Esterase-8 (Es-8): Characterization,polymorphism, and linkage of an erythrocyte esterase locus on chromosome 7 of Mus musculus

An electrophoretic polymorphism of an erythrocyte esterase, esterase-8, specific for the substrates α- and β-naphthyl acetate has been observed in the Asian house mouse, Mus musculus castaneus. M. m. castaneus is interfertile with inbred strains of mice, and F₁ hybrids (C57BL/6J × castaneus)F₁ and (SWR/J × castaneus)F₁ show a double-banded phenotype similar to a mixture of parental forms. This pattern suggests codominant expression of a structural gene difference. In backcrosses, ES-8 segregated as a single autosomal gene, designated Es-8, linked to Gpi-1 on chromosome 7. A gene order of Es-8, Gpi-1, c, Mod-2, and Hbb was determined from a series of crosses.     

Strain-Dependent Developmental Relaxation of Imprinting of an Endogenous Mouse Gene, Kvlqt1

Genomic imprinting is an epigenetic modification of the gamete or zygote leading to parental origin-specific differential expression of the two alleles of a gene in somatic cells of the offspring. We previously reported that the human K_VLQT1 gene is imprinted and disrupted in patients with germline balanced chromosomal rearrangements and Beckwith–Wiedemann syndrome. In human, the gene is imprinted in most fetal tissues except the heart, and K_VLQT1 is part of a 1-Mb cluster of imprinted genes on human chromosome 11p15.5. We sought to determine whether the mouse K_vlqt1 gene is imprinted, by performing interspecific crosses of 129/SvEv mice with CAST/E_i(Mus musculus castaneus). We identified a transcribed polymorphism that distinguishes the two parental alleles in F₁offspring. Examination of embryonic, neonatal, and postnatal tissues revealed that K_vlqt1 is imprinted in mouse early embryos, in both female 129 × male CS and female CS × male 129 offspring, with preferential expression of the maternal allele, like the human homologue. Surprisingly, imprinting was developmentally relaxed, and the developmental stage and tissue specificity of relaxation of imprinting was strain-dependent. To our knowledge, this is the first example of an endogenous gene that shows strain-dependent developmental relaxation of imprinting.     

Molecular evolution of the leptin exon 3 in some species of the family Canidae

The structure of the leptin gene seems to be well conserved. The polymorphism of this gene in four species belonging to the Canidae family (the dog (Canis familiaris) – 16 different breeds, the Chinese racoon dog (Nyctereutes procyonoides procyonoides), the red fox (Vulpes vulpes) and the arctic fox (Alopex lagopus)) were studied with the use of single strand conformation polymorphism (SSCP), restriction fragment length polymorphism (RFLP) and DNA sequencing techniques. For exon 2, all species presented the same SSCP pattern, while in exon 3 some differences were found. DNA sequencing of exon 3 revealed the presence of six nucleotide substitutions, differentiating the studied species. Three of them cause amino acid substitutions as well. For all dog breeds studied, SSCP patterns were identical.     

Quantitative Changes in Gimap3 and Gimap5 Expression Modify Mitochondrial DNA Segregation in Mice

Mammalian mitochondrial DNA (mtDNA) is a high-copy maternally inherited genome essential for aerobic energy metabolism. Mutations in mtDNA can lead to heteroplasmy, the co-occurence of two different mtDNA variants in the same cell, which can segregate in a tissue-specific manner affecting the onset and severity of mitochondrial dysfunction. To investigate mechanisms regulating mtDNA segregation we use a heteroplasmic mouse model with two polymorphic neutral mtDNA haplotypes (NZB and BALB) that displays tissue-specific and age-dependent selection for mtDNA haplotypes. In the hematopoietic compartment there is selection for the BALB mtDNA haplotype, a phenotype that can be modified by allelic variants of Gimap3. Gimap3 is a tail-anchored member of the GTPase of the immunity-associated protein (Gimap) family of protein scaffolds important for leukocyte development and survival. Here we show how the expression of two murine Gimap3 alleles from Mus musculus domesticus and M. m. castaneus differentially affect mtDNA segregation. The castaneus allele has incorporated a uORF (upstream open reading frame) in-frame with the Gimap3 mRNA that impairs translation and imparts a negative effect on the steady-state protein abundance. We found that quantitative changes in the expression of Gimap3 and the paralogue Gimap5, which encodes a lysosomal protein, affect mtDNA segregation in the mouse hematopoietic tissues. We also show that Gimap3 localizes to the endoplasmic reticulum and not mitochondria as previously reported. Collectively these data show that the abundance of protein scaffolds on the endoplasmic reticulum and lysosomes are important to the segregation of the mitochondrial genome in the mouse hematopoietic compartment.     

Prdm9 Polymorphism Unveils Mouse Evolutionary Tracks

PR/SET domain containing 9 (Prdm9) mediates histone modifications such as H3K4me3 and marks hotspots of meiotic recombination. In many mammalian species, the Prdm9 gene is highly polymorphic. Prdm9 polymorphism is assumed to play two critical roles in evolution: to diversify the spectrum of meiotic recombination hotspots and to cause male hybrid sterility, leading to reproductive isolation and speciation. Nevertheless, information about Prdm9 sequences in natural populations is very limited. In this study, we conducted a comprehensive population survey on Prdm9 polymorphism in the house mouse, Mus musculus. Overall M. musculus Prdm9 displays an extraordinarily high level of polymorphism, particularly in regions encoding zinc finger repeats, which recognize recombination hotspots. Prdm9 alleles specific to various M. musculus subspecies dominate in subspecies territories. Moreover, introgression into other subspecies territories was found for highly divergent Prdm9 alleles associated with t-haplotype. The results of our phylogeographical analysis suggest that the requirement for hotspot diversity depends on geographical range and time span in mouse evolution, and that Prdm9 polymorphism has not been maintained by a simple balanced selection in the population of each subspecies.     

Large-scale profiling and identification of potential regulatory mechanisms for allelic gene expression in colorectal cancer cells

Allelic variation in gene expression is common in humans and this variation is associated with phenotypic variation. In this study, we employed high-density single nucleotide polymorphism (SNP) chips containing 13,900 exonic SNPs to identify genes with allelic gene expression in cells from colorectal cancer cell lines. We found 2 monoallelically expressed genes (ERAP2 and MYLK4), 32 genes with an allelic imbalance in their expression, and 13 genes showing allele substitution by RNA editing. Among a total of 34 allelically expressed genes in colorectal cancer cells, 15 genes (44.1%) were associated with cis-acting eQTL, indicating that large portions of allelically expressed genes are regulated by cis-acting mechanisms of gene expression. In addition, potential regulatory variants present in the proximal promoter regions of genes showing either monoallelic expression or allelic imbalance were not tightly linked with coding SNPs, which were detected with allelic gene expression. These results suggest that multiple rare variants could be involved in the cis-acting regulatory mechanism of allelic gene expression. In the comparison with allelic gene expression data from Centre d'Etude du Polymorphisme Humain (CEPH) family B cells, 12 genes showed B-cell specific allelic imbalance and 1 noncoding SNP showed colorectal cancer cell-specific allelic imbalance. In addition, different patterns of allele substitution were observed between B cells and colorectal cancer cells. Overall, our study not only indicates that allelic gene expression is common in colorectal cancer cells, but our study also provides a better understanding of allele-specific gene expression in colorectal cancer cells.     

The Early-Onset Myocardial Infarction Associated PHACTR1 Gene Regulates Skeletal and Cardiac Alpha-Actin Gene Expression

The phosphatase and actin regulator 1 (PHACTR1) locus is a very commonly identified hit in genome-wide association studies investigating coronary artery disease and myocardial infarction (MI). However, the function of PHACTR1 in the heart is still unknown. We characterized the mechanisms regulating Phactr1 expression in the heart, used adenoviral gene delivery to investigate the effects of Phactr1 on cardiac function, and analyzed the relationship between MI associated PHACTR1 allele and cardiac function in human subjects. Phactr1 mRNA and protein levels were markedly reduced (60%, P<0.01 and 90%, P<0.001, respectively) at 1 day after MI in rats. When the direct myocardial effects of Phactr1 were studied, the skeletal α-actin to cardiac α-actin isoform ratio was significantly higher (1.5-fold, P<0.05) at 3 days but 40% lower (P<0.05) at 2 weeks after adenovirus-mediated Phactr1 gene delivery into the anterior wall of the left ventricle. Similarly, the skeletal α-actin to cardiac α-actin ratio was lower at 2 weeks in infarcted hearts overexpressing Phactr1. In cultured neonatal cardiac myocytes, adenovirus-mediated Phactr1 overexpression for 48 hours markedly increased the skeletal α-actin to cardiac α-actin ratio, this being associated with an enhanced DNA binding activity of serum response factor. Phactr1 overexpression exerted no major effects on the expression of other cardiac genes or LV structure and function in normal and infarcted hearts during 2 weeks’ follow-up period. In human subjects, MI associated PHACTR1 allele was not associated significantly with cardiac function (n = 1550). Phactr1 seems to regulate the skeletal to cardiac α-actin isoform ratio.     

Phactr4: A new integrin modulator required for directional migration of enteric neural crest cells

The enteric nervous system (ENS) is critically important for many intestinal functions such as peristalsis and secretion. Defects in the embryonic formation of the ENS cause Hirschsprung disease (HSCR) or megacolon, a severe birth defect that affects approximately 1 in 5,000 newborns. One of the least understood aspects of ENS development are the cellular and molecular mechanisms that control chain migration of the ENS cells during their migration into and along the embryonic gut. We recently reported a mouse model of HSCR in which mutant embryos carrying a hypomorphic allele of the Phactr4 gene show an embryonic gastrointestinal defect due to loss of enteric neurons in the colon. We found that Phactr4 modulates integrin signaling and cofilin activity to coordinate the forces that drive enteric neural crest cell (ENCC) migration in the mammalian embryo. In this extra view, we briefly summarize the current knowledge on integrin signaling in ENCC migration and introduce the Phactr protein family. Employing the ENS as a model, we shed some light on the mechanisms by which Phactr4 regulates integrin signaling and controls the cell polarity required for directional ENCC migration in the mouse developing gut.     

Significance of MEFV gene R202Q polymorphism in Turkish familial Mediterranean fever patients

Objective

Familial Mediterranean fever (FMF) is an autosomal recessive disorder characterized by recurrent attacks of fever and inflammation in the peritoneum, synovium, or pleura, accompanied by pain. The disease is associated with mutations in the Mediterranean fever (MEFV) gene, which encodes for the pyrin protein. The aim of this study was to explore the frequency and clinical significance of the R202Q (c.605G>A) polymorphism in exon 2 of the MEFV gene in a cohort of Turkish patients with FMF.

Methods

The study included 191 patients with FMF and 150 healthy controls. Genomic DNA was isolated and genotyped using polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) assay for the MEFV gene R202Qpolymorphism.

Results

The genotype and allele frequencies of R202Q polymorphism showed a statistically significant difference between FMF patients and controls (p < 0.0001 and p = 0.0004, respectively) and especially the homozygous AA genotype was significantly higher in FMF patients than healthy controls (p = 0.0002; odds ratio = 6.27; 95% CI = 2.1–18.3). However no significant association was observed between clinical and demographic features of FMF patients and R202Qpolymorphism.

Conclusion

The results of this study showed that there was a high association between MEFV gene R202Q polymorphism and FMF. R202Q polymorphism should be included in routine molecular diagnosis of FMF patients.     

Genetic analysis of OCT1 gene polymorphisms in an Indian population

BACKGROUND:

Genetic variants of the organic cation transporter (OCT1) gene could influence interindividual variation in clinical response to metformin therapy. The genetic basis for the single-nucleotide polymorphism (SNP) of OCT1 gene has been established in other populations, but it remains to be elucidated in the Indian population. This study is focused on OCT1 gene variants rs2282143 (P341L, 1022C>T), rs628031 (M408V, 1222A>G) and rs622342 (1386C>A) frequency distributions in the South Indian Tamilian population.

MATERIALS AND METHODS:

A total of 112 unrelated healthy subjects of South Indian Tamilian origin, aged 18–60 years, of either sex were recruited for the study. Genotyping was determined using the quantitative real time-polymerase chain reaction and polymerase chain reaction followed by restriction fragment length polymorphism methods.

RESULTS:

Allele frequencies of rs2282143, rs628031and rs622342 polymorphisms were 8.9%, 80.3% and 24.5%, respectively. Interethnic differences in the genotype and allele frequencies of OCT1 gene polymorphism were observed when compared with other major populations. The SNPs rs2282143, T allele and rs628031, G allele were more common in Asians (5.5–16.8% and 76.2–81%) and African Americans (8.2% and 73.5%) than in Caucasians (0–2% and 57.4–60%).

CONCLUSION:

This is the first time the frequency of OCT1 gene polymorphism was determined in the Indian population, and is similar to the frequencies observed in African-Americans and other Asian populations but different from those in Caucasians. The data observed in this study would justify further pharmacogenetic studies to potentially evaluate the role of OCT1 gene polymorphism in the therapeutic efficacy of metformin.     

Significant association of interleukin-4 gene intron 3 VNTR polymorphism with susceptibility to knee osteoarthritis

Objective

Interleukin-4 (IL-4) is a strong chondroprotective cytokine and polymorphisms within this gene may be a risk factor for osteoarthritis (OA). We aimed to investigate genotype and allele frequencies of IL-4 gene intron 3 variable number of tandem repeats (VNTR) polymorphism in patients with knee OA in a Turkish population.

Methods

The study included 202 patients with knee OA and 180 healthy controls. Genomic DNA was isolated and IL-4 gene 70 bp VNTR polymorphism determined by using polymerase chain reaction (PCR) with specific primers followed by restriction fragment length polymorphism (RFLP) analysis.

Results

Our result show that there was statistically significant difference between knee OA patients and control group with respect to IL-4 genotype distribution and allele frequencies (p = 0.000, OR: 0.20, 95% CI: 0.10–0.41, OR: 0.22, 95% CI: 0.12–0.42, respectively).

Conclusions

Our findings suggest that there is an association of IL-4 gene intron 3 VNTR polymorphism with susceptibility of a person for development of knee OA. As a result, IL-4 gene intron 3 VNTR polymorphism could be a genetic marker in OA in a Turkish study population. This is the first association study that evaluates the associations between IL-4 gene VNTR polymorphism and knee OA.     

Association between 1603C>T polymorphism of DBH gene and bipolar disorder in a Turkish population

Objectives

Dopamine-β-hydroxylase (DBH) is the enzyme responsible for the conversion of dopamine (DA) to norepinephrine (NE, noradrenaline) which is a key neurotransmitter in the central and peripheral nervous systems. Bipolar disorder is a major psychiatric disorder. The present study was designed to explore the associations of polymorphisms of DBH gene in Turkish patients with bipolar disorder.

Methods

− 1021C>T (rs1611115) polymorphism in promoter region, 444G>A (rs1108580) polymorphism in exon 2 and 1603C>T (rs6271; C535R) polymorphism in exon11 of DBH gene were analyzed in 106 patients with bipolar disorder and 106 healthy subjects by using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis.

Results

The results showed statistically significant associations for genotypic and allelic distribution between the 1603C>T polymorphism and bipolar disease (p = 0.0012 and p = 0.034, respectively). There was no association observed between the genotype and allelic frequencies for − 1021C>T and 444G>A polymorphisms and bipolar disorder.

Conclusions

Our data suggests that the 1603C>T polymorphism of the DBH gene is associated with susceptibility to bipolar disorder in a Turkish population.     

The telocentric tandem repeat at the p-arm is not conserved in Mus musculus subspecies

Mouse chromosomes, with the exception of the Y chromosome, are telocentric. The telomere at the p-arm is separated from the centromere by the tL1 sequence and TLC tandem repeats. A previous report showed that the TLC array was also conserved in other strains of the subgenus Mus. These results suggest that the TLC arrays promote the stable evolutionary maintenance of a telocentric karyotype in the subgenus Mus. In this study, we investigated the degree of conservation of TLC arrays among a variety of wild-derived inbred strains, all of which are descendants of wild mice captured in several areas of the world. Genomic PCR analysis indicates that the sequential order of telomere-tL1 is highly conserved in all strains, whereas tL1-TLC is not. Next, Southern blot analysis of DNAs isolated from a panel of mouse subspecies showed both Mus musculus domesticus and Mus musculus castaneus subspecies possess TLC arrays. Unexpectedly, this repeat appears to be lost in almost all Mus musculus musculus and Mus musculus molossinus subspecies, which show a clear geographic divide. These results indicate that either other unknown sequences were replaced by the TLC repeat or almost all M. m. musculus and M. m. molossinus subspecies do not have any sequence between the telomere and minor satellites. Our observation suggests that the TLC array might be evolutionarily unstable and not essential for murine chromosomal conformation. This is the first example of the subspecies-specific large genome alterations in mice.     

A Novel Statistical Approach for Jointly Analyzing RNA-Seq Data from F1 Reciprocal Crosses and Inbred Lines

RNA sequencing (RNA-seq) not only measures total gene expression but may also measure allele-specific gene expression in diploid individuals. RNA-seq data collected from F₁ reciprocal crosses in mice can powerfully dissect strain and parent-of-origin effects on allelic imbalance of gene expression. In this article, we develop a novel statistical approach to analyze RNA-seq data from F₁ and inbred strains. Method development was motivated by a study of F₁ reciprocal crosses derived from highly divergent mouse strains, to which we apply the proposed method. Our method jointly models the total number of reads and the number of allele-specific reads of each gene, which significantly boosts power for detecting strain and particularly parent-of-origin effects. The method deals with the overdispersion problem commonly observed in read counts and can flexibly adjust for the effects of covariates such as sex and read depth. The X chromosome in mouse presents particular challenges. As in other mammals, X chromosome inactivation silences one of the two X chromosomes in each female cell, although the choice of which chromosome to be silenced can be highly skewed by alleles at the X-linked X-controlling element (Xce) and stochastic effects. Our model accounts for these chromosome-wide effects on an individual level, allowing proper analysis of chromosome X expression. Furthermore, we propose a genomic control procedure to properly control type I error for RNA-seq studies. A number of these methodological improvements can also be applied to RNA-seq data from other species as well as other types of next-generation sequencing data sets. Finally, we show through simulations that increasing the number of samples is more beneficial than increasing the library size for mapping both the strain and parent-of-origin effects. Unless sample recruiting is too expensive to conduct, we recommend sequencing more samples with lower coverage.     

Multiplex allele-specific PCR combined with PCR-RFLP analysis for rapid detection of gyrA gene fluoroquinolone resistance mutations in Mycobacterium tuberculosis

A combined use of MAS-PCR (multiplex allele-specific PCR) and PCR-RFLP (PCR restriction fragment length polymorphism), was established to detect mutations in codons 90, 91 and 94 of the gyrA gene in Mycobacterium tuberculosis (M. tuberculosis). With conventional phenotypic drug susceptibility testing as a reference standard, the sensitivity, specificity and accuracy of the modified method for gyrA gene mutation detection were 70.8%, 100% and 84.8% respectively.