Genotyping of the porcine ryanodine receptor 1 (RYR1) and estrogen receptor 1 (ESR1) genes by high resolution melting (HRM) approach

During the last decade, DNA mutations in the porcine ryanodine receptor 1 gene (RYR1, C1843T) and the estrogen receptor 1 gene (ESR1, T1665G), have been widely used in marker-assisted selection (MAS) for the pig industry. These 2 well-characterized SNPs in RYR1 and ESR1 are responsible for porcine stress syndrome (PSS) and litter size, respectively. Here, we describe a reliable, high-efficiency method for the genotyping of these 2 genes using the high-resolution melting (HRM) method. The HRM approach exhibited high-accuracy and repeatability, comparable with the classic PCR-restriction fragment length polymorphism (PCR-RFLP) approach, and is potentially suitable for large-scale genotyping in commercial pig farms.     

Association between estrogen receptor alpha gene polymorphisms and bone mineral density in Polish female patients with Graves' disease

Graves' (GD) hyperthyroidism leads to reduced bone mineral density (BMD) accompanied by accelerated bone turnover. Ample studies have identified association between estrogen receptor (ESR1) gene polymorphism and decreased BMD and osteoporosis. In contrast, number of publications that link ESR1, BMD and Graves' disease is limited. The purpose of this study was to identify the association between ESR1 polymorphisms and BMD in premenopausal women with GD and to determine whether ESR1 polymorphic variants can predispose to GD. The study included 75 women aged 23-46 years with GD and 163 healthy controls. BMD was measured at lumbar spine and femoral neck. We investigated two SNPs in the ESR1 gene and analyzed genetic variants in the form of haplotypes reconstructed by statistical method. Three out of four possible haplotypes of the PvuII and XbaI restriction fragment length polymorphisms were found in GD patients: px (55.3 %), PX (33.3 %) and Px (11.4 %). Women homozygous for xx of XbaI and for pp of PvuII had the lowest BMD at lumbar spine. Moreover, the px haplotype predisposed to reduced lumbar BMD. No associations were observed for femoral neck BMD. No statistically significant relationship were found between ESR1 polymorphisms or their haplotypes and GD. These results indicate that the PvuII and the XbaI polymorphisms of ESR1 gene are associated with bone mineral density in premenopausal women with GD and may help to estimate the risk of bone loss particularly at lumbar spine. However, none of the ESR1 gene alleles predict the risk of GD in Polish female patients.     

Relation between Ava I polymorphism within the estrogen receptor gene (ESR) and meatiness in Polish Large White boars

It is currently debated whether identification of ESR (estrogen receptor) genotypes should be introduced into breeding programs of Large White pigs. The aim of this study was to evaluate the possible relations between ESR/Ava I polymorphism and carcass performance traits in Polish Large White boars. We examined 103 boars originating from one herd in NE Poland. ESR/Ava I genotypes were determined by the PCR-RFLP method. By the use of the Duncan test, we found highly significant differences (P < 0.01) between WW and MW genotypes, as well as significant differences (P < 0.05) between WW and MM genotypes for meatiness. No significant differences were found for daily gain and selection index.     

Simultaneous detection of malignant hyperthermia and genetic predisposition for improved litter size in pigs by multiplex PCR-RFLP

The ryanodine receptor gene (RYR1) and the estrogen receptor gene (ESR) are the best commercially used markers for predisposition of stress susceptibility (malignant hyperthermia--MH) and increased litter size, respectively. A simplified method of simultaneous detection of MH and ESR genotypes has been developed. The method is based on simultaneous amplification of fragments of two genes by multiplex PCR and subsequent digestion of the products with two restriction enzymes. The PCR and the digestion could be performed in a single tube and all genotypes could be detected by electrophoretic separation on the same agarose gel. Thus, the development of the method can decrease the cost of the sample analysis and increase the speed and efficiency of the analysis. In our study, frequencies of mutated T allele of the RYR1 gene in Large White (LW), White Meaty (WM) and Landrace (L) were 0.11, 0.13, and 0.15, respectively. Frequencies of the preferred B allele of the ESR gene in the same breeds were 0.35, 0.26, and 0.06, respectively.     

Polymorphisms and deduced amino acid substitutions in the coding sequence of the ryanodine receptor (RYR1) gene in individuals with malignant hyperthermia.

Twenty-one polymorphic sequence variants of the RYR1 gene, including 13 restriction fragment length polymorphisms (RFLPs), were identified by sequence analysis of human ryanodine receptor (RYR1) cDNAs from three individuals predisposed to malignant hyperthermia (MH). All RFLPs were detectable in PCR-amplified products, and their segregation was consistent with our initial finding of linkage to MH in the nine families previously informative for one or more intragenic markers (MacLennan et al., 1990, Nature 343:559-561). Four amino acid substitutions were identified in the study: Arg for Gly248, Cys for Arg470, Leu for Pro1785, and Cys for Gly2059. Of 45 families tested, a single family presented the Arg for Gly248 substitution where it segregated with malignant hyperthermia, making it a candidate mutation for predisposition to MH in man. The other three polymorphic substitutions failed to segregate with malignant hyperthermia in those families in which they occurred, implying that they represent polymorphisms with little or no effect on the function of the RYR1 gene.     

Microsatellite polymorphism in locus OMHC1 (MHC Class I) in Polish Heath Sheep and Polish Lowland Sheep (Zelazna variety)

Our study aimed at comparative analysis of microsatellite polymorphism in locus OMHC1 (MHC Class I) in Polish Heath Sheep and Polish Lowland Sheep (Zelazna variety). The study was conducted on 100 ewes of each breed. We identified 13 alleles of the gene in Polish Heath Sheep and 9 in Polish Lowland Sheep. We found marked differences in frequency of OMHC1 alleles between both breeds. The heterozygosity coefficient and PIC, amounting to 0.79 and 0.77 for Polish Heath Sheep, and 0.82 and 0.80 for Polish Lowland Sheep, respectively, suggest considerable variability in both breeds. Additionally, the values of both coefficients indicate that OMHC1 locus can be used as a genetic marker.     

Association between MCT1 T1470A polymorphism and climbing status in Polish and Japanese climbers

Sport climbing will become an official event at the 2020 Tokyo Olympics; it is a popular wilderness sport among athletes and amateurs. Our previous study suggested that the T1470A polymorphism (rs1049434) of the monocarboxylate transporter 1 (MCT1) gene is associated with athletic performance and physiological phenotypes. The purpose of this study was to investigate the frequency of MCT1 T1470A polymorphism in Polish and Japanese climbers using a case-control study. Our sample consisted of 226 climbers (Japanese: n = 100, 64 male and 36 female; Polish: n = 126, 97 male and 29 female) and 1028 non-athletic controls (Japanese, n = 407; Polish = 621) who were genotyped for the MCT1 T1470A polymorphism (rs1049434) using the TaqMan SNP genotyping assay or restriction enzyme. The frequency of the TT genotype and T allele was significantly higher in climbers than in controls among the Polish subjects (genotype: p = 0.030, allele: p = 0.010); however, there were no significant differences in the genotype and allelic frequencies between the Japanese climbers and controls (genotype: p = 0.968; allele: p = 0.803). Our results suggested that the frequency of the T allele (TT+TA genotype) in the MCT1 T1470A polymorphism is over-represented in Polish climbers but not in Japanese climbers. In addition, the frequency of the T allele and TT genotype in Polish lead climbers is higher than that in controls.     

Restriction fragment length polymorphism of exon 2 Ovar-DRB1 gene in Polish Heath Sheep and Polish Lowland Sheep

Exon 2 of the Ovar-DR gene is known to encode the MHC outer domain (alpha or beta chain) that forms the binding area to antigens presented. The study was aimed at analysing exon 2 Ovar -DRB1 gene polymorphism in Polish Heath Sheep and Polish Lowland Sheep (Zelazna variety). A total of 101 and 99 ewes of the respective breeds were included in this study. We identified 65 different haplotypes in Polish Heath Sheep and 68 in Polish Lowland Sheep. The PCR-RFLP method and PCR products sequencing made it possible to identify two new sequences of exon 2 Ovar-DRB1 gene (AY230000 and AY248695). A distinct polymorphism in the exon 2 sequence presents possibilities for immune response toward a great variety of pathogens.     

Characterization of an angiotensin type-1 receptor partial cDNA from rat kidney: evidence for a novel AT1B receptor subtype.

We sought to determine if multiple forms of mRNA for the angiotensin type-1 (AT1) receptor could be detected in rat kidney using the polymerase chain reaction (PCR) procedure. Amplification of rat kidney cDNA with oligonucleotide primers derived from the second and sixth transmembrane domains of the rat AT1 receptor yielded a single cDNA fragment 528bp in size. Sequence analysis indicated, however, that the cDNA fragment was a mixture of two highly similar gene products: the first cDNA was identical to the previously cloned AT1 receptor (termed here AT1A) whereas the second cDNA (termed here AT1B) was 92% identical at the nucleotide level and 96% identical at the amino acid level. Nucleotide substitutions were dispersed throughout the cDNA and 80% (33 of 41) were conservative. Significant levels of AT1A and AT1B mRNA were detected by PCR amplification of kidney poly(A)+ RNA and restriction enzyme analysis. These results indicate that at least two distinct AT1 receptor genes are expressed in rat kidney.     

微卫星复杂结构对分型的影响：以熊UamD116 和UamB1 为例

目的:微卫星是基因组上的短串联重复序列,具有高度多态性,表现为核心序列中重复单位的重复次数的变化,这种变化造成不同等位基因核心序列的长度不同。因此,其基因型主要依靠PCR扩增片段长度来判定。在各类研究中,人们更倾向于使用4碱基重复的微卫星以减少2碱基微卫星的stutter等问题的影响。但是4碱基微卫星核心序列结构复杂时,就会对分型的正确性产生影响,从而影响到下游分析的正确性。在很多野生动物的研究中,这一问题常常被忽略。本文以亚洲黑熊(Ursus thibetanus)的2个四碱基微卫星位点UamD116和UamB1为例,揭示内部结构对分型的影响。方法:我们选用96份亚洲黑熊样品(包括血液、肌肉组织和毛发等样品)进行微卫星分型研究,通过荧光标记的PCR扩增和毛细管电泳分型,比较了基于扩增片段长度的分型和基于序列核心结构的分型效果的差异。结果:UamD116核心序列结构除了含有多种不同的重复单位外,还在重复单位之间有碱基插入,出现单碱基T、二碱基TC和三碱基AAG插入;并在一类等位基因下游侧翼序列有1个GA缺失。基于序列结构的分型中可以将不同的等位基因分开,而在基于片段长度的分型中,容易将不同的等位基因合并为1个等位基因。在位点UamB1共发现两种类型的等位基因,在一类等位基因中出现一个3bp的插入,使等位基因之间的差异不再是4bp,而是1bp。在仅依据片段长度分型时,相差1bp的等位基因被认定为1个。此外,还有不同等位基因核心序列不同,但是二者长度完全一致。依据片段长度分型共发现8个等位基因,而经过序列分型确定的等位基因数为12个,相应地基因频率及其他遗传学参数都发生相应的改变。结论:对于核心序列结构复杂的微卫星必须通过等位基因测序来矫正片段长度分型的结果,才能得到可靠的群体遗传学结论。     

PCR方法在HSF1基因敲除小鼠基因型分析中的应用

目的　为HSF1基因敲除鼠探索快速、简单的基因型PCR检测方法。方法　设计两对引物扩增野生型HSF1基因和HSF1缺陷突变基因的DNA片段 ,用PCR仪梯度方案测试最佳退火温度 ,并将所得基因型结果与经典的Southernblot方法比较。结果　野生型仅在 5 6 2bp处有一条条带 ,突变纯合子仅在 377bp处有一条条带 ,杂合子则在377bp和 5 6 2bp处出现两条条带。用PCR方法获得的HSF1基因分析结果与经典的Southernblot方法获得的结果完全一致。结论　用PCR方法分析HSF1基因敲除鼠的基因型具有快速、简单、廉价和适用的特点     

麦长管蚜烟碱型乙酰胆碱受体基因的克隆和序列分析

昆虫烟碱型乙酰胆碱受体(nicotinic acetylcholine receptor, nAChR)是杀虫剂的重要作用靶标之一。本研究利用简并引物PCR和半巢式PCR技术从麦长管蚜Sitobion avenae (Fabricius)中克隆nAChR基因,成功地获得了5个α型nAChR亚基的cDNA片段。根据5个α亚基片段设计特异引物,结合快速扩增cDNA末端(RACE)技术,成功克隆了5个α型亚基的全长,并发现α5亚基有两种存在形式,它们仅在胞外区有一段175 bp的片段有差异。序列分析发现,这些基因均具有nAChR基因家族的典型特征,并与已报道的其他昆虫的烟碱型乙酰胆碱受体的相应亚基具有很高的同源性。该研究为进一步利用基因表达技术研究昆虫nAChR的天然亚基组成,以及分析麦长管蚜对新烟碱类杀虫剂的靶标抗性,奠定了基础。     

Prevalence of estrogen receptor alpha PvuII and XbaI polymorphism in population of Polish postmenopausal women

Numerous data indicate that polymorphism of estrogen receptor alpha (ERalpha) may predict lipid levels, lipid response to hormone replacement therapy (HRT), myocardial infarction risk, bone fracture risk, bone mineral density (BMD) and changes in BMD over time. In this study we aimed to evaluate distribution of ERalpha PvuII and XbaI genotypes in population of Polish postmenopausal women qualified to different protocols of HRT. Subject of the study were 64 consecutive postmenopausal women aged from 45 to 65 years (mean 56.6) assigned to HRT. ERalpha PvuII and XbaI polymorphism was determined by PCR-restriction fragment length polymorphism (RFLP). The absence of PvuII and XbaI restriction sites were indicated by "P" and "X" and presence by "p" and "x", respectively. PvuII genotype was distributed as follows: PP 17.2% (n=11), Pp 50% (n=32), pp 32.83% (n=21). Frequency of XbaI genotype was: XX 6.25% (n=4), Xx 34.4% (n=22), xx 59.4% (n=38). Four haplotypes with following frequencies were recognized: PX 17.3%, px 47.4%, Px 24.4% and pX 10.9%. Prevalence of estrogen receptor alpha PvuII and XbaI polymorphisms in Polish women is similar to previously studied population.     

Molecular Characterization of Acanthamoeba spp. Occurring in Water Bodies and Patients in Poland and Redefinition of Polish T16 Genotype

Acanthamoeba genus is divided into 20 genotypes (T1–T20) on the basis of the gene encoding 18S rRNA sequence. Using of at least 2 kbp gene fragments is strongly recommended to identify new genotypes and 5% difference is commonly used as a criterion of new genotypes, however, this value is questionable. In this paper, Polish Acanthamoeba strains described earlier on the basis of ~850 bp Ami fragment of 18S rRNA gene as T4, T11 and a new T16 genotype, have been analyzed using near‐complete sequence of the gene. This analysis was needed because the Ami fragment does not reveal full variability within 18S rRNA gene. Phylogenetic analysis based on Ami fragment is biased by artifacts in the construction of the tree, so the fragment should not be used for identification of new putative Acanthamoeba genotypes. The analysis confirmed that the Polish sequences represent T4 and T11 genotypes and that the strains described earlier as T16 genotype are in fact a new subgroup of the T20 genotype and that this genotype should be divided into two subgroups: T20a (two strains described by [J. Eukaryot. Microbiol. 62 (2015) 69]) and T20b (11 Polish strains described in this study). The T20b subgroup was isolated from both clinical samples and water bodies used by people as bathing places and there is a risk of infection for humans during contact with water.     

Deletion/insertion polymorphism of the prion protein gene (<Emphasis Type="Italic">PRNP</Emphasis>) in Polish Red cattle,Polish White-backed cattle and European bison (<Emphasis Type="Italic">Bison bonasus</Emphasis> L., 1758)

The aim of the present study was to identify deletion/insertion polymorphism of the bovine prion protein (PRNP) gene within the promoter sequence (23 bp indel), intron 1 (12 bp indel) and the 3′ end untranslated region (14 bp indel). The experiment was performed on three groups of animals protected under a genetic resources conservation program: 139 Polish Red (PR) cows, 79 Polish White-backed cows and 50 European bison (Bison bonasus L., 1758). White-backed cattle were characterized by a higher frequency of ins/del heterozygotes and a relatively lower frequency of ins/ins homozygotes within the promoter sequence region (23 bp indel), compared to Polish Red cattle. At the polymorphic locus of intron 1 (12 bp indel) the genetic structure of both cattle populations was similar. Monomorphism, expressed by the occurrence of one genotype variant in each of the analyzed sequence regions, was observed in European bison. Five haplotypes were found in Polish White-backed cows, four haplotypes in Polish Red cows and only one in analyzed group of bison. Differences between the observed and expected number of PRNP haplotypes were recorded in Polish Red cattle. The article is published in the original.     

拟黑多刺蚁雌激素相关受体基因cDNA的克隆及生物信息学分析

雌激素相关受体（estrogen related receptors, ERRs）能够直接与类固醇激素受体共激活子（steroidhormone receptor coactivator,SRC）结合,激活靶基因的表达,与众多生理和发育过程有关.采用RT-PCR、RACE等方法从拟黑多刺蚁Polyrhachis vicina Roger克隆得到了雌激素相关受体基因的cDNA克隆,命名为pvERR （GenBank 登录号为EF474463）,并采用生物信息学方法对其cDNA和编码的蛋白质的理化性质、蛋白质二级及三级结构、分子系统进化关系等进行了预测和推断.结果表明：pvERR基因cDNA全长1 935 bp,包含一个1 305 bp的开放阅读框、245 bp的5′-UTR 和385 bp的3′-UTR,编码一个由434个氨基酸组成的蛋白质.pvERR的配体结合区（ligand binding domain,LBD）主要由两个β折叠和11个α螺旋（H1, H3～H12, 缺少H2）构成,这与哺乳类动物已知晶体结构的ERR_γ的配体结合区结构非常相似.pvERR氨基酸序列与其他昆虫ERRs氨基酸序列同源性很高,它与西方蜜蜂Apis mellifera雌激素相关受体amERR氨基酸序列相似性达到89.9 %;在进化关系上,pvERR与人类Homo sapiens ERRs的关系比果蝇Drosophila melanogaster雌激素相关受体dERR与人类的更近.本文可为进一步研究pvERR在昆虫发育中的功能提供有价值的信息.     

Genetic variability among freshwater mussel Anodonta woodiana (Lea, 1834) (Bivalvia: Unionidae) populations recently introduced in Poland

The alien Chinese mussel Anodonta woodiana was first reported in Poland in the system of heated lakes near Konin in 1993. Genetic studies with use of three molecular techniques (isoenzyme electrophoresis, PCR-RFLP and sequence analysis of a COI gene fragment) were carried out on the Polish first populations of A. woodiana. The studies have revealed low genetic variation between the populations (Nei's genetic distance for 12 loci ranged 0.000 to 0.007) as well as their considerable polymorphism. Each population averaged 2.28 alleles per locus, 2.72 alleles per polymorphic locus, and 75% polymorphic loci. Restriction analysis of the COI gene fragment have not revealed variability between the analysed specimens, including males and females. Restriction enzymes, ScrFI, Csp6I, and EcoRI used in the COI gene fragment PCR-RFLP generate distinct restriction patterns, which can be molecular markers for A. woodiana. The sequence obtained for COI fragment was the same in the examined female and male specimens and represents F mitotype (DNA was isolated from somatic tissues). The divergence between A. woodiana F and M mitotypes is high (34%), however it remains within the range of the general character of the DUI (doubly uniparental inheritance) phenomenon in freshwater bivalves (Unionidae).     

Identification of novel mutations in the ryanodine-receptor gene (RYR1) in malignant hyperthermia: genotype-phenotype correlation.

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle that is triggered in genetically predisposed individuals by common anesthetics and muscle relaxants. The ryanodine receptor (RYR1) is mutated in a number of MH pedigrees, some members of which also have central core disease (CCD), an inherited myopathy closely associated with MH. Mutation screening of 6 kb of the RYR1 gene has identified four adjacent novel mutations, C6487T, G6488A, G6502A, and C6617T, which result in the amino acid alterations Arg2163Cys, Arg2163His, Val2168Met, and Thr2206Met, respectively. Collectively, these mutations account for 11% of MH cases and identify the gene segment 6400-6700 as a mutation hot spot. Correlation analysis of the in vitro contracture-test data available for pedigrees bearing these and other RYR1 mutations showed an exceptionally good correlation between caffeine threshold and tension values, whereas no correlation was observed between halothane threshold and tension values. This finding has important ramifications for assignment of the MH-susceptible phenotype, in genotyping studies, and indicates that assessment of recombinant individuals on the basis of caffeine response is justified, whereas assessment on the basis of halothane response may be problematic. Interestingly, the data suggest a link between the caffeine threshold and tension values and the MH/CCD phenotype.     

Association of the estrogen receptor 1 (ESR1) gene with body height in adult males from two Swedish population cohorts

Human body height is a complex genetic trait with high heritability. We performed an association study of 17 candidate genes for height in the Uppsala Longitudinal Study of Adult Men (ULSAM) that consists of 1153 elderly men of age 70 born in the central region of Sweden. First we genotyped a panel of 137 single nucleotide polymorphism (SNPs) evenly distributed across the candidate genes in the ULSAM cohort. We identified 4 SNPs in the estrogen receptor gene (ESR1) on chromosome 6q25.1 with suggestive signals of association (p<0.05) with standing body height. This result was followed up by genotyping the same 25 SNPs in the ESR1 gene as in ULSAM in a second population cohort, the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) cohort that consist of 507 males and 509 females of age 70 from the same geographical region as ULSAM. One SNP, rs2179922 located in intron 4 of ESR1 showed and association signal (p = 0.0056) in the male samples from the PIVUS cohort. Homozygote carriers of the G-allele of the SNP rs2179922 were on average 0.90 cm taller than individuals with the two other genotypes at this SNP in the ULSAM cohort and 2.3 cm taller in the PIVUS cohort. No association was observed for the females in the PIVUS cohort.