A PstI polymorphism at 3′UTR of goat POU1F1 gene and its effect on cashmere production

POU1F1 is a positive regulator for prolactin (PRL) whose metabolites may directly or indirectly affect some aspects of the hair growth cycle, therefore, POU1F1 gene is an important candidate gene for cashmere traits selection through marker-assisted selection (MAS). Hence, in this study, the PCR-RFLP method was applied to detect a T>C transition determining a PstI polymorphism at the 3′UTR of POU1F1 locus and evaluate its associations with cashmere traits in 847 Inner Mongolia White Cashmere goats. In the analyzed population, the allelic frequencies for the T and C alleles are 0.959 and 0.041, respectively and the genotypic frequencies are in Hardy-Weinberg equilibrium (P > 0.05). Moreover, significant statistical relationships between the PstI polymorphism of POU1F1 gene and goat cashmere yields were found (*P < 0.05). When compared with TC genotype, TT genotype was associated with superior cashmere yields in 2, 4, and 5 years old individuals, as well as with average cashmere yield. Hence, TT genotype is suggested to be a molecular marker for senior cashmere yield. X. Y. Lan and J. H. Shu have contributed equally to this article.     

Identification of novel SNPs in the Sarda breed goats POU1F1 gene and their association with milk productive performance

The aim of the study was to detect polymorphism in the POU1F1 gene in Sarda breed goat, as well as to establish if SNPs could be associated with milk productive traits. The research was conducted on 129 Sarda breed goats from 4 to 5 years old, multiparous, lactating and in their third to fifth lactation. We report nine exonic and seven non-coding regions SNPs within the Sarda goat POU1F1 gene, namely, Ex 1 61 G>C; Ex 1 108 G>A; Ex 3 C>T; Ex 3 92 C>T; Ex 4 110 A>G; Ex 5 34 G>A resulting in Arg213Lys change; IVS4 641 G>A, IVS4 643 A>C, IVS4 659 G>A, IVS4 677 A>C, IVS4 G699Del, IVS4 709 C>G, Ex 6 17 G>A resulting in Arg228Ser change, Ex 6 58 G>T, Ex 6 172 T>C, 3′UTR 110 T>C. A statistically significant association was found between genotype TT, in position 17 of the exon 6 (3.1 % of frequency), and increased milk yield (P < 0.01) while genotype GT (25.6 % of frequency) was associated with a higher fat content. Genotype TT in position 58 of the exon 6 (3.9 % of frequency) was found to be associated with a higher fat (P < 0.01) and protein content (P < 0.05). Twenty-eight haplotypes were detected, but no significant association between the haplotypes and the milk production traits have been found. Our data, as well as providing new SNPs extending the POU1F1 gene characterization, evidence a relationship between polymorphism and milk production traits in Sarda goat breed.     

Analysis of caprine pituitary specific transcription factor-1 gene polymorphism in indigenous Chinese goats

Since mutations on POU1F1 gene possibly resulted in deficiency of GH, PRL, TSH and POU1F1, this study revealed the polymorphism of goat POU1F1-AluI locus and analyzed the distribution of alleles on 13 indigenous Chinese goat breeds. The PCR-RFLP analysis showed the predominance of TT genotype and the frequencies of allele T varied from 0.757 to 0.976 in the analyzed populations (SBWC, Bo, XH and HM). Further study, distributions of genotypic and allelic frequencies at this locus were found to be significantly different among populations based on a χ²-test (P < 0.001), suggesting that the breed factor significantly affected the molecular genetic character of POU1F1 gene. The genetic diversity analysis revealed that Chinese indigenous populations had a wide spectrum of genetic diversity in goat POU1F1-AluI locus. However, the ANOVA analysis revealed no significant differences for gene homozygosty, gene heterozygosty, effective allele numbers and PIC (polymorphism information content) among meat, dairy and cashmere utility types (P > 0.05), suggesting that goat utility types had no significant effect on the spectrum of genetic diversity. X. Y. Lan and M. J. Li equally contributed to this work.     

Two new mutations of the human BCHE gene (IVS3-14T>C and L574fsX576)

The genetic variation of human butyrylcholinesterase is associated with the majority of prolonged cases of apnea in patients submitted to the muscle relaxant succinylcholine. The present study reports two new mutations of the BCHE gene in 346 Euro-Brazilians: IVS3-14T>C found in five heterozygotes (allele frequency: 0.72 ± 0.32%) and L574fsX576 found in one heterozygote (allele frequency: 0.14 ± 0.14%). These two variants were not found in 85 Guarani Amerindians. It is not expected that the IVS3-14T>C mutation may interfere in the splicing process and that the mutation found in exon 4 (L574fsX576) may disturb BChE tetramerization and activity.     

Identification of SNPs within the sheep <Emphasis Type="Italic">PROP1</Emphasis> gene and their effects on wool traits

Regarding mutations of PROP1 (Prophet of POU1F1) gene significantly associating with combined pituitary hormone deficiency (CPHD) in human patients and animals, PROP1 gene is a novel important candidate gene for detecting genetic variation and growth, reproduction, metabolism traits selection and breeding. The aim of this study was to detect PROP1 gene mutation of the exon 1–3 and its association with wool traits in 345 Chinese Merino sheep. In this study, on the basis of PCR-SSCP and DNA sequencing methods, ten novel SNPs within the sheep PROP1 gene, namely, AY533708: g.45A > G resulting in Glu15Glu, g.1198A > G, g.1341G > C resulting in Arg63Ser, g.1389G > A resulting in Ala79Ala, g.1402C > T resulting in Leu84Leu, g.1424A > G resulting in Asn91Ser, g.1522C > T, g.1556A > T, g.1574T > C, g.2430C > G were reported. In addition, association analysis showed that three genotypes of P4 fragment were significantly associated with fiber diameter in the analyzed population (P = 0.044). These results strongly suggested that polymorphisms of the PROP1 gene could be a useful molecular marker for sheep breeding and genetics through marker-assisted selection (MAS).     

A novel SNP of the <Emphasis Type="Italic">Hesx1</Emphasis> gene in bovine and its associations with average daily gain

As an essential repressor, the homeobox gene Hesx1/HESX1 is required within the anterior neural plate for normal forebrain development. Mutations within the Hesx1 gene have been associated with GH deficiency or combined pituitary hormone deficiency. We detected the polymorphism of Hesx1 gene by PCR-SSCP and DNA sequencing methods in 702 individuals from four Chinese cattle breeds. A novel single nucleotide polymorphism (SNP) (IVS1 + 382T > C) was detected. The frequencies of genotype TC in four breeds were 0.000–0.222. Polymorphism of the Hesx1 gene was shown to be associated with growth in the Nanyang breed. Individuals with genotype TC was significantly lower average daily gain than TT at 18 months (P < 0.05).     

Estimation of genetic parameters for post-weaning traits of Kermani sheep

The objective of the present study was to estimate genetic parameters for post-weaning traits in Kermani sheep. Traits were included 6-month weight (6MW), 9-month weight (9MW), yearling weight (YW), greasy fleece weight at first shearing (GFW) and greasy fleece weights at various shearings (RFW). Data and pedigree information used in this research were collected at Breeding Station of Kermani sheep during 1993–2004. Genetic parameters were estimated with single- and multi-traits analysis using restricted maximum likelihood (REML) procedures, under animal models. Log likelihood ratio test indicated the most appropriate model for 6MW and 9MW should included direct additive genetic effects as well as maternal permanent environmental effects. However the most appropriate model for YW and GFW had only the direct additive genetic effects. The effects of sex, age of dam and year of birth were significant on body weight traits (P < 0.01). GFW was influenced significantly by sex and year of birth (P < 0.01) but was not affected by age of dam (P > 0.05). Type of birth was no significant effect on studied traits (P > 0.05). Also, the age of lamb at weighing time was a significant influence on 6MW, 9MW and YW. Direct heritability estimates for 6MW, 9MW, YW and GFW were 0.32, 0.03, 0.15 and 0.15, respectively. Maternal permanent environmental estimates of 0.09 were obtained for 6MW and 9MW. Genetic correlation estimates between mentioned traits ranged from 0.51 to 0.99. Phenotypic correlations were generally lower than those of genetic correlation and varied from 0.05 to 0.79 for various traits. The environmental correlations estimates between GFW with growth traits were low, but between other traits were positive and high, ranged from 0.54 to 0.72. The value of repeatability estimated for greasy fleece weight was 0.22.     

Effects of genetic variability of the carpine homeobox transcription factor <Emphasis Type="Italic">HESX1</Emphasis> gene on performance traits

HESX1 plays a key role in the development of the forebrain and pituitary gland and produces potential effects on performance traits. The objective of this study was to detect and assess the associations of the possible polymorphisms of six loci within HESX1 gene with performance traits in Chinese 1,119 goats. Only one novel SNP (NM_001494116:g.307049A > G) locating on IVS1 + 348A > G was identified and detected by HaeIII forced-RFLP-PCR. The frequencies of allele “G” varied from 0.025 to 0.245 in analyzed populations with the Hardy–Weinberg equilibrium (P > 0.05). Genotypic and allelic frequencies were found to be significantly different in four breeds (χ² = 147.674, df = 6, P < 0.001; χ² = 157.250, df = 3, P < 0.001, respectively), implying that the distribution of genotypic and allelic frequencies of goat HESX1 gene was significantly associated with different goat utilities (cashmere, meat and dairy). Association analysis results revealed no significant effects of caprine HESX1 gene on body sizes in XNSN population (P > 0.05) and cashmere traits in IMWC population (P > 0.05). Significant statistical of HESX1 gene with body weight was found (*P < 0.05). The genotype AA showed significantly higher body weight than those of AG in 2-year-old age (*P < 0.05), while the AA genotype was senior to AG genotype in 4-year-old body weight trait (*P < 0.05). These suggestions indicated that the HESX1 gene has significant effect on goat body weight depending on ages, which is accordance with the function repressor of the HESX1.     

The polymorphism of a novel 30 bp-deletion mutation at KAP9.2 locus in the cashmere goat

The keratins and keratin-associated proteins (KAPs) are a large heterogeneous group of proteins that make up about 90% of the cashmere fiber. Keratin-associated proteins 9.2 gene (KAP9.2) is one of the ultra high sulfur KAPs, which might play an important role in the bundling of intermediate filaments. In this study, the deletion/insertion mutation of KAP9.2 gene in 997 cashmere goat samples was firstly detected, at the same time, parts of these samples were sequenced. The results showed that two alleles were detected at this KAP9.2P1 locus, named allele W and D. The frequencies of the KAP9.2-W allele in Inner Mongolia White cashmere (n = 785) and Shaanbei White cashmere goat breeds (n = 212) were 0.878 and 0.790, respectively. The χ²-test showed that the genotype distributions in these two cashmere goat breeds were not in agreement with Hardy–Weinberg equilibrium. According to the classification of polymorphism information content (PIC), Shaanbei White cashmere goat was more polymorphic at this locus. Moreover a 30 bp-deletion mutation was described at KAP9.2P2 locus for the first time and no deletion/insertion was described at KAP9.2P1 locus. The results possibly revealed that the size polymorphism existed in the two Chinese cashmere goat and the 30 bp-deletion mutation was possibly caused by variations in the number of the decapeptide repeat structures.     

Analysis of polymorphism within <Emphasis Type="Italic">POU1F1</Emphasis> gene in relation to milk production traits in dairy Sarda sheep breed

The ovine POU1F1 gene is localized on chromosome 1 and it contains five introns and six exons. In different mammalian species some mutations in different exons are associated with different production traits. The aim of our research was to study the POU1F1 gene nucleotide sequence to detect possible polymorphisms and their relationships with milk productive traits in Sarda breed sheep. The study had been conducted on 140 ewes, 4 or 5 years old coming from a farm located in Sardinia. All the animals were multiparous, lactating and in their third to fifth lactation. Individual milk yield had been recorded monthly and for each sample fat, protein, casein, lactose, and somatic cell count values were analysed. A jugular blood sample was collected from each ewe to perform genomic DNA extraction. PCR, SSCP and sequencing analysis were carried out to examine the six exons to highlight possible SNPs. One-way ANOVA was used to analyse association of variants with milk yield and/or its composition. Two novel SNP were found: 121 C>T in the 5′UTR of the fourth intron fragment and 249 G>A in the 3′UTR of the sixth exon fragment. The statistical analysis did not shown association between milk productive traits and the found polymorphisms. However, further investigations about the promoter region or the prophet genes, like the PROP-1, could clarify its exact role in regulating the productive traits in sheep.     

Identification of two novel mutations in POU4F3 gene associated with autosomal dominant hearing loss in Chinese families

Autosomal dominant non‐syndromic hearing loss is genetically heterogeneous with 47 genes identified to date, including POU4F3. In this study, by using a next‐generation sequencing panel targeting 127 deafness genes, we identified a pathogenic frameshift mutation c.704_705del and a missense mutation c.593G>A in two three‐generation Chinese families with late‐onset progressive ADNSHL, respectively. The novel mutations of POU4F3 co‐segregated with the deafness phenotype in these two families. c.704_705del caused a frameshift p.T235fs and c.593G>A caused an amino acid substitution of p.R198H. Both mutations led to an abnormal and incomplete protein structure. POU4F3 with either of the two mutations was transiently transfected into HEI‐OC1 and HEK 293 cell lines and immunofluorescence assay was performed to investigate the subcellular localization of mutated protein. The results indicated that both c.704_705del (p.T235fs) and c.593G>A (p.R198H) could impair the nuclear localization function of POU4F3. The p.R198H POU4F3 protein was detected as a weak band of the correct molecular weight, indicating that the stability of p.R198H POU4F3 differed from that of the wild‐type protein. While, the p.T235fs POU4F3 protein was expressed with a smaller molecular weight, implying this mutation result in a frameshift and premature termination of the POU4F3 protein. In summary, we report two novel mutations of POU4F3 associated with progressive ADNSHL and explored their effects on POU4F3 nuclear localization. These findings expanded the mutation spectrum of POU4F3 and provided new knowledge for the pathogenesis of POU4F3 in hearing loss.     

Genetic analysis of hereditary multiple exostoses in Tunisian families: a novel frame-shift mutation in the <Emphasis Type="Italic">EXT1</Emphasis> gene

Hereditary multiple exostoses (HME) is an autosomal dominant orthopaedic disorder most frequently caused by mutations in the EXT1 gene. The aim of the present study is to determine the underlying molecular defect of HME in two multigenerational Tunisian families with 21 affected members and to examine the degree of intrafamilial variability. Linkage analysis was performed using three microsatellite markers encompassing the EXT1 locus and mutation screening was carried out by direct sequencing. In family 1, evidence for linkage to EXT1 was obtained on the basis of a maximum LOD score of 4.26 at θ = 0.00 with D8S1694 marker. Sequencing of the EXT1 revealed a heterozygous G > T transversion (c.1019G>T) in exon 2, leading to a missense mutation at the codon 340 (p.Arg340Leu). In family 2 we identified a novel heterozygous 1 bp deletion in the exon 1 (c.529_531delA) leading to a premature codon stop and truncated EXT1 protein expression (p.Lys177LysfsX15). This mutation was associated with the evidence of an intrafamilial clinical variability and considered to be a novel disease-causing mutation in the EXT1 gene. These findings provide additional support for the involvement of EXT1 gene in the HME disease.     

Identification of a missense mutation in the bovine ATP2A1 gene in congenital pseudomyotonia of Chianina cattle: an animal model of human Brody disease

Congenital pseudomyotonia in Chianina cattle is a muscle function disorder very similar to that of Brody disease in humans. Mutations in the human ATP2A1 gene, encoding SERCA1, cause Brody myopathy. The analysis of the collected Chianina pedigree data suggested monogenic autosomal recessive inheritance and revealed that all 17 affected individuals traced back to a single founder. A deficiency of SERCA1 function in skeletal muscle of pseudomyotonia affected Chianina cattle was observed as SERCA1 activity in affected animals was decreased by about 70%. Linkage analysis showed that the mutation was located in the ATP2A1 gene region on BTA25 and subsequent mutation analysis of the ATP2A1 exons revealed a perfectly associated missense mutation in exon 6 (c.491G > A) leading to a p.Arg164His substitution. Arg164 represents a functionally important and strongly conserved residue of SERCA1. This study provides a suitable large animal model for human Brody disease.     

Associations of <Emphasis Type="Italic">GHSR</Emphasis> gene polymorphisms with chicken growth and carcass traits

Ghrelin receptor (GHSR), or growth hormone secretagogue receptor, modulates many physiological effects by binding to its ligand and therefore is a candidate gene for chicken production performance. In this study, five polymorphisms (four SNP and a ‘GGTACA’ indel) of GHSR gene were genotyped in a F₂ full sib chicken population to investigate their associations with production traits. Results showed that c.739 + 726T > C (M2) was significantly associated with body weight (BW) at 28 days (BW28), BW90, dressed weight, eviscerated weight, eviscerated weight with giblet, breast muscle weight and leg muscle weight (P < 0.05). Meanwhile, T allele rather than C was positive for chicken body weight gain as individuals with CC had the lowest value of all traits. Otherwise, no significant association of c.264G > A (M1), c.3211-196_3211-181delGGTACA (M3), c.3211 + 75C > T (M4), and c.3211 + 150C > T (M5) with any growth and carcass traits was found. Haplotypes based on five polymorphisms were significantly associated with hatch weight, BW7, BW14, BW21 and breast angle (P < 0.05), as well as BW28 (P < 0.01). Therefore, it was concluded that M2 of the GHSR gene and the analyzed haplotypes were associated with some chicken growth and carcass traits.     

Comparison of some novel polyculture and traditional monoculture vermicomposting reactors to decompose organic wastes

Efforts were made to evaluate the decomposition potentials of traditional monoculture and some novel polyculture vermireactors. Three earthworm species, i.e. Eisenia fetida (E. f.), Perionyx excavatus (P. ex.) and Lampito mauritii (L. m.), representing two different ecological categories: epigeic (E. fetida and P. excavatus) and anecic (L. mauritii), were used to design seven different vermireactors, i.e. Mono-(E. f.), Mono-(P. ex.), Mono-(L. m.), Poly-(E. f. + P. ex.), Poly-(P. ex. + L. m.), Poly-(E. f. + L. m.) and Poly-(E. f. + P. ex. + L. m.). The microbial load of vermireactors was evaluated through measuring dehydrogenises activities (DH-ase) and microbial biomass-N, while mineralization rate was measured in respect to changed level of some important nutrients in vermicomposted substrate. The vermicomposting caused decrease in pH (67.0–15.0%), organic C (46.1–28.4%) and C:N ratio (72.2–57.1%) and increase in total N (137.7–67.8%) as well as available P (107.9–16.9%) contents, at the end. The carbon and nitrogen mineralization rate showed the order: Poly-(E. f. + P. ex. + L. m.) > Poly-(E. f. + L. m.) > Poly-(P. ex. + L. m.) > Poly-(E. f. + P. ex.) > Mono-(E. f.) > Mono-(P. ex.) > Mono-(L. m.) for this study. The Poly-(E. f. + P. ex. + L. m.) vermireactor showed the maximum level of DH-ase activity 1926 ± 245 μg g⁻¹ substrate 24 h as well as microbial biomass-N 3059.1 ± 242.3 mg N g⁻¹ substrate, during experimentation. This study clearly suggests that burrowing earthworms in vermireactor not only promote the microbial colonization, but at the same time also accelerate the mineralization rate in decomposing waste. The polyculture vermicomposting, using burrowing earthworms with epigeics, could be more efficient than traditional monoculture vermireactors to decompose organic waste resources.     

Over-expression in E. coli and purification of the human OCTN1 transport protein

The hOCTN1 amplified from skin fibroblast RNA was cloned in pET-28a(+) or in pH6EX3 plasmid. The encoded recombinant hOCTN1 resulted in a 6-His tagged fusion protein with a 34 or 21 amino acid extra N-terminal sequence in the pET-28a(+)-hOCTN1 or in the pH6EX3-hOCTN1 constructs, respectively. Both constructs were used to express the hOCTN1 in Escherichia coli Rosetta(DE3)pLysS. The best over-expression was obtained with the pH6EX3-hOCTN1 after 6 h of induction with IPTG at 28 °C. The expressed protein with an apparent molecular mass of 54 kDa, was collected in the insoluble fraction of the cell lysate. Further improvement was obtained using the E. coli RosettaGami2(DE3)pLysS strain to express the protein encoded by pH6EX3-hOCTN1. After 6 h of induction with IPTG at 28 °C, hOCTN1 accounted for 30% of the total protein in the insoluble pellet. This protein fraction was washed with Triton X-100 and deoxycholate, solubilized with a buffer containing 0.8% Sarkosyl, 3 M urea and applied to a Ni²⁺-chelating chromatography column. The homogeneously purified hOCTN1 was eluted with a buffer containing 50 mM imidazole, 0.1% Triton X-100 and 50 mM 2-mercaptoethanol. A yield of about 3 mg purified protein per liter of cell culture was obtained.     

Effect of Genetic Variations of the POU1F1 Gene on Growth Traits of Nanyang Cattle

PCR-RFLP was applied to analyze the effect of the genetic variations of the POU1F1 gene on growth traits of 100 Nanyang cattle. The results showed that the 451 bp PCR product digested with Hinf I demonstrated polymorphism in the population, which was at Hardy-Weinberg equilibrium. Moreover, the frequencies of alleles A/B in the Nanyang population were 0.465/0.535. The association of the variations of the POU1F1 gene with the growth traits in the population was analyzed. The following parameters were greater in individuals with a genotype BB than in those with an genotype AB: birth weight, average weight increase before ablactation, body height at 12 months, body weight, body length, and chest girth at 6 months and 12 months (P<0.05). The body weight at 12 months was higher in the BB individuals than in the AA individuals (P <0.05). The body weight and body sizes also showed a trend of allele B> allele A in the other age groups. Therefore, the genotype BB maybe a dominant genotype and the allele B may be a dominant allele. These results imply that the allele B of the POU1F1 gene is likely to positively affect the growth traits.     

IL2-330G polymorphic allele is associated with decreased risk of Helicobacter pylori infection in adulthood

We evaluated whether polymorphisms in genes coding molecules linked to the innate and adaptive immune response are associated with susceptibility to Helicobacter pylori infection. IL1B-511C → T, IL1B-31 T → C, IL1RN allele 2, IL2-330 T → G, TNFA-307 G → A, TLR2Arg677Trp, TLR2Arg753Gln, TLR4Asp299Gly, and TLR5^392STOP polymorphisms were determined in 541 blood donors. IL2-330 T → G allele carriers had a decreased H. pylori infection risk (OR = 0.63, 95% CI = 0.43–0.93) after adjustment for demographic and environmental factors. Hence, we investigated whether the polymorphism is functional by evaluating IL-2 serum concentration in 150 blood donors and 100 children. IL-2 pro-inflammatory and anti-inflammatory properties were indirectly investigated by determining serum IFN-γ and IL-10/TGF-β levels. The polymorphism was associated with increased mean IL-2 levels in H. pylori-positive adults (2.65 pg/mL vs. 7.78 pg/mL) and children (4.19 pg/mL vs. 8.03 pg/mL). Increased IL-2 was associated with pro-inflammatory activity in adults (IFN-γ = 18.61 pg/mL vs. 25.71 pg/mL), and with anti-inflammatory activity in children (IL-10 = 6.99 vs. 14.17 pg/mL, TGF-β = 45.88 vs. 93.44 pg/mL) (p < 10⁻³ for all). In conclusion, in the context of H. pylori infection, IL2-330 T → G polymorphism is functional and is associated with decreased risk of infection in adults.