Second Case of Gonadal Mosaicism and a Novel Nonsense NR2F1 Gene Variant as the Cause of Bosch-Boonstra-Schaaf Optic Atrophy Syndrome.

Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS) is an autosomal dominant disease characterized by developmental delay, intellectual disability, and optic atrophy with a variable expression of other clinical features (dysmorphic features, autistic behaviour, corpus callosum hypoplasia and seizures). To date, approximately a hundred cases of the syndrome have been described, with an estimated prevalence of 1 in 100 000-250 000. BBSOAS is caused by the loss of function of the NR2F1 gene (nuclear receptor subfamily 2 group F member 1), which encodes the COUP-TFI (Chicken ovalbumin upstream promotor-transcription factor 1). COUP-TFI functions as a homodimer and is one of the major transcriptional regulators directing cortical arealization, cell differentiation and maturation. Most cases of BBSOAS occur de novo, and one case was previously described in which the disease resulted from gonadal mosaicism. In the present case, we report two sisters with BBSOAS, a novel nonsense mutation in the NR2F1 gene and potential gonadal mosaicism as the cause of this rare disease, making it the second such case described in the literature.

Loss-of-Function Mutations in NR4A2 Cause Dopa-Responsive Dystonia Parkinsonism.

BACKGROUND: The group of dystonia genes is expanding, and mutations of these genes have been associated with various combined dystonia syndromes. Among the latter, the cause of some dystonia parkinsonism cases remains unknown. OBJECTIVE: To report patients with early-onset dystonia parkinsonism as a result of loss-of-function mutations in nuclear receptor subfamily 4 group A member 2. METHODS: Phenotypic characterization and exome sequencing were carried out in 2 families. RESULTS: The 2 patients reported here both had a history of mild intellectual disability in childhood and subsequently developed dystonia parkinsonism in early adulthood. Brain magnetic resonance imaging was normal, and DATscan suggested bilateral dopaminergic denervation. Two frameshift mutations in NR4A2 were identified: a de novo insertion (NM_006186.3; c.326dupA) in the first case and another small insertion (NM_006186.3; c.881dupA) in the second. CONCLUSIONS: NR4A2 haploinsufficiency mutations have been recently reported in neurodevelopmental phenotypes. Our findings indicate that dystonia and/or parkinsonism may appear years after initial symptoms. Mutations in NR4A2 should be considered in patients with unexplained dystonia parkinsonism. © 2020 International Parkinson and Movement Disorder Society.

Comprehensive use of extended exome analysis improves diagnostic yield in rare disease: a retrospective survey in 1,059 cases.

PurposeWe sought to determine the analytical sensitivity of several extended exome variation analysis approaches in terms of their contribution to diagnostic yield and their clinical feasibility.MethodsWe retrospectively analyzed the results of genetic testing in 1,059 distinct cases referred for exome sequencing to our institution. In these, we routinely employed extended exome analysis approaches in addition to basic variant analysis, including (i) copy-number variation (CNV) detection, (ii) nonconsensus splice defect detection, (ii) genomic breakpoint detection, (iv) homozygosity mapping, and (v) mitochondrial variant analysis.ResultsExtended exome analysis approaches assisted in identification of causative genetic variant in 44 cases, which represented a 4.2% increase in diagnostic yield. The greatest contribution was associated with CNV analysis (1.8%) and splice variant prediction (1.2%), and the remaining approaches contributed an additional 1.2%. Analysis of workload has shown that on average nine additional variants per case had to be interpreted in the extended analysis.ConclusionWe show that extended exome analysis approaches improve the diagnostic yield of heterogeneous genetic disorders and result in considerable increase of diagnostic yield of exome sequencing with a minor increase of interpretative workload.

Functional single nucleotide polymorphisms of matrix metalloproteinase 7 and 12 genes in idiopathic recurrent spontaneous abortion.

PURPOSE: The aim of this study was to investigate the potential association of matrix metalloproteinase 7 (MMP7) -181 A/G and MMP12 -82 A/G functional single nucleotide polymorphisms (SNP) with idiopathic recurrent spontaneous abortion (IRSA) in Slovenian reproductive couples. METHODS: A case-control study was conducted on 149 couples with 3 or more consecutive idiopathic spontaneous pregnancy loses and 149 women and men with at least 2 live births and no history of pregnancy complications. Genotyping of MMP7 -181 A/G and MMP12 -82 A/G SNPs was performed using polymerase chain reaction and restriction fragment length polymorphism methods. RESULTS: There were no statistically significant differences in the distribution of MMP7 -181 A/G and MMP12 -82 A/G genotype, allele, or haplotype frequencies between IRSA patients and controls, as well as patients' primary and secondary IRSA. We also found no association of MMP7 -181 A/G and MMP12 -82 A/G genotypes, alleles, and haplotypes with IRSA. CONCLUSIONS: We found no evidence to support the association between IRSA and MMP7 -181 A/G and MMP12 -82 A/G SNPs in Slovenian reproductive couples.

Phenotype-driven gene target definition in clinical genome-wide sequencing data interpretation.

PURPOSE: Genome-wide sequencing approaches are increasingly being used in place of disease gene panel sequencing approaches. Despite the well-recognized benefits of these approaches, they also carry with them an increased burden of analyzing overwhelmingly large gene targets and an increased possibility of detecting incidental findings. METHODS: We propose a novel approach for design of individualized phenotype gene panels using the set of signs and symptoms observed and selecting relevant genes on the basis of known phenotype-gene associations. RESULTS: We used results of diagnostic exome sequencing in 405 cases submitted to our institution to show retrospectively that using the phenotype gene panel increases the sensitivity of masked exome analysis (increase from 25.4 to 29.7% in overall diagnostic yield). We also show that such a strategy enables the possibility of masked analysis of genome-wide sequencing data in patients with poorly defined and multifaceted clinical presentations. Ultimately, we show that this approach enables control over the incidental findings rate (0.25% in phenotype gene panels). Finally, we provide a Web tool for customized phenotype panel creation (available at http://www.kimg.eu/generator). CONCLUSION: In conclusion, we present a novel approach to a phenotype-driven diagnostic process of genome scale sequencing data that harnesses the sensitivity of these approaches while restricting the analysis to genes relevant to clinical presentation in patient.Genet Med 18 11, 1102-1110.

Genetic variation in leptin and leptin receptor genes is a risk factor for idiopathic recurrent spontaneous abortion.

AIM: To determine whether maternal leptin (LEP) and leptin receptor (LEPR) gene polymorphisms are associated with idiopathic recurrent spontaneous abortion (IRSA). METHODS: This case-control association study conducted from 2010 to 2012 at the Department of Gynecology and Obstetrics, University Hospital Center Osijek and Clinical Institute of Medical Genetics Ljubljana included 178 women with a history of three or more IRSAs before the 22nd week of gestation and 145 women with at least two live births and no history of pathologic pregnancies during reproductive period. Polymorphisms of maternal LEP (rs7799039, rs2122627, rs11761556, rs10244329) and LEPR (rs1137101, rs7516341, rs1186403, rs12062820) were assessed by allele specific real-time polymerase chain reaction. Genotype distribution, allele frequencies, and frequency of haplotypes at LEP and LEPR genetic loci were determined. RESULTS: We observed more frequent genotype for rs7516341 (nominal P=0.034, odds ratio [OR] 0.61, 95% confidence interval [CI] 0.38-0.97) and rs1137101 (nominal P=0.048, OR 1.66, 95% CI 1.00-2.80) in the LEPR gene in patients than in controls, but these results did not remain significant after correction for multiple testing according to Bonferroni (adjusted P value threshold was set at 0.05). We did not observe differential distribution of genotype frequencies in the LEP gene between cases and controls. In patients with IRSA, GTCC haplotype in the LEPR gene locus was significantly less frequent than in controls (PP=0.00865, OR 0.45), contrary to ACTC haplotype (PP=0.0087, OR 1.98). CONCLUSIONS: We showed that genetic variability in the LEPR gene was associated with IRSA, warranting confirmation on a greater number of patients and pathogenesis investigation.

The -2549 insertion/deletion polymorphism in the promoter region of the VEGFA gene in couples with idiopathic recurrent spontaneous abortion.

PURPOSE: The vascular endothelial growth factor A (VEGFA) is crucial for normal vasculogenesis and angiogenesis during pregnancy, and alterations in the VEGFA gene expression were detected in women with idiopathic recurrent spontaneous abortion (IRSA) and spontaneously aborted conceptuses. Our aim was to evaluate whether there is an association between the functional -2549 insertion/deletion (I/D) polymorphism in the promoter region of the VEGFA gene and IRSA in reproductive couples. METHODS: We performed a case-control study involving 149 women and their 140 partners with three or more IRSA and 149 control women and men. Allele-specific polymerase chain reaction was used for genotyping. RESULTS: We found no association of the -2549 I/D polymorphism with IRSA in women. However, men with the DD genotype have a 1.75-fold increased risk of IRSA compared with men carrying the ID and II genotypes (95 % confidence interval (CI) = 1.05-2.93, P = 0.032). In addition, the D allele in men contributes to a 1.42-fold increased risk of IRSA (95 % CI = 1.02-1.97, P = 0.036) compared to men carrying the I allele. CONCLUSIONS: Our results indicate that the -2549 I/D polymorphism in the VEGFA gene in men might be associated with IRSA. Additional genetic association studies including both partners, as well as expression studies, are needed to elucidate the role of this polymorphism in IRSA.

Insight into the complexity of male infertility: a multi-omics review.

Male infertility is a reproductive disorder, accounting for 40-50% of infertility. Currently, in about 70% of infertile men, the cause remains unknown. With the introduction of novel omics and advancement in high-throughput technology, potential biomarkers are emerging. The main purpose of our work was to overview different aspects of omics approaches in association with idiopathic male infertility and highlight potential genes, transcripts, non-coding RNA, proteins, and metabolites worth further exploring. Using the Gene Ontology (GO) analysis, we aimed to compare enriched GO terms from each omics approach and determine their overlapping. A PubMed database screening for the literature published between February 2014 and June 2022 was performed using the keywords: male infertility in association with different omics approaches: genomics, epigenomics, transcriptomics, ncRNAomics, proteomics, and metabolomics. A GO enrichment analysis was performed using the Enrichr tool. We retrieved 281 global studies: 171 genomics (DNA level), 21 epigenomics (19 of methylation and two histone residue modifications), 15 transcriptomics, 31 non-coding RNA, 29 proteomics, two protein posttranslational modification, and 19 metabolomics studies. Gene ontology comparison showed that different omics approaches lead to the identification of different molecular factors and that the corresponding GO terms, obtained from different omics approaches, do not overlap to a larger extent. With the integration of novel omics levels into the research of idiopathic causes of male infertility, using multi-omic systems biology approaches, we will be closer to finding the potential biomarkers and consequently becoming aware of the entire spectrum of male infertility, their cause, prognosis, and potential treatment.

Case Report: Non-ossifying fibromas with pathologic fractures in a patient with NONO-associated X-linked syndromic intellectual developmental disorder.

The NONO gene encodes a nuclear protein involved in transcriptional regulation, RNA synthesis and DNA repair. Hemizygous loss-of function, de novo or maternally inherited variants in NONO have been associated with an X-linked syndromic intellectual developmental disorder-34 (OMIM # 300967), characterized by developmental delay, intellectual disability, hypotonia, macrocephaly, elongated face, structural abnormalities of corpus callosum and/or cerebellum, congenital heart defect and left ventricular non-compaction cardiomyopathy. Few patients have been described in the literature and the phenotype data are limited. We report a 17-year-old boy with dolihocephaly, elongated face, strabismus, speech and motor delay, intellectual disability, congenital heart defect (ASD, VSD and Ebstein's anomaly), left ventricular non-compaction cardiomyopathy, bilateral inguinal hernia and cryptorchidism. Additional features included recurrent fractures due to multiple non-ossifying fibromas, thrombocytopenia, and renal anomalies. Exome sequencing revealed a de novo pathogenic variant (NM_001145408.2: c.348+2_ 348+15del) in intron 5 of the NONO gene. Renal anomalies and thrombocytopenia have been rarely reported in patients with NONO-X-linked intellectual disability syndrome, while recurrent fractures due to multiple non-ossifying fibromas have not previously been associated with this syndrome. The phenotypic spectrum of NONO-X-linked intellectual disability syndrome may be broader than currently known.

Transcriptomic signatures for human male infertility.

Introduction: Male infertility is a common, complex disorder. A better understanding of pathogenesis and etiology is needed for timely diagnosis and treatment. The aim of this study, therefore, was to identify genes involved in the pathogenesis of idiopathic male infertility based on data from transcriptomic level supported with data from genomic level. Materials and methods: First, we performed whole gene expression analysis in 20 testis biopsy samples of patients with severely impaired (10) and normal spermatogenesis (10). Further, we have performed systematic review of comparable male infertility studies and overlapped the most significantly expressed genes identified in our study with the most differentially expressed genes from selected studies. Gene Ontology analysis and KEGG functional enrichment have been performed with Enrichr analysis tool. Additionally, we have overlapped these genes with the genes where rare variants have been identified previously. Results: In 10 patients with severely impaired spermatogenesis and 10 controls, we identified more than 1,800 differentially expressed genes (p < 0.001). With the systematic review of three previously performed microarray studies that have met inclusion criteria we identified 257 overlapped differentialy expressed genes (144 downregulated and 113 upregulated). Intersection of genes from transcriptomic studies with genes with identified rare variants revealed a total of 7 genes linked with male infertility phenotype (CYP11A1, CYP17A1, RSPH3, TSGA10, AKAP4, CCIN, NDNF). Conclusion: Our comprehensive study highlighted the role of four genes in pathogenesis of male infertility and provided supporting evidence for three promising candidate genes which dysfunction may result in a male infertility disorder.

A novel splice-site FHOD3 founder variant is a common cause of hypertrophic cardiomyopathy in the population of the Balkans-A cohort study.

Founder variants in sarcomere protein genes account for a significant proportion of disease-causing variants in patients with hypertrophic cardiomyopathy (HCM). However, information on founder variants in non-sarcomeric protein genes, such as FHOD3, which have only recently been associated with HCM, remains scarce. In this study, we conducted a retrospective analysis of exome sequencing data of 134 probands with HCM for recurrent pathogenic variants. We discovered a novel likely pathogenic variant c.1646+2T>C in FHOD3 in heterozygous state in eight probands with HCM and confirmed its presence in seven additional relatives. Individuals with this variant had a wide range of ages at onset of the disease (4-63 years). No adverse cardiac events were observed. Haplotype analysis revealed that the individuals with this variant shared a genomic region of approximately 5 Mbp surrounding the variant, confirming the founder effect of the variant. FHOD3 c.1646+2T>C is estimated to have arisen 58 generations ago (95% CI: 45-81) in a common ancestor living on the Balkans. A founder FHOD3 c.1646+2T>C variant is the second most common genetic variant in our cohort of patients with HCM, occurring in 16% of probands with a known genetic cause of HCM, which represents a substantially higher proportion than the currently estimated 0.5-2% for causal FHOD3 variants. Our study broadens the understanding of the genetic causes of HCM and may improve the diagnosis of this condition, particularly in patients from the Balkans.

Genetic variation in the maternal vitamin D receptor FokI gene as a risk factor for recurrent pregnancy loss.

PURPOSE: Recurrent pregnancy loss (RPL) is a reproductive disorder defined as the loss of two or more pregnancies before 24 weeks of gestation. Despite the fact that several mechanisms have been previously described for the pathogenesis of RPL, the causes of ∼50% of cases remain unknown. However, recent studies indicate association of vitamin D deficiency with adverse pregnancy outcome, including RPL. The vitamin D receptor (VDR) is a crucial mediator of the pleiotropic cellular effects of vitamin D. Its function is influenced by several single nucleotide polymorphisms (SNPs). The main objective of this study is to assess whether maternal VDR SNPs are associated with the risk of RPL in Slovenian and Croatian women. METHODS: A case-control study including 320 women with RPL and control women is designed to examine the potential association of VDR polymorphisms (FokI rs222857, Cdx2 rs11568820, and Taq1 rs731236) with RPL. Genotyping is performed using polymerase chain reaction and restriction fragment length polymorphism methods. RESULTS: We find a statistically significant higher frequency of the rs222857 CC genotype (χ2 = 6.61, p = .036) and C allele (χ2 = 5.93, p = .015) in RPL women compared to controls. Subsequently, the odds for RPL for the rs222857 are increased under the recessive (CCvsCT + TT: OR = 1.78; 95% CI = 1.12-2.82; p = .015) and the codominant (CCvsTT: OR = 2.21; 95% CI = 1.08-4.53; p = .029; CCvsCT: OR = 1.68; 95% CI = 1.04-2.72; p = .036) genetic models. The other two analyzed polymorphisms did not show any statistical significant result. CONCLUSIONS: Our results suggest that variations in the maternal VDR FokI gene might be associated with RPL in Slovenian and Croatian women.

De novo mutations in idiopathic male infertility-A pilot study.

STUDY QUESTION: Are de novo mutations in the human genome associated with male infertility? SUMMARY ANSWER: We identified de novo mutations in five candidate genes: SEMA5A, NEURL4, BRD2, CD1D, and CD63. WHAT IS KNOWN ALREADY: Epidemiological and genetic studies have consistently indicated contribution of genetic factors to the etiology of male infertility, suggesting that more than 1500 genes are involved in spermatogenesis. STUDY DESIGN, SIZE, DURATION: First, we searched for de novo mutations in patients with idiopathic azoospermia with whole-exome sequencing (WES). To evaluate the potential functional impact of de novo identified mutations, we analyzed their expression differences on independent testis samples with normal and impaired spermatogenesis. In the next step, we tested additional group of azoospermic patients for mutations in identified genes with de novo mutations. In addition to the analysis of de novo mutations in patients with idiopathic azoospermia, we considered other models of inheritance and searched for candidate genes harboring rare maternally inherited variants and biallelic autosomal and X-chromosome hemizygous variants. PARTICIPANTS/MATERIALS, SETTING, METHODS: We performed WES in 13 infertile males with idiopathic azoospermia and their parents. Potential functional impact of de novo identified mutations was evaluated by global gene expression profiling on 20 independent testis samples. To replicate the results, we performed WES in further 16 independent azoospermic males, which were screened for the variants in the same genes. Library preparation was performed with Nextera Coding Exome Capture Kit (Illumina), with subsequent sequencing on Illumina HiSeq 2500 platform. MAIN RESULTS AND THE ROLE OF CHANCE: We identified 11 de novo mutations in 10 genes of which 5 were considered potentially associated with azoospermia: SEMA5A, NEURL4, BRD2, CD1D, and CD63. All candidate genes showed significant differential expression in testis samples composed of patients with severely impaired and normal spermatogenesis. Additionally, we identified rare, potentially pathogenic mutations in the genes previously implicated in male infertility-a maternally inherited heterozygous frameshift variant in FKBPL gene and inframe deletion in UPF2 gene, homozygous frameshift variant in CLCA4 gene, and a heterozygous missense variant NR0B1 gene, which represent promising candidates for further clinical implication. LIMITATIONS OF THE STUDY, REASONS FOR CAUTION: We provided limited functional support for involvement of de novo identified genes in pathogenesis of male infertility, based on expression analysis. Additionally, the sample size was limited. WIDER IMPLICATIONS OF THE FINDINGS: We provide support that de novo mutations might contribute to male infertility and propose five genes as potentially implicated in its pathogenesis.

Vaginal Microbiome Signature Is Associated With Spontaneous Preterm Delivery.

Background: Preterm delivery (PTD) represents an important public health and therapeutic challenge. Despite the reported link between the composition of vaginal microbiome and PTD, previous studies were inconsistent in their conclusions and utilized non-uniform designs. We performed an independent case-control study carried out on the Slovenian population, where we re-evaluated the role of the vaginal microbiome in PTD. Methods: Vaginal microbiomes of pregnant women who delivered preterm were compared to those delivered at term to examine differences in the microbial richness, diversity, and differential abundance of specific taxa. We obtained vaginal swab samples from 155 Caucasian women who were classified as either term (≥380/7 weeks, n = 107) or preterm (≤366/7 weeks, n = 48) in exclusion of any other medical or obstetric conditions. The vaginal microbiomes of these women were characterized by 16S ribosomal RNA (rRNA) gene sequencing of the V3-V4 region on the MiSeq platform. Results: Women who experienced PTD had a higher microbial richness (Chao1, P = 0.011) and alpha diversity (Shannon, P = 0.00059) than women with term deliveries. We report that overall vaginal microbial community composition (beta-diversity) was significantly different by delivery gestational age category (P WeightedUnifrac < 0.001). Women who delivered preterm had decreased Lactobacilli spp. abundance as well as increased abundance of Gardnerella and other bacterial vaginosis (BV) and aerobic vaginitis (AV) associated genera including Atopobium, Sneathia, Gemella, Megasphaera, Dorea, Streptococcus, and Escherichia/Shigella. Conclusions: In the present study, we provide further evidence that vaginal microbiome composition is associated with PTD.

Genetic variation in the CLOCK gene is associated with idiopathic recurrent spontaneous abortion.

Physiological studies in animals and human support an important role of circadian system in reproduction. The aim of this study was to investigate the potential association of CLOCK gene polymorphisms with idiopathic recurrent spontaneous abortion (IRSA). We performed a case-control study. The study group consisted of 268 women with a history of three or more idiopathic recurrent spontaneous abortions and 284 women with at least two live births and no history of pathologic pregnancies all from Slovenia and Serbia. Two SNPs in the CLOCK gene were chosen and genotyped. The results showed a statistically significant difference in genotype distribution between the two groups in the CLOCK gene for rs6850524 and rs11932595. Our analysis showed that G allele under dominant model (GG+GC/CC) for rs6850524 (p = 2∙10-4, OR = 2.28, 95%CI = 1.46-3.56) as well as G allele under dominant model (GA+AA/AA) for rs11932595 (p = 0.04, OR = 1.47, 95%CI = 1.01-2.04) might be risk factors against IRSA. Our data suggest that genetic variability in the CLOCK gene is associated with IRSA warranting further confirmation and mechanistic investigations.

Association of circadian rhythm genes ARNTL/BMAL1 and CLOCK with multiple sclerosis.

Prevalence of multiple sclerosis varies with geographic latitude. We hypothesized that this fact might be partially associated with the influence of latitude on circadian rhythm and consequently that genetic variability of key circadian rhythm regulators, ARNTL and CLOCK genes, might contribute to the risk for multiple sclerosis. Our aim was to analyse selected polymorphisms of ARNTL and CLOCK, and their association with multiple sclerosis. A total of 900 Caucasian patients and 1024 healthy controls were compared for genetic signature at 8 SNPs, 4 for each of both genes. We found a statistically significant difference in genotype (ARNTL rs3789327, P = 7.5·10-5; CLOCK rs6811520 P = 0.02) distributions in patients and controls. The ARNTL rs3789327 CC genotype was associated with higher risk for multiple sclerosis at an OR of 1.67 (95% CI 1.35-2.07, P = 0.0001) and the CLOCK rs6811520 genotype CC at an OR of 1.40 (95% CI 1.13-1.73, P = 0.002). The results of this study suggest that genetic variability in the ARNTL and CLOCK genes might be associated with risk for multiple sclerosis.

Transcriptome Profiling Uncovers Potential Common Mechanisms in Fetal Trisomies 18 and 21.

Human trisomies have recently been investigated using transcriptomics approaches to identify the gene expression (GE) signatures characteristic of each of these specific aneuploidy conditions. We hypothesized that the viability of cells with gross genomic imbalances might be associated with the activation of resilience mechanisms that are common to different trisomies and that are reflected by specific shared GE patterns. We report in this article our microarray GE analyses of amniocytes from fetuses with viable trisomy conditions, trisomy 21 or trisomy 18, to detect such common expression signatures. Comparative analysis of significantly differentially expressed genes in trisomies 18 and 21 revealed six dysregulated genes common to both: OTUD5, ADAMTSL1, TADA2A, PPID, PIAS2, and MAPRE2. These genes are involved in ubiquitination, protein folding, cell proliferation, and apoptosis. Pathway-based enrichment analyses demonstrated that both trisomies showed dysregulation of the PI3K/AKT pathway, cell cycle G2/M DNA damage checkpoint regulation, and cell death and survival, as well as inhibition of the upstream regulator TP53. Our data collectively suggest that trisomies 18 and 21 share common functional GE signatures, implying that common mechanisms of resilience might be activated in aneuploid cells to resist large genomic imbalances. To the best of our knowledge, this is the first study to use global GE profiling data to identify potential common mechanisms in fetal trisomies. Studies of other trisomies using transcriptomics and multiomics approaches might further clarify mechanisms activated in trisomy syndromes.

A Single Nucleotide Polymorphism of DNA methyltransferase 3B gene is a risk factor for recurrent spontaneous abortion.

PROBLEM: Aberrant DNA methylation has been suggested as a potential cause of recurrent spontaneous abortion (RSA). Considering the growing evidence on the important roles of DNA methylation in gametogenesis and early pregnancy, we investigated the potential association of DNA methyltransferase gene polymorphisms (DNMT1 rs2228611, DNMT3A rs1550117, DNMT3B rs1569686) with RSA in Slovenian reproductive couples. METHOD OF STUDY: A total of 146 couples with ≥3 consecutive spontaneous abortions and 149 control women and men with ≥2 normal pregnancies were included. Genotyping was performed using PCR-RFLP methods. RESULTS: We found a statistically significant higher frequency of the DNMT3B rs1569686 GG genotype (X2 =7.37;P = .025) and G allele (X2  = 6.33;P = .012) in RSA women compared with controls. Moreover, the odds for RSA in women were increased under the recessive genetic model (GGvsTG+TT: OR=1.92; 95% CI=1.18-3.09; P = .008). CONCLUSION: DNMT3B rs1569686 gene polymorphism in women might be a genetic marker for the susceptibility to RSA.

The lack of association between angiotensin-converting enzyme gene insertion/deletion polymorphism and nicotine dependence in multiple sclerosis.

OBJECTIVE: Blood-borne angiotensin II is generated from angiotensinogen via cleavage by renin and angiotensin-converting enzyme (ACE), an enzymatic cascade known as the renin-angiotensin system (RAS). Several lines of evidence indicate that ACE, beyond its classical role of mediating blood pressure regulation, might contribute to the etiology of substance addictions by influencing dopaminergic signaling. A functional insertion/deletion (I/D) polymorphism of the ACE gene was associated with risk for being a smoker among individuals with depression and with smoking severity in studies comprising patients with depression and healthy controls. Several reports have described significantly increased ACE activity in cerebrospinal fluid and serum among MS patients. Furthermore, in our previous work with MS patients from Croatian and Slovenian populations, we demonstrated that the ACE-I/D polymorphism contributes to an elevated MS risk among male patients. Here we investigated whether the ACE-I/D polymorphism might influence smoking behavior among patients with MS. PATIENTS AND METHODS: Genotyping was performed in 521 patients (males/females: 139/382) using polymerase chain reaction. RESULTS: We revealed no significant differences in ACE genotype and allele frequencies between smokers and nonsmokers and no significant association between the ACE-I/D polymorphism and either pack-year smoking history or number of cigarettes smoked daily (p > .05, respectively). CONCLUSION: The ACE-I/D polymorphism does not contribute either to risk for nicotine dependence or to smoking severity among MS patients. In the context of reports on the ACE-I/D polymorphism and nicotine dependence among healthy controls and patients with depression, we may speculate that the mechanism by which this polymorphism influences nicotine dependence risk differs in MS compared to depression, although not compared to a healthy population. In addition to angiotensin II, other potential ACE substrates, such as substance P and neurotensin, which also influence dopaminergic neurotransmission (and are proposed to be associated with MS), may deserve study in future.

Genetic variation in circadian rhythm genes CLOCK and ARNTL as risk factor for male infertility.

BACKGROUND: The circadian system has a major role in maintaining homeostasis and proper body functions including reproductive capacity. The aim of this study was to examine whether there is an association between genetic variability in the primary clock genes CLOCK and ARNTL and male infertility in humans. METHODOLOGY/PRINCIPAL FINDINGS: We performed a case-control study, where we searched for an association between polymorphisms of CLOCK and ARNTL genes and male infertility in 961 Slovenian and Serbian Caucasian men. The study group consisted of 517 patients with idiopathic infertility and a control group of 444 fertile men. A statistically significant difference was found in genotype distribution between the two groups in the CLOCK gene: rs11932595 (p = 6·10(-5), q = 4·10(-4), OR equaled 1.9 with 95% CI 1.4-2.7), rs6811520 (p = 2·10(-3), q = 8·10(-3), OR = 1.7 with 95% CI 1.2-2.2) and rs6850524 (p = 0.01, q = 0.02, OR = 1.4 with 95% CI 1.1-1.9). Further analyses of haplotypes were consistent with genotyping results. CONCLUSIONS/SIGNIFICANCE: We provide evidence that genetic variability in the CLOCK gene might be associated with male infertility warranting further confirmation and mechanistic investigations.