Immortalized human myotonic dystrophy type 1 muscle cell lines to address patient heterogeneity.

Historically, cellular models have been used as a tool to study myotonic dystrophy type 1 (DM1) and the validation of therapies in said pathology. However, there is a need for in vitro models that represent the clinical heterogeneity observed in patients with DM1 that is lacking in classical models. In this study, we immortalized three DM1 muscle lines derived from patients with different DM1 subtypes and clinical backgrounds and characterized them at the genetic, epigenetic, and molecular levels. All three cell lines display DM1 hallmarks, such as the accumulation of RNA foci, MBNL1 sequestration, splicing alterations, and reduced fusion. In addition, alterations in early myogenic markers, myotube diameter and CTCF1 DNA methylation were also found in DM1 cells. Notably, the new lines show a high level of heterogeneity in both the size of the CTG expansion and the aforementioned molecular alterations. Importantly, these immortalized cells also responded to previously tested therapeutics. Altogether, our results show that these three human DM1 cellular models are suitable to study the pathophysiological heterogeneity of DM1 and to test future therapeutic options.

Phosphorylated neurofilament heavy chain in cerebrospinal fluid and plasma as a Nusinersen treatment response marker in childhood-onset SMA individuals from Serbia.

Introduction: Biomarkers capable of reflecting disease onset and short- and long-term therapeutic effects in individuals with spinal muscular atrophy (SMA) are still an unmet need and phosphorylated neurofilament heavy chain (pNF-H) holds significant promise. Methods: We conducted a longitudinal prospective study to evaluate pNF-H levels in the cerebrospinal fluid (CSF) and plasma of 29 individuals with childhood-onset SMA treated with Nuinersen (SMA type 1: n = 6, 2: n = 17, 3: n = 6). pNF-H levels before and during treatment were compared with the levels of controls (n = 22), patients with Duchenne muscular dystrophy (n = 17), myotonic dystrophy type 1 (n = 11), untreated SMA individuals with chronic type 3 disease (n = 8), and children with presymptomatic SMA (n = 3). Results: SMA type 1 showed the highest mean CSF pNF-H levels before treatment initiation. All Nusinersen-treated individuals (types 1, 2, and 3) showed significantly elevated mean baseline CSF pNF-H compared to controls, which inversely correlated with age at disease onset, age at first dose, disease duration and the initial CHOP INTEND result (SMA type 1 and 2). During 22 months of treatment, CSF pNF-H levels declined during loading doses, stabilizing at reduced levels from the initial maintenance dose in all individuals. Baseline plasma pNF-H levels in type 1 and 2 SMA were significantly increased compared to other cohorts and decreased notably in type 1 after 2 months of treatment and type 2 after 14 months. Conversely, SMA type 3, characterized by lower baseline pNF-H levels, did not show significant fluctuations in plasma pNF-H levels after 14 months of treatment. Conclusion: Our findings suggest that CSF pNF-H levels in untreated SMA individuals are significantly higher than in controls and that monitoring of CSF pNF-H levels may serve as an indicator of rapid short-term treatment response in childhood-onset SMA individuals, irrespective of the subtype of the disease, while also suggesting its potential for assessing long-term suppression of neurodegeneration. Plasma pNF-H may serve as an appropriate outcome measure for disease progression and/or response to treatment in types 1 and 2 but not in type 3. Presymptomatic infants with SMA may show elevated pNF-H levels, confirming early neuronal degeneration.

Main features and disease outcome of congenital myotonic dystrophy - experience from a single tertiary center.

Congenital myotonic dystrophy type 1 (CDM1) is a rare neuromuscular disease. The aim of our study was to evaluate clinical variability of CDM1 and factors that may influence survival in CDM1. Research included 24 pediatric patients with CDM1. Most of our patients had some form of hypoxic ischemic encephalopathy (HIE) (74 %), from mild to severe. Prolonged and complicated deliveries (75 %), high percentage of children resuscitated at birth (57 %) and respiratory insufficiency (46 %) with consequent hypoxia were the main reasons that could explain high percentage of HIE. Therapeutic hypothermia was applied in three children with poor outcome. Median survival of all CDM1 was 14.2 ± 1.5 years. Six patients had a fatal outcome (25 %). Their mean age of death was 3.0 ± 2.8 years. Poor prognostic factors for the survival of our CDM1 patients were: preterm delivery, resuscitation at birth, severe HIE, hypothermia treatment and permanent mechanical ventilation. Respiratory insufficiency was the main life-threatening factor. Our data clearly indicates the need to develop natural history studies in CDM1 in order to enhance the standards of care and to develop clinical trials investigating causative therapies in pediatric patients with CDM1.

Association between Cytotoxic T-Lymphocyte-Associated Antigen 4 (CTLA-4) Locus and Early-Onset Anti-acetylcholine Receptor-Positive Myasthenia Gravis in Serbian Patients.

Genome-wide association studies (GWAS) have provided strong evidence that early- and late-onset MG have different genetic backgrounds. Recent in silico analysis based on GWAS results revealed rs231735 and rs231770 variants within CTLA-4 locus as possible MG causative genetic factors. We aimed to explore the association of rs231735 and rs231770 with MG in a representative cohort of Serbian patients. We conducted an age-, sex-, and ethnicity-matched case-control study. Using TaqMan allele discrimination assays, the frequency of rs231735 and rs231770 genetic variants was examined in 447 AChR-MG patients and 447 matched controls. There was no significant association of rs231735 and rs231770 with the entire MG cohort (P > 0.05). Nevertheless, when stratifying patients into early-onset (n = 183) and late-onset MG (n = 264), we found early-onset patients had a significantly lower frequency of the rs231735 allele T compared to controls (OR = 0.734, 95% CI = 0.575-0.938, p10e6 permutation < 0.05), and rs231735 genotype TT and rs231770 genotype TT had a protective effect on early-onset MG (OR = 0.548, 95% CI = 0.339-0.888, and OR = 0.563, 95% CI = 0.314-1.011, p10e6 permutation < 0.05). Consequently, we found that individuals with the rs231735-rs231770 haplotype GC had a higher risk for developing early-onset MG (OR = 1.360, P = 0.027, p10e6 permutation < 0.05). Our results suggest that CTLA-4 rs231735 and rs231770 may be risk factors only for patients with early-onset MG in Serbian population.

Whole exome sequencing in Serbian patients with hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) is a group of neurodegenerative diseases with a high genetic and clinical heterogeneity. Numerous HSP patients remain genetically undiagnosed despite screening for known genetic causes of HSP. Therefore, identification of novel variants and genes is needed. Our previous study analyzed 74 adult Serbian HSP patients from 65 families using panel of the 13 most common HSP genes in combination with a copy number variation analysis. Conclusive genetic findings were established in 23 patients from 19 families (29%). In the present study, nine patients from nine families previously negative on the HSP gene panel were selected for the whole exome sequencing (WES). Further, 44 newly diagnosed adult HSP patients from 44 families were sent to WES directly, since many studies showed WES may be used as the first step in HSP diagnosis. WES analysis of cohort 1 revealed a likely genetic cause in five (56%) of nine HSP families, including variants in the ETHE1, ZFYVE26, RNF170, CAPN1, and WASHC5 genes. In cohort 2, possible causative variants were found in seven (16%) of 44 patients (later updated to 27% when other diagnosis were excluded), comprising six different genes: SPAST, SPG11, WASCH5, KIF1A, KIF5A, and ABCD1. These results expand the genetic spectrum of HSP patients in Serbia and the region with implications for molecular genetic diagnosis and future causative therapies. Wide HSP panel can be the first step in diagnosis, alongside with the copy number variation (CNV) analysis, while WES should be performed after.

Exosomal Prostate-Specific Membrane Antigen (PSMA) and Caveolin-1 as Potential Biomarkers of Prostate Cancer-Evidence from Serbian Population.

Prostate-specific membrane antigen (PSMA) and caveolin-1 are membrane proteins that are overexpressed in prostate cancer (PCa) and are involved in tumor growth and increase in aggressiveness. The aim of the present study is therefore to evaluate PSMA and caveolin-1 proteins from plasma exosomes as effective liquid biopsy biomarkers for PCa. This study included 39 patients with PCa and 33 with benign prostatic hyperplasia (BPH). The shape and size of the exosomes were confirmed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analysis. Immunogold analysis showed that PSMA is localized to the membrane of exosomes isolated from the plasma of both groups of participants. The relative protein levels of PSMA and caveolin-1 in the plasma exosomes of PCa and BPH patients were determined by Western blot analysis. The relative level of the analyzed plasma exosomal proteins was compared between PCa and BPH patients and the relevance of the exosomal PSMA and caveoin-1 level to the clinicopathological parameters in PCa was investigated. The analysis performed showed an enrichment of exosomal PSMA in the plasma of PCa patients compared to the exosomes of men with BPH. The level of exosomal caveolin-1 in plasma was significantly higher in PCa patients with high PSA levels, clinical-stage T3 or T4 and in the group of PCa patients with aggressive PCa compared to favorable clinicopathological features or tumor aggressiveness. Plasma exosomes may serve as a suitable object for the identification of potential biomarkers for the early diagnosis and prognosis of PCa as well as carriers of therapeutic agents in precision medicine of PCa treatment.

Single-molecule RNA sizing enables quantitative analysis of alternative transcription termination.

Transcription, a critical process in molecular biology, has found many applications in RNA synthesis, including mRNA vaccines and RNA therapeutics. However, current RNA characterization technologies suffer from amplification and enzymatic biases that lead to loss of native information. Here, we introduce a strategy to quantitatively study both transcription and RNA polymerase behaviour by sizing RNA with RNA nanotechnology and nanopores. To begin, we utilize T7 RNA polymerase to transcribe linear DNA lacking termination sequences. Surprisingly, we discover alternative transcription termination in the origin of replication sequence. Next, we employ circular DNA without transcription terminators to perform rolling circle transcription. This allows us to gain valuable insights into the processivity and transcription behaviour of RNA polymerase at the single-molecule level. Our work demonstrates how RNA nanotechnology and nanopores may be used in tandem for the direct and quantitative analysis of RNA transcripts. This methodology provides a promising pathway for accurate RNA structural mapping by enabling the study of full-length RNA transcripts at the single-molecule level.

Clinical score for early diagnosis of myotonic dystrophy type 2.

INTRODUCTION: Myotonic dystrophy type 2 (DM2) is a rare, multisystemic, autosomal dominant disease with highly variable clinical presentation. DM2 is considered to be highly underdiagnosed. OBJECTIVE: The aim of this study was to determine which symptoms, signs, and diagnostic findings in patients referred to neurological outpatient units are the most indicative to arouse suspicion of DM2. We tried to make a useful and easy-to-administer clinical scoring system for early diagnosis of DM2-DM2 early diagnosis score (DM2-EDS). PATIENTS AND METHODS: Two hundred ninety-one patients with a clinical suspicion of DM2 were included: 69 were genetically confirmed to have DM2, and 222 patients were DM2 negative. Relevant history, neurological, and paraclinical data were obtained from the electronic medical records. RESULTS: The following parameters appeared as significant predictors of DM2 diagnosis: cataracts (beta = 0.410, p < 0.001), myotonia on needle EMG (beta = 0.298, p < 0.001), hand tremor (beta = 0.211, p = 0.001), positive family history (beta = 0.171, p = 0.012), and calf hypertrophy (beta = 0.120, p = 0.043). In the final DM2-EDS, based on the beta values, symptoms were associated with the following values: cataracts (present 3.4, absent 0), myotonia (present 2.5, absent 0), tremor (present 1.7, absent 0), family history (positive 1.4, negative 0), and calf hypertrophy (present 1.0, absent 0). A cut-off value on DM2-EDS of 3.25 of maximum 10 points had a sensitivity of 84% and specificity of 81% to diagnose DM2. CONCLUSION: Significant predictors of DM2 diagnosis in the neurology outpatient unit were identified. We made an easy-to-administer DM2-EDS score for early diagnosis of DM2.

MiR-375 and miR-21 as Potential Biomarkers of Prostate Cancer: Comparison of Matching Samples of Plasma and Exosomes.

MiR-21 and miR-375 have been reported as dysregulated in prostate cancer (PCa) in multiple previous studies. Still, variable or even opposing data for the expression of these microRNAs in PCa were found, and their potential biomarker properties remain elusive. In an attempt to clarify their significance as PCa biomarkers, as well as to compare different types of specimens as a source of relevant microRNAs, we used plasma and matching plasma-derived exosomes from patients with PCa and patients with benign prostatic hyperplasia (BPH). Plasma and exosomes were obtained from 34 patients with PCa and 34 patients with BPH, and their levels of expression of miR-21 and miR-375 were determined by RT-qPCR. We found no significant difference in the level of expression of these microRNAs in plasma and exosomes between patients with PCa and BPH. The level of exosomal miR-21 was elevated in PCa patients with high serum PSA values, as well as in patients with aggressive PCa, while for plasma samples, the results remained insignificant. For miR-375, we did not find an association with the values of standard prognostic parameters of PCa, nor with cancer aggressiveness. Therefore, our results support the potential prognostic role of exosomal miR-21 expression levels in PCa.

Autoimmune Diseases in Patients With Myotonic Dystrophy Type 2.

Introduction: Myotonic dystrophy type 2 (DM2) is a rare autosomal dominant multisystemic disease with highly variable clinical presentation. Several case reports and one cohort study suggested a significant association between DM2 and autoimmune diseases (AIDs). Aim: The aim of this study is to analyze the frequency and type of AIDs in patients with DM2 from the Serbian DM registry. Patients and Methods: A total of 131 patients with DM2 from 108 families were included, [62.6% women, mean age at DM2 onset 40.4 (with standard deviation 13) years, age at entering the registry 52 (12.8) years, and age at analysis 58.4 (12.8) years]. Data were obtained from Akhenaten, the Serbian registry for DM, and through the hospital electronic data system. Results: Upon entering the registry, 35 (26.7%) of the 131 patients with DM2 had AIDs including Hashimoto thyroiditis (18.1%), rheumatoid arthritis, diabetes mellitus type 1, systemic lupus, Sjogren's disease, localized scleroderma, psoriasis, celiac disease, Graves's disease, neuromyelitis optica, myasthenia gravis, and Guillain-Barre syndrome. At the time of data analysis, one additional patient developed new AIDs, so eventually, 36 (28.8%) of 125 DM2 survivors had AIDs. Antinuclear antibodies (ANAs) were found in 14 (10.7%) of 63 tested patients, including 12 without defined corresponding AID (all in low titers, 1:40 to 1:160). Antineutrophil cytoplasmic antibodies (ANCAs) were negative in all 50 tested cases. The percentage of women was significantly higher among patients with AIDs (82.9% vs. 55.2%, p <0.01). Conclusion: AIDs were present in as high as 30% of the patients with DM2. Thus, screening for AIDs in DM2 seems reasonable. Presence of AIDs and/or ANAs may lead to under-diagnosis of DM2.

LTBP4, SPP1, and CD40 Variants: Genetic Modifiers of Duchenne Muscular Dystrophy Analyzed in Serbian Patients.

BACKGROUND: Clinical course variability in Duchenne muscular dystrophy (DMD) is partially explained by the mutation location in the DMD gene and variants in modifier genes. We assessed the effect of the SPP1, CD40, and LTBP4 genes and DMD mutation location on loss of ambulation (LoA). METHODS: SNPs in SPP1-rs28357094, LTBP4-rs2303729, rs1131620, rs1051303, rs10880, and CD40-rs1883832 were genotyped, and their effect was assessed by survival and hierarchical cluster analysis. RESULTS: Patients on glucocorticoid corticosteroid (GC) therapy experienced LoA one year later (p = 0.04). The modifying effect of SPP1 and CD40 variants, as well as LTBP4 haplotypes, was not observed using a log-rank test and multivariant Cox regression analysis. Cluster analysis revealed two subgroups with statistical trends in differences in age at LoA. Almost all patients in the cluster with later LoA had the protective IAAM LTBP4 haplotype and statistically significantly fewer CD40 genotypes with harmful T allele and "distal" DMD mutations. CONCLUSIONS: The modifying effect of SPP1, CD40, and LTBP4 was not replicated in Serbian patients, although our cohort was comparable in terms of its DMD mutation type distribution, SNP allele frequencies, and GC-positive effect with other European cohorts. Cluster analysis may be able to identify patient subgroups carrying a combination of the genetic variants that modify LoA.

Cognitive assessment in patients with myotonic dystrophy type 2.

Myotonic dystrophy type 2 (DM2) is an autosomal dominant multisystemic disorder. Previous studies conducted on small cohorts of DM2 patients indicated presence of a cognitive dysfunction. We aimed to assess cognitive functions in a larger cohort of Serbian DM2 patients using an extensive battery of neuropsychological tests. The study included 76 patients with a genetically confirmed DM2, 68 of whom had all tests for different cognitive domains performed. Patients underwent clinical and neuropsychological testing, including cognitive screening and assessment of general intellectual level, attention, executive and visuospatial abilities, memory, and language functions. Only 6% of patients achieved a below-average score on the general intellectual level test. Cognitive screening tests indicated presence of cognitive deficits in 5.5% of patients according to the Mini Mental State Examination test and 25.8% according to the Addenbrooke's Cognitive Examination Revised test. Twenty-four (35.3%) patients had a cognitive impairment (being two standard deviations out of norm in at least two cognitive domains). Around one quarter of DM2 patients had a significant cognitive impairment that interfered with their everyday functioning. Patients with significant cognitive impairment were older at testing and at disease onset, less educated, and had more severe muscle weakness.

Copy number variants within AZF region of Y chromosome and their association with idiopathic male infertility in Serbian population.

Results of numerous studies gave contradictory conclusions when analysing associations between copy number variants (CNVs) within the azoospermia factor (AZF) locus of the Y chromosome and idiopathic male infertility. The aim of this study was to identify the presence and possible association of CNVs in the AZF region of Y chromosome with idiopathic male infertility in the Serbian population. Using the multiplex ligation-dependent probe amplification technique, we were able to detect CNVs in 24 of 105 (22.86%) infertile men and in 11 of 112 (9.82%) fertile controls. The results of Fisher's exact test showed a statistically significant difference between cases and controls after merging g(reen)-r(ed)/g(reen)-r(ed) and b(lue)2/b(lue)3 partial deletions identified in the AZFc region (p = 0.024). At the same time, we observed a trend towards statistical significance for a deletion among gr/gr amplicons (p = 0.053). In addition to these, we identified a novel complex CNV involving inversion of r2/r3 amplicons, followed by b2/b3 duplication and b3/b4 deletion, respectively. Additional analyses on a larger study group would be necessary to draw meaningful conclusions about associations among CNVs that presented with higher frequency in the infertile men than the fertile controls.

Association between genetic variants in DICER1 and cancer risk: An updated meta-analysis.

Dysfunctions in mechanisms of gene regulation based on RNA interference are recognized as a common feature of the molecular basis of cancer pathogenesis. Therefore, as one of the crucial components of the machinery involved in the biogenesis of both siRNAs and microRNA molecules, DICER was recognized as one of the candidates for the research in the field of carcinogenesis. Due to their potential functional properties, several genetic variants located within DICER1 gene were analyzed for their possible association with the susceptibility to cancer through case-control studies. In order to elucidate their effect on the overall cancer risk, we conducted an updated meta-analysis of all eligible association studies. The publications were selected based on PubMed database search, while OpenMeta-analyst and MetaGenyo software were used for quantitative data synthesis. Statistically significant results were found for the association of rs1057035 with the overall cancer risk under multiple genetic models (PCT vs. TT < 0.001, ORCT vs. TT = 0.870, 95% CI = 0.812-0.933; Pallelic = 0.009, ORallelic = 0.896, 95% CI = 0.825-0.973; Pdom < 0.001, ORdom = 0.874, 95% CI = 0.817-0.934; Poverdom = 0.004, ORoverdom = 0.858, 95% CI = 0.773-0.953). Other selected genetic variants within DICER1, rs13078, rs1209904 and rs3742330, did not show the association with the overall susceptibility to malignant diseases. We conclude that rs1057035 may represent a potential biomarker associated with the risk of developing cancer, which requires a confirmation in a larger set of studies.

Molecular and Clinical Implications of Variant Repeats in Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1) is one of the most variable monogenic diseases at phenotypic, genetic, and epigenetic level. The disease is multi-systemic with the age at onset ranging from birth to late age. The underlying mutation is an unstable expansion of CTG repeats in the DMPK gene, varying in size from 50 to >1000 repeats. Generally, large expansions are associated with an earlier age at onset. Additionally, the most severe, congenital DM1 form is typically associated with local DNA methylation. Genetic variability of DM1 mutation is further increased by its structural variations due to presence of other repeats (e.g., CCG, CTC, CAG). These variant repeats or repeat interruptions seem to confer an additional level of epigenetic variability since local DNA methylation is frequently associated with variant CCG repeats independently of the expansion size. The effect of repeat interruptions on DM1 molecular pathogenesis is not investigated enough. Studies on patients indicate their stabilizing effect on DMPK expansions because no congenital cases were described in patients with repeat interruptions, and the age at onset is frequently later than expected. Here, we review the clinical relevance of repeat interruptions in DM1 and genetic and epigenetic characteristics of interrupted DMPK expansions based on patient studies.

Association of KLK3, VAMP8 and MDM4 Genetic Variants within microRNA Binding Sites with Prostate Cancer: Evidence from Serbian Population.

A growing number of studies have suggested that genetic variants affecting the micro-RNA- binding mechanisms (miRSNPs) constitute a promising novel class of biomarkers for prostate cancer (PCa) biology. Among the most extensively studied miRSNPs in the context of cancer is the variation rs4245739 in the MDM4 gene, while a recent large-scale analysis revealed significant differences in genotype distributions between aggressive and non-aggressive disease for rs1058205 in KLK3 and rs1010 in VAMP8. In this study, we examined a total of 1083 subjects for these three variants using Taqman® SNP Genotyping Assays. Three hundred and fifty-five samples of peripheral blood were obtained from patients with PCa and 358 samples from patients with benign prostatic hyperplasia (BPH). The control group consisted of 370 healthy volunteers. Comparisons of genotype distributions among PCa and BPH patients, as well as between PCa patients and healthy controls, yielded no evidence of association between the analyzed genetic variants and the risk of developing PCa. However, all three tested genetic variants have shown the association with the parameters of PCa progression. For KLK3 variant rs1058205, minor allele C was found to associate with the lower serum PSA score in PCa patients (PSA > 20 ng/ml vs. PSA < 10 ng/ml comparison, Prec = 0.038; ORrec = 0.20, 95%CI 0.04-1.05). The obtained results point out the potential relevance of the tested genetic variants for the disease aggressiveness assessment.

Association between genetic variants in genes encoding Argonaute proteins and cancer risk: A meta-analysis.

With the accumulation of evidence of the involvement of small-RNA-based regulatory mechanisms in carcinogenesis, genes encoding Ago proteins emerged as candidates for case-control studies on cancer. Since the data from association studies on various cancer types was not previously meta-analyzed, the potential effect of these variants on cancer risk in general was not previously evaluated. Therefore, we conducted a meta-analysis of all eligible studies, testing multiple genetic models of association. The identification of publication was based on PubMed database search, while OpenMeta-analyst, as well as MetaGenyo software, were used for quantitative data synthesis. AGO1 genetic variant rs636832 was found to associate with the overall cancer risk, assuming the overdominant genetic model (P =  0.030; ORoverdom = 0.865, 95%CI 0.759-0.986). For the same genetic variant, statistical significance was reached for the association with solid tumors, as well as with lung cancer susceptibility. Similar results were found in the Asians cohort for another AGO1 variant, rs595961. For rs4961280, none of the meta-analyses yielded statistically significant results. We conclude that genetic variants rs636832 and rs595961 located within AGO1 may represent susceptibility variants for specific types of cancer, while the association with malignant diseases was not determined for AGO2 variant rs4961280.

Analysis of association of potentially functional genetic variants within genes encoding miR-34b/c, miR-378 and miR-143/145 with prostate cancer in Serbian population.

MiRNA-associated genetic variants occurring in regulatory regions can affect the efficiency of transcription and potentially modify pri-miRNA or pre-miRNA processing. Since miRNA-based mechanisms are shown to be involved in the pathogenesis of prostate cancer (PCa), the aim of the present study was to evaluate the effect of rs4938723, rs1076064 and rs4705343 occurring in regulatory regions of miR-34b/c, miR-143/145 and miR-378, respectively, on PCa risk and progression in Serbian population. We examined a total of 1060 subjects, of which 350 were patients with PCa, 354 were patients with benign prostatic hyperplasia (BPH), while 356 healthy volunteers were included in the control group. Genotyping of rs4938723, rs1076064 and rs4705343 was performed by using Taqman® SNP Genotyping Assays. Allele C of rs4705342 was found to increase the risk of PCa (P=0.031 for codominant model, P=0.0088 for recessive model). Rs1076064 minor allele G was found to associate with serum PSA score, as well as with PCa T category and disease aggressiveness. For rs4938723 minor allele C was shown to be associated with the lower PCa T category (Pdom=0.0046; OR=0.36, 95 % CI 0.17-0.76) in T2 vs. T1 comparison. Rs4705342 was identified as PCa susceptibility variant in Serbian population, while for rs1076064 and rs4938723 association with PCa progression parameters was found.

Body composition analysis in patients with myotonic dystrophy types 1 and 2.

INTRODUCTION: To date, there are only several reports on body composition in myotonic dystrophy type 1 (DM1) and there are no data for myotonic dystrophy type 2 (DM2). The aim was to analyze body composition of patients with DM1 and DM2, and its association with socio-demographic and clinical features of the diseases. METHODS: There were no statistical differences in sociodemographic features between 20 DM1 patients and 12 DM2 patients. Body composition was assessed by DEXA (dual-energy x-ray absorptiometry). A three-compartment model was used: bone mineral content (BMC), fat mass (FM), and lean tissue mass (LTM). RESULTS: Patients with DM1 and DM2 had similar total body mass (TBM), BMC, FM, and LTM. Patients with DM1 had higher trunk-limb fat index (TLFI) in comparison to DM2 patients which indicates visceral fat deposition in DM1 (1.16 ± 0.32 for DM1 vs. 0.87 ± 0.23 for DM2, p < 0.05). Right ribs bone mineral density was lower in DM2 group (0.68 ± 0.07 g/cm2 vs. 0.61 ± 0.09 g/cm2, p < 0.05). Higher percentage of FM in legs showed correlation with lower strength of the upper leg muscles in DM1 (ρ = - 0.47, p < 0.05). Higher muscle strength in DM2 patients was in correlation with higher bone mineral density (ρ = + 0.62, p < 0.05 for upper arm muscles, ρ = + 0.87, p < 0.01 for lower arm muscles, ρ = + 0.72, p < 0.05 for lower leg muscles). CONCLUSION: DM1 patients had visceral obesity, and percentage of FM correlated with a degree of muscle weakness in upper legs. In DM2 patients, degree of muscle weakness was in correlation with higher FM index and lower bone mineral density.

Repeat Interruptions Modify Age at Onset in Myotonic Dystrophy Type 1 by Stabilizing DMPK Expansions in Somatic Cells.

CTG expansions in DMPK gene, causing myotonic dystrophy type 1 (DM1), are characterized by pronounced somatic instability. A large proportion of variability of somatic instability is explained by expansion size and patient's age at sampling, while individual-specific differences are attributed to additional factors. The age at onset is extremely variable in DM1, and inversely correlates with the expansion size and individual-specific differences in somatic instability. Three to five percent of DM1 patients carry repeat interruptions and some appear with later age at onset than expected for corresponding expansion size. Herein, we characterized somatic instability of interrupted DMPK expansions and the effect on age at onset in our previously described patients. Repeat-primed PCR showed stable structures of different types and patterns of repeat interruptions in blood cells over time and buccal cells. Single-molecule small-pool PCR quantification of somatic instability and mathematical modeling showed that interrupted expansions were characterized by lower level of somatic instability accompanied by slower progression over time. Mathematical modeling demonstrated that individual-specific differences in somatic instability had greater influence on age at onset in patients with interrupted expansions. Therefore, repeat interruptions have clinical importance for disease course in DM1 patients due to stabilizing effect on DMPK expansions in somatic cells.