Genetic Dissection of Quantitative Trait Loci for Hemostasis and Thrombosis on Mouse Chromosomes 11 and 5 Using Congenic and Subcongenic Strains

Susceptibility to thrombosis varies in human populations as well as many inbred mouse strains. Only a small portion of this variation has been identified, suggesting that there are unknown modifier genes. The objective of this study was to narrow the quantitative trait locus (QTL) intervals previously identified for hemostasis and thrombosis on mouse distal chromosome 11 (Hmtb6) and on chromosome 5 (Hmtb4 and Hmtb5). In a tail bleeding/rebleeding assay, a reporter assay for hemostasis and thrombosis, subcongenic strain (6A-2) had longer clot stability time than did C57BL/6J (B6) mice but a similar time to the B6-Chr11^A/J consomic mice, confirming the Hmtb6 phenotype. Six congenic and subcongenic strains were constructed for chromosome 5, and the congenic strain, 2A-1, containing the shortest A/J interval (16.6 cM, 26.6 Mbp) in the Hmtb4 region, had prolonged clot stability time compared to B6 mice. In the 3A-2 and CSS-5 mice bleeding time was shorter than for B6, mice confirming the Hmtb5 QTL. An increase in bleeding time was identified in another congenic strain (3A-1) with A/J interval (24.8 cM, 32.9 Mbp) in the proximal region of chromosome 5, confirming a QTL for bleeding previously mapped to that region and designated as Hmtb10. The subcongenic strain 4A-2 with the A/J fragment in the proximal region had a long occlusion time of the carotid artery after ferric chloride injury and reduced dilation after injury to the abdominal aorta compared to B6 mice, suggesting an additional locus in the proximal region, which was designated Hmtb11 (5 cM, 21.4 Mbp). CSS-17 mice crossed with congenic strains, 3A-1 and 3A-2, modified tail bleeding. Using congenic and subcongenic analysis, candidate genes previously identified and novel genes were identified as modifiers of hemostasis and thrombosis in each of the loci Hmtb6, Hmtb4, Hmtb10, and Hmtb11.     

Quantitative trait locus analysis for hemostasis and thrombosis

Susceptibility to thrombosis varies in human populations as well as many in inbred mouse strains. The objective of this study was to characterize the genetic control of thrombotic risk on three chromosomes. Previously, utilizing a tail-bleeding/rebleeding assay as a surrogate of hemostasis and thrombosis function, three mouse chromosome substitution strains (CSS) (B6-Chr5^A/J, Chr11^A/J _, Chr17^A/J) were identified (Hmtb1, Hmtb2, Hmtb3). The tail-bleeding/rebleeding assay is widely used and distinguishes mice with genetic defects in blood clot formation or dissolution. In the present study, quantitative trait locus (QTL) analysis revealed a significant locus for rebleeding (clot stability) time (time between cessation of initial bleeding and start of the second bleeding) on chromosome 5, suggestive loci for bleeding time (time between start of bleeding and cessation of bleeding) also on chromosomes 5, and two suggestive loci for clot stability on chromosome 17 and one on chromosome 11. The three CSS and the parent A/J had elevated clot stability time. There was no interaction of genes on chromosome 11 with genes on chromosome 5 or chromosome 17. On chromosome 17, twenty-three candidate genes were identified in synteny with previously identified loci for thrombotic risk on human chromosome 18. Thus, we have identified new QTLs and candidate genes not previously known to influence thrombotic risk. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

EMILIN2 Regulates Platelet Activation,Thrombus Formation,and Clot Retraction

Thrombosis, like other cardiovascular diseases, has a strong genetic component, with largely unknown determinants. EMILIN2, Elastin Microfibril Interface Located Protein2, was identified as a candidate gene for thrombosis in mouse and human quantitative trait loci studies. EMILIN2 is expressed during cardiovascular development, on cardiac stem cells, and in heart tissue in animal models of heart disease. In humans, the EMILIN2 gene is located on the short arm of Chromosome 18, and patients with partial and complete deletion of this chromosome region have cardiac malformations. To understand the basis for the thrombotic risk associated with EMILIN2, EMILIN2 deficient mice were generated. The findings of this study indicate that EMILIN2 influences platelet aggregation induced by adenosine diphosphate, collagen, and thrombin with both EMILIN2-deficient platelets and EMILIN2-deficient plasma contributing to the impaired aggregation response. Purified EMILIN2 added to platelets accelerated platelet aggregation and reduced clotting time when added to EMILIN2-deficient mouse and human plasma. Carotid occlusion time was 2-fold longer in mice with platelet-specific EMILIN2 deficiency, but stability of the clot was reduced in mice with both global EMILIN2 deficiency and with platelet-specific EMILIN2 deficiency. In vitro clot retraction was markedly decreased in EMILIN2 deficient mice, indicating that platelet outside-in signaling was dependent on EMILIN2. EMILIN1 deficient mice and EMILIN2:EMILIN1 double deficient mice had suppressed platelet aggregation and delayed clot retraction similar to EMILIN2 mice, but EMILIN2 and EMILIN1 had opposing affects on clot retraction, suggesting that EMILIN1 may attenuate the effects of EMILIN2 on platelet aggregation and thrombosis. In conclusion, these studies identify multiple influences of EMILIN2 in pathophysiology and suggest that its role as a prothrombotic risk factor may arise from its effects on platelet aggregation and platelet mediated clot retraction.     

Fine-mapping alleles for body weight in LG/J �� SM/J F2 and F34 advanced intercross lines

The present study measured variation in body weight using a combined analysis in an F₂ intercross and an F₃₄ advanced intercross line (AIL). Both crosses were derived from inbred LG/J and SM/J mice, which were selected for large and small body size prior to inbreeding. Body weight was measured at 62 (±5) days of age. Using an integrated GWAS and forward model selection approach, we identified 11 significant QTLs that affected body weight on ten different chromosomes. With these results we developed a full model that explained over 18% of the phenotypic variance. The median 1.5-LOD support interval was 5.55 Mb, which is a significant improvement over most prior body weight QTLs. We identified nonsynonymous coding SNPs between LG/J and SM/J mice in order to further narrow the list of candidate genes. Three of the genes with nonsynonymous coding SNPs (Rad23b, Stk33, and Anks1b) have been associated with adiposity, waist circumference, and body mass index in human GWAS, thus providing evidence that these genes may underlie our QTLs. Our results demonstrate that a relatively small number of loci contribute significantly to the phenotypic variance in body weight, which is in marked contrast to the situation in humans. This difference is likely to be the result of strong selective pressure and the simplified genetic architecture, both of which are important advantages of our system.     

Emilin1 deficiency causes structural and functional defects of lymphatic vasculature

Lymphatic-vasculature function critically depends on extracellular matrix (ECM) and on its connections with lymphatic endothelial cells (LECs). However, the composition and the architecture of ECM have not been fully taken into consideration in studying the biology and the pathology of the lymphatic system. EMILIN1, an elastic microfibril-associated protein, is highly expressed by LECs in vitro and colocalizes with lymphatic vessels in several mouse tissues. A comparative study between WT and Emilin1-/- mice highlighted the fact that Emilin1 deficiency in both CD1 and C57BL/6 backgrounds results in hyperplasia, enlargement, and frequently an irregular pattern of superficial and visceral lymphatic vessels and in a significant reduction of anchoring filaments. Emilin1-deficient mice also develop larger lymphangiomas than WT mice. Lymphatic vascular morphological alterations are accompanied by functional defects, such as mild lymphedema, a highly significant drop in lymph drainage, and enhanced lymph leakage. Our findings demonstrate that EMILIN1 is involved in the regulation of the growth and in the maintenance of the integrity of lymphatic vessels, a fundamental requirement for efficient function. The phenotype displayed by Emilin1(-/-) mice is the first abnormal lymphatic phenotype associated with the deficiency of an ECM protein and identifies EMILIN1 as a novel local regulator of lymphangiogenesis.     

H2 control of natural T regulatory cell frequency in the lymph node correlates with susceptibility to day 3 thymectomy-induced autoimmune disease

Day 3 thymectomy (D3Tx) results in a loss of peripheral tolerance mediated by natural regulatory T cells (nTregs) and development of autoimmune ovarian dysgenesis (AOD) and autoimmune dacryoadenitis (ADA) in A/J and (C57BL/6J × A/J) F(1) hybrids (B6A), but not in C57BL/6J (B6) mice. Previously, using quantitative trait locus (QTL) linkage analysis, we showed that D3Tx-AOD is controlled by five unlinked QTL (Aod1-Aod5) and H2. In this study, using D3Tx B6-Chr(A/J)/NaJ chromosome (Chr) substitution strains, we confirm that QTL on Chr16 (Aod1a/Aod1b), Chr3 (Aod2), Chr1 (Aod3), Chr2 (Aod4), Chr7 (Aod5), and Chr17 (H2) control D3Tx-AOD susceptibility. In addition, we also present data mapping QTL controlling D3Tx-ADA to Chr17 (Ada1/H2), Chr1 (Ada2), and Chr3 (Ada3). Importantly, B6-ChrX(A/J) mice were as resistant to D3Tx-AOD and D3Tx-ADA as B6 mice, thereby excluding Foxp3 as a susceptibility gene in these models. Moreover, we report quantitative differences in the frequency of nTregs in the lymph nodes (LNs), but not spleen or thymus, of AOD/ADA-resistant B6 and AOD/ADA-susceptible A/J, B6A, and B6-Chr17(A/J) mice. Similar results correlating with experimental allergic encephalomyelitis and orchitis susceptibility were seen with B10.S and SJL/J mice. Using H2-congenic mice, we show that the observed difference in frequency of LN nTregs is controlled by Ada1/H2. These data support the existence of an LN-specific, H2-controlled mechanism regulating the prevalence of nTregs in autoimmune disease susceptibility.     

Functional Analyses of Endometriosis-Related Polymorphisms in the Estrogen Synthesis and Metabolism-Related Genes

Endometriosis is determined by genetic factors, and the prevalence of genetic polymorphisms varies greatly depending on the ethnic group studied. The objective of this study was to investigate the relationship between single nucleotide polymorphisms (SNPs) of 9 genes involved in estrogen biosynthesis and metabolism and the risks of endometriosis. Three hundred patients with endometriosis and 337 non-endometriotic controls were recruited. Thirty four non-synonymous SNPs, which change amino acid residues, were analyzed using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). The functions of SNP-resulted amino acid changes were analyzed using multiple web-accessible databases and phosphorylation predicting algorithms. Among the 34 NCBI-listed SNPs, 22 did not exhibit polymorphism in this study of more than 600 Taiwanese Chinese women. However, homozygous and heterozygous mutants of 4 SNPs - rs6165 (genotype GG+GA, 307^Ala/Ala+307^Ala/Thr) of FSHR, rs 6166 (genotype GG+GA, 680^Ser/Asn+680^Ser/Ser) of FSHR, rs2066479 (genotype AA+AG, 289^Ser/Ser+289^Ser/Gly) of HSD17B3 and rs700519 (genotype TT+TC, 264^Cys/Cys+264^Cys/Arg) of CYP19, alone or in combination, were significantly associated with decreased risks of endometriosis. Bioinformatics results identified 307^Thr of FSHR to be a site for O-linked glycosylation, 680^Ser of FSHR a phosphorylated site by protein kinase B, and 289^Ser of HSD17B3 a phosphorylated site by protein kinase B or ribosomal protein S6 kinase 1. Results of this study suggest that non-synonymous polymorphisms of FSHR, HSD17B3 and CYP19 genes may modulate the risk of endometriosis in Taiwanese Chinese women. Identification of the endometrosis-preferential non-synonymous SNPs and the conformational changes in those proteins may pave the way for the development of more disease-specific drugs.     

Paternal Allele Influences High Fat Diet-Induced Obesity

C57BL/6J (B6) mice are susceptible to high-fat diet (HFD)-induced obesity and have been used in metabolism research for many decades. However, the genetic component of HFD-induced obesity has not yet been elucidated. This study reports evidence for a paternal transmission of HFD-induced obesity and a correlated expression of Igf2 and Peg3 (paternal expressed gene 3) imprinted genes. We found that PWK mice are resistant to HFD-induced obesity compared to C57BL/6J mice. Therefore, we generated and analyzed reciprocal crosses between these mice, namely; (PWK×B6) F1 progeny with B6 father and (B6×PWK) F1 progeny with PWK father. The (PWK×B6) F1 mice were more sensitive to diet-induced obesity compared to (B6×PWK) F1 mice, suggesting a paternal transmission of diet-induced obesity. Expression analysis of imprinted genes in adipocytes revealed that HFD influences the expression of some of the imprinted genes in adipose tissue in B6 and PWK mice. Interestingly, Igf2 and Peg3, which are paternally expressed imprinted genes involved in the regulation of body fat accumulation, were down-regulated in B6 and (PWK×B6) F1 mice, which are susceptible to HFD-induced obesity, but not in PWK and (B6×PWK) F1 mice, which are resistant. Furthermore, in vitro analysis showed that Igf2, but not Peg3, had an anti-inflammatory effect on TNF-α induced MCP-1 expression in adipocytes. Taken together, our findings suggest that the down-regulation of Igf2 and Peg3 imprinted genes in adipocytes may be involved in the paternal transmission of HFD-induced obesity.     

Androgen metabolism and JAK/STAT pathway genes and prostate cancer risk

Background: Prostate cancer (PC) is the most frequently diagnosed solid tumor in U.S. men. Genome-wide association studies (GWAS) have identified over 40 risk-associated single nucleotide polymorphisms (SNPs), including variants in androgen pathway genes (e.g., KLK3 and AR). Androgens are important in PC and genes involved in this pathway are therefore candidates for conferring susceptibility to PC. Methods: In this hypothesis-testing study, we evaluated PC risk in association with SNPs in 22 candidate genes involved in androgen metabolism or interactions with the androgen receptor (AR). A total of 187 SNPs were genotyped in 1458 cases and 1351 age-matched controls from a population-based study. PC risk was estimated using adjusted unconditional logistic regression and multinomial regression models. Results: Single SNP analyses showed evidence (p < 0.05) for associations with 14 SNPs in 9 genes: NKX3.1, HSD17B3, AKR1C3, SULT2A1, CYP17A1, KLK3, JAK2, NCOA4 and STAT3. The most significant result was observed for rs2253502 in HSD17B3 (odds ratio, OR = 0.57, 95% CI: 0.39–0.84). In addition, five SNPs in four genes (CYP17A1, HSD17B4, NCOA4, and SULT2A1) were associated with more aggressive disease (p < 0.01). Conclusions: Our results replicate previously reported associations for SNPs in CYP17A1, HSD17B3, ARK1C3, NKX3.1, NCOA4 and KLK3. In addition, novel associations were observed for SNPs in JAK2, HSD17B4, and SULT2A1. These results will require replication in larger studies.     

TGF-β mediates early angiogenesis and latent fibrosis in an Emilin1-deficient mouse model of aortic valve disease

Aortic valve disease (AVD) is characterized by elastic fiber fragmentation (EFF), fibrosis and aberrant angiogenesis. Emilin1 is an elastin-binding glycoprotein that regulates elastogenesis and inhibits TGF-β signaling, but the role of Emilin1 in valve tissue is unknown. We tested the hypothesis that Emilin1 deficiency results in AVD, mediated by non-canonical (MAPK/phosphorylated Erk1 and Erk2) TGF-β dysregulation. Using histology, immunohistochemistry, electron microscopy, quantitative gene expression analysis, immunoblotting and echocardiography, we examined the effects of Emilin1 deficiency (Emilin1^−/−) in mouse aortic valve tissue. Emilin1 deficiency results in early postnatal cell-matrix defects in aortic valve tissue, including EFF, that progress to latent AVD and premature death. The Emilin1^−/− aortic valve displays early aberrant provisional angiogenesis and late neovascularization. In addition, Emilin1^−/− aortic valves are characterized by early valve interstitial cell activation and proliferation and late myofibroblast-like cell activation and fibrosis. Interestingly, canonical TGF-β signaling (phosphorylated Smad2 and Smad3) is upregulated constitutively from birth to senescence, whereas non-canonical TGF-β signaling (phosphorylated Erk1 and Erk2) progressively increases over time. Emilin1 deficiency recapitulates human fibrotic AVD, and advanced disease is mediated by non-canonical (MAPK/phosphorylated Erk1 and Erk2) TGF-β activation. The early manifestation of EFF and aberrant angiogenesis suggests that these processes are crucial intermediate factors involved in disease progression and therefore might provide new therapeutic targets for human AVD.KEY WORDS: Elastic fibers, Extracellular matrix, Aortic valve, Fibrosis, Angiogenesis     

Genetic control of mitogen-induced B-cell hyperproliferation in SM/J mice

Previous studies have shown that B cells from SM/J mice exhibit hyperproliferative responsiveness to several bacterial-derived B-cell mitogens. This hyperresponse trait was found to be under autosomal, polygenic control by non-H-2 genes. We have now estimated the number of genes involved by statistical analysis of the proliferative responses of splenocytes from SM/J and low-responder C57BL/6J strains, and progeny from the (B6 × SM)F₁, F₂ and (F₁ × B6) crosses. The number of loci involved was ascertained using two different statistical approaches. An estimate of two loci was determined using chi-squared statistics. The second approach, based on an additive model in the natural log scale, also pointed to a lower bound of two genes. We conclude that the hyperresponse to B-cell mitogens in SM/J mice is determined by two autosomal genes which are not linked to the H-2 major histocompatibility complex.Abbreviations used in this paper LPS a bacterial lipopolysaccharide - AVIS a mitogenic preparation from Actinomyces viscosus - B6 C57BL/6J mice - ¹²⁵IUdR ¹²⁵Iodo-deoxyuridine     

Searching for Genomic Region of High-Fat Diet-Induced Type 2 Diabetes in Mouse Chromosome 2 by Analysis of Congenic Strains

SMXA-5 mice are a high-fat diet-induced type 2 diabetes animal model established from non-diabetic SM/J and A/J mice. By using F2 intercross mice between SMXA-5 and SM/J mice under feeding with a high-fat diet, we previously mapped a major diabetogenic QTL (T2dm2sa) on chromosome 2. We then produced the congenic strain (SM.A-T2dm2sa (R0), 20.8–163.0 Mb) and demonstrated that the A/J allele of T2dm2sa impaired glucose tolerance and increased body weight and body mass index in the congenic strain compared to SM/J mice. We also showed that the combination of T2dm2sa and other diabetogenic loci was needed to develop the high-fat diet-induced type 2 diabetes. In this study, to narrow the potential genomic region containing the gene(s) responsible for T2dm2sa, we constructed R1 and R2 congenic strains. Both R1 (69.6–163.0 Mb) and R2 (20.8–128.2 Mb) congenic mice exhibited increases in body weight and abdominal fat weight and impaired glucose tolerance compared to SM/J mice. The R1 and R2 congenic analyses strongly suggested that the responsible genes existed in the overlapping genomic interval (69.6–128.2 Mb) between R1 and R2. In addition, studies using the newly established R1A congenic strain showed that the narrowed genomic region (69.6–75.4 Mb) affected not only obesity but also glucose tolerance. To search for candidate genes within the R1A genomic region, we performed exome sequencing analysis between SM/J and A/J mice and extracted 4 genes (Itga6, Zak, Gpr155, and Mtx2) with non-synonymous coding SNPs. These four genes might be candidate genes for type 2 diabetes caused by gene-gene interactions. This study indicated that one of the genes responsible for high-fat diet-induced diabetes exists in the 5.8 Mb genomic interval on mouse chromosome 2.     

High-Resolution Mapping of a Genetic Locus Regulating Preferential Carbohydrate Intake,Total Kilocalories,and Food Volume on Mouse Chromosome 17

The specific genes regulating the quantitative variation in macronutrient preference and food intake are virtually unknown. We fine mapped a previously identified mouse chromosome 17 region harboring quantitative trait loci (QTL) with large effects on preferential macronutrient intake-carbohydrate (Mnic1), total kilcalories (Kcal2), and total food volume (Tfv1) using interval-specific strains. These loci were isolated in the [C57BL/6J.CAST/EiJ-17.1-(D17Mit19-D17Mit50); B6.CAST-17.1] strain, possessing a ∼40.1 Mb region of CAST DNA on the B6 genome. In a macronutrient selection paradigm, the B6.CAST-17.1 subcongenic mice eat 30% more calories from the carbohydrate-rich diet, ∼10% more total calories, and ∼9% more total food volume per body weight. In the current study, a cross between carbohydrate-preferring B6.CAST-17.1 and fat-preferring, inbred B6 mice was used to generate a subcongenic-derived F₂ mapping population; genotypes were determined using a high-density, custom SNP panel. Genetic linkage analysis substantially reduced the 95% confidence interval for Mnic1 (encompassing Kcal2 and Tfv1) from 40.1 to 29.5 Mb and more precisely established its boundaries. Notably, no genetic linkage for self-selected fat intake was detected, underscoring the carbohydrate-specific effect of this locus. A second key finding was the separation of two energy balance QTLs: Mnic1/Kcal2/Tfv1 for food intake and a newly discovered locus regulating short term body weight gain. The Mnic1/Kcal2/Tfv1 QTL was further de-limited to 19.0 Mb, based on the absence of nutrient intake phenotypes in subcongenic HQ17IIa mice. Analyses of available sequence data and gene ontologies, along with comprehensive expression profiling in the hypothalamus of non-recombinant, cast/cast and b6/b6 F₂ controls, focused our attention on candidates within the QTL interval. Zfp811, Zfp870, and Btnl6 showed differential expression and also contain stop codons, but have no known biology related to food intake regulation. The genes Decr2, Ppard and Agapt1 are more appealing candidates because of their involvement in lipid metabolism and down-regulation in carbohydrate-preferring animals.     

Ion Torrent sequencing for conducting genome-wide scans for mutation mapping analysis

Mutation mapping in mice can be readily accomplished by genome wide segregation analysis of polymorphic DNA markers. In this study, we showed the efficacy of Ion Torrent next generation sequencing for conducting genome-wide scans to map and identify a mutation causing congenital heart disease in a mouse mutant, Bishu, recovered from a mouse mutagenesis screen. The Bishu mutant line generated in a C57BL/6J (B6) background was intercrossed with another inbred strain, C57BL/10J (B10), and the resulting B6/B10 hybrid offspring were intercrossed to generate mutants used for the mapping analysis. For each mutant sample, a panel of 123 B6/B10 polymorphic SNPs distributed throughout the mouse genome was PCR amplified, bar coded, and then pooled to generate a single library used for Ion Torrent sequencing. Sequencing carried out using the 314 chip yielded >600,000 usable reads. These were aligned and mapped using a custom bioinformatics pipeline. Each SNP was sequenced to a depth >500×, allowing accurate automated calling of the B6/B10 genotypes. This analysis mapped the mutation in Bishu to an interval on the proximal region of mouse chromosome 4. This was confirmed by parallel capillary sequencing of the 123 polymorphic SNPs. Further analysis of genes in the map interval identified a splicing mutation in Dnaic1 ^c.204+1G>A, an intermediate chain dynein, as the disease causing mutation in Bishu. Overall, our experience shows Ion Torrent amplicon sequencing is high throughput and cost effective for conducting genome-wide mapping analysis and is easily scalable for other high volume genotyping analyses.     

Genomic analysis of the appearance of testicular oocytes in MRL/MpJ mice

Mammals produce sperm or oocytes depending on their sex; however, newborn MRL/MpJ (MRL) male mice produce oocytes within their testes. We previously reported that one of the genes responsible for this phenotype is present on the MRL-type Y chromosome (Y^MRL), and that multiple genes, probably autosomal, are also required for the development of this phenotype. In this study we focused on the autosomal genes and examined their relationship with this phenotype by analyzing the progeny from crosses between MRL mice and other strains. We first observed the male F1 progeny from the crosses between female A/J, C57BL/6 (B6), BALB/c, C3H/He, or DBA/2 mice and male MRL mice, and two consomic strains, male B6-Y^MRL and MRL-Y^B6. Testicular oocytes that were morphologically similar to those of MRL mice were detected in all mouse strains except BALBMRLF1; however, the incidence of testicular oocytes was significantly lower than that in MRL mice. The appearance of testicular oocytes in MRL-Y^B6 mice indicates that this phenotype is strongly affected by genomic factors present on autosomes, and that there is at least one other causative gene on the MRL-type autosomes (MRL testicular oocyte production, mtop) other than that on Y^MRL. Furthermore, a quantitative trait locus (QTL) analysis using N2 backcross progeny from crosses between female MRLB6F1 and male MRL mice revealed the presence of susceptibility loci for the appearance of testicular oocytes at 8?C17?cM on Chr 15. These findings demonstrate that the appearance of testicular oocytes is regulated by the genetic factors on Chr 15 and on Y^MRL.     

MYP2 locus genes: Sequence variations,genetic association studies and haplotypic association in patients with High Myopia

High Myopia (HM) is a common complex-trait eye disorder. There is essential evidence that genetic factors play a significant role in the development of nonsyndromic high myopia. Identification of susceptibility genes of high myopia will shed light on the pathophysiological mechanism underlying their genesis. This was a case control study examining the prospect of association of DLGAP1, EMILIN2 & MYOM1 genes on MYP2 locus in purely ethnic (Kashmiri) population representing a homogeneous cohort. Genomic DNA was extracted using phenol chloroform and salting out method. Extracted DNA was genotyped for polymorphic variations in MYOM1, EMILIN2 and DLGAP1 genes involving Sanger di-deoxy method. Allele frequencies were tested for Hardy-Weinberg disequilibrium in 224 cases and compared with 220 emmetropic controls. In DLGAP1, documented single nucleotide polymorphism (SNP); Pro517Pro was observed. A previously reported Asn451Asn SNP was observed in EMILIN2. MYOM1 showed five polymorphic variations; two in coding region (Gly333Gly & Gly341Ala) and three intronic (c.1022+23, G>A; c.3418+44 G>T & c.3418+65; C>G). All of the elucidated SNPs were having statistical significant role in increasing or decreasing the risk of disease. Although not statistically significant, a novel Glu507Lys SNP was observed in DLGAP1 (P>0.05). In silico predictions showed MYOM1 Gly341Ala to be benign & tolerated substitution while as DLGAP1 Glu507Lys to be possibly damaging substitution. The studied SNPs followed Over-Dominant, Recessive and Co-Dominant mode of inheritance with specific haplotypes associated with the disease. Our study reveals the involvement of MYP2 locus candidate gene polymorphism in the pathogenesis of HM.