Awassi ewes with an opportunity to be introgressed to other breeds using large-scale association analysis.

Genetic variants and altered expression of SERPINF1 confer disease susceptibility in patients with otosclerosis.

Otosclerosis (OTSC) is a focal and diffuse bone disorder of the human middle ear characterized by abnormal bone growth and deposition at the stapes' footplate. This hinders the transmission of acoustic waves to the inner ear leading to subsequent conductive hearing loss. The plausible convections for the disease are genetic and environmental factors with yet an unraveled root cause. Recently, exome sequencing of European individuals with OTSCrevealed rare pathogenic variants in the Serpin Peptidase Inhibitor, Clade F (SERPINF1)gene. Here, we sought to investigate the causal variants of SERPINF1 in the Indian population. The gene and protein expression was also evaluated in otosclerotic stapes to ameliorate our understanding of the potential effect of this gene in OTSC. A total of 230 OTSC patients and 230 healthy controls were genotyped by single-strand conformational polymorphism and Sanger sequencing methods. Bycomparing the case controls, we identified five rare variants (c.72 C > T, c.151 G > A, c.242 C > G, c.823 A > T, and c.826 T > A) only in patients. Four variants c.390 T > C (p = 0.048), c.440-39 C > T (p = 0.007), c.643 + 9 G > A (p = 0.035), and c.643 + 82 T > C (p = 0.005) were found to be significantly associated with the disease. Down-regulation of SERPINF1 transcript level in otosclerotic stapes was quantified by qRT-PCR, ddPCR and further validated by in situ hybridization. Similarly, reduced protein expression was observed by immunohistochemistry and immunofluorescence in otosclerotic stapes that corroborate with immunoblotting of patients' plasma samples. Our findings identified that SERPINF1 variants are associated with the disease. Furthermore, reduced expression of SERPINF1 in otosclerotic stapes might contribute to OTSC pathophysiology.