Molecular Genetics and Pathogenesis of the Floating Harbor Syndrome: Case Report of Long-Term Growth Hormone Treatment and a Literature Review.

Introduction: Floating Harbor syndrome (FHS) is an extremely rare disorder, with slightly more than a hundred cases reported worldwide. FHS is caused by heterozygous mutations in the SRCAP gene; however, little is known about the pathogenesis of FHS or the effectiveness of its treatment. Methods: Whole-exome sequencing (WES) was performed for the definitive molecular diagnosis of the disease. Identified variants were validated using Sanger sequencing. In addition, systematic literature and public data on genetic variation in SRCAP and the effects of growth hormone (GH) treatment was conducted. Results: We herein report the first case of FHS in the Russian Federation. The male proband presented with most of the typical phenotypic features of FHS, including short stature, skeletal and facial features, delayed growth and bone age, high pitched voice, and intellectual impairment. The proband also had partial growth hormone deficiency. We report the history of treatment of the proband with GH, which resulted in modest improvement in growth prior to puberty. WES revealed a pathogenic c.7466C>G (p.Ser2489*) mutation in the last exon of the FHS-linked SRCAP gene. A systematic literature review and analysis of available genetic variation datasets highlighted an unusual distribution of pathogenic variants in SRCAP and confirmed the lack of pathogenicity for variants outside of exons 33 and 34. Finally, we suggested a new model of FHS pathogenesis which provides possible basis for the dominant negative nature of FHS-causing mutations and explains limited effects of GH treatment in FHS. Conclusion: Our findings expand the number of reported FHS cases and provide new insights into disease genetics and the efficiency of GH therapy for FHS patients.

Identification of Genetic Risk Factors of Severe COVID-19 Using Extensive Phenotypic Data: A Proof-of-Concept Study in a Cohort of Russian Patients.

The COVID-19 pandemic has drawn the attention of many researchers to the interaction between pathogen and host genomes. Over the last two years, numerous studies have been conducted to identify the genetic risk factors that predict COVID-19 severity and outcome. However, such an analysis might be complicated in cohorts of limited size and/or in case of limited breadth of genome coverage. In this work, we tried to circumvent these challenges by searching for candidate genes and genetic variants associated with a variety of quantitative and binary traits in a cohort of 840 COVID-19 patients from Russia. While we found no gene- or pathway-level associations with the disease severity and outcome, we discovered eleven independent candidate loci associated with quantitative traits in COVID-19 patients. Out of these, the most significant associations correspond to rs1651553 in MYH14p = 1.4 × 10-7), rs11243705 in SETX (p = 8.2 × 10-6), and rs16885 in ATXN1 (p = 1.3 × 10-5). One of the identified variants, rs33985936 in SCN11A, was successfully replicated in an independent study, and three of the variants were found to be associated with blood-related quantitative traits according to the UK Biobank data (rs33985936 in SCN11A, rs16885 in ATXN1, and rs4747194 in CDH23). Moreover, we show that a risk score based on these variants can predict the severity and outcome of hospitalization in our cohort of patients. Given these findings, we believe that our work may serve as proof-of-concept study demonstrating the utility of quantitative traits and extensive phenotyping for identification of genetic risk factors of severe COVID-19.

Cytogenomic Profile of Uterine Leiomyoma: In Vivo vs. In Vitro Comparison.

We performed a comparative cytogenomic analysis of cultured and uncultured uterine leiomyoma (UL) samples. The experimental approach included karyotyping, aCGH, verification of the detected chromosomal abnormalities by metaphase and interphase FISH, MED12 mutation analysis and telomere measurement by Q-FISH. An abnormal karyotype was detected in 12 out of 32 cultured UL samples. In five karyotypically abnormal ULs, MED12 mutations were found. The chromosomal abnormalities in ULs were present mostly by complex rearrangements, including chromothripsis. In both karyotypically normal and abnormal ULs, telomeres were ~40% shorter than in the corresponding myometrium, being possibly prerequisite to chromosomal rearrangements. The uncultured samples of six karyotypically abnormal ULs were checked for the detected chromosomal abnormalities through interphase FISH with individually designed DNA probe sets. All chromosomal abnormalities detected in cultured ULs were found in corresponding uncultured samples. In all tumors, clonal spectra were present by the karyotypically abnormal cell clone/clones which coexisted with karyotypically normal ones, suggesting that chromosomal abnormalities acted as drivers, rather than triggers, of the neoplastic process. In vitro propagation did not cause any changes in the spectrum of the cell clones, but altered their ratio compared to uncultured sample. The alterations were unique for every UL. Compared to its uncultured counterpart, the frequency of chromosomally abnormal cells in the cultured sample was higher in some ULs and lower in others. To summarize, ULs are characterized by both inter- and intratumor genetic heterogeneity. Regardless of its MED12 status, a tumor may be comprised of clones with and without chromosomal abnormalities. In contrast to the clonal spectrum, which is unique and constant for each UL, the clonal frequency demonstrates up or down shifts under in vitro conditions, most probably determined by the unequal ability of cells with different genetic aberrations to exist outside the body.

Telomere Length in Chromosomally Normal and Abnormal Miscarriages and Ongoing Pregnancies and Its Association with 5-hydroxymethylcytosine Patterns.

The present study investigates telomere length (TL) in dividing chorionic cytotrophoblast cells from karyotypically normal and abnormal first trimester miscarriages and ongoing pregnancies. Using Q-FISH, we measured relative TLs in the metaphase chromosomes of 61 chorionic villous samples. Relative TLs did not differ between karyotypically normal samples from miscarriages and those from ongoing pregnancies (p = 0.3739). However, among the karyotypically abnormal samples, relative TLs were significantly higher in ongoing pregnancies than in miscarriages (p < 0.0001). Relative TLs were also significantly higher in chorion samples from karyotypically abnormal ongoing pregnancies than in those from karyotypically normal ones (p = 0.0018) in contrast to miscarriages, where relative TL values were higher in the karyotypically normal samples (p = 0.002). In the karyotypically abnormal chorionic cytotrophoblast, the TL variance was significantly lower than in any other group (p < 0.05). Assessed by TL ratios between sister chromatids, interchromatid TL asymmetry demonstrated similar patterns across all of the chorion samples (p = 0.22) but significantly exceeded that in PHA-stimulated lymphocytes (p < 0.0001, p = 0.0003). The longer telomere was predominantly present in the hydroxymethylated sister chromatid in chromosomes featuring hemihydroxymethylation (containing 5-hydroxymethylcytosine in only one sister chromatid)-a typical sign of chorionic cytotrophoblast cells. Our results suggest that the phenomena of interchromatid TL asymmetry and its association to 5hmC patterns in chorionic cytotrophoblast, which are potentially linked to telomere lengthening through recombination, are inherent to the development programme. The TL differences in chorionic cytotrophoblast that are associated with karyotype and embryo viability seem to be determined by heredity rather than telomere elongation mechanisms. The inheritance of long telomeres by a karyotypically abnormal embryo promotes his development, whereas TL in karyotypically normal first-trimester embryos does not seem to have a considerable impact on developmental capacity.

Telomere Length in Metaphase Chromosomes of Human Triploid Zygotes.

The human lifespan is strongly influenced by telomere length (TL) which is defined in a zygote-when two highly specialised haploid cells form a new diploid organism. Although TL is a variable parameter, it fluctuates in a limited range. We aimed to establish the determining factors of TL in chromosomes of maternal and paternal origin in human triploid zygotes. Using Q-FISH, we examined TL in the metaphase chromosomes of 28 human triploid zygotes obtained from 22 couples. The chromosomes' parental origin was identified immunocytochemically through weak DNA methylation and strong hydroxymethylation in the sperm-derived (paternal) chromosomes versus strong DNA methylation and weak hydroxymethylation in the oocyte-derived (maternal) ones. In 24 zygotes, one maternal and two paternal chromosome sets were identified, while the four remaining zygotes contained one paternal and two maternal sets. For each zygote, we compared mean relative TLs between parental chromosomes, identifying a significant difference in favour of the paternal chromosomes, which attests to a certain "imprinting" of these regions. Mean relative TLs in paternal or maternal chromosomes did not correlate with the respective parent's age. Similarly, no correlation was observed between the mean relative TL and sperm quality parameters: concentration, progressive motility and normal morphology. Based on the comparison of TLs in chromosomes inherited from a single individual's gametes with those in chromosomes inherited from different individuals' gametes, we compared intraindividual (intercellular) and interindividual variability, obtaining significance in favour of the latter and thus validating the role of heredity in determining TL in zygotes. A comparison of the interchromatid TL differences across the chromosomes from sets of different parental origin with those from PHA-stimulated lymphocytes showed an absence of a significant difference between the maternal and paternal sets but a significant excess over the lymphocytes. Therefore, interchromatid TL differences are more pronounced in zygotes than in lymphocytes. To summarise, TL in human zygotes is determined both by heredity and parental origin; the input of other factors is possible within the individual's reaction norm.

Uterine Leiomyomas with an Apparently Normal Karyotype Comprise Minor Heteroploid Subpopulations Differently Represented in vivo and in vitro.

In the present study, we aimed to check whether uterine leiomyomas (ULs) with an apparently normal karyotype in vitro comprise "hidden" cell subpopulations with numerical chromosome abnormalities (heteroploid cells). A total of 32 ULs obtained from 32 patients were analyzed in the study. Each UL was sampled for in vivo and in vitro cytogenetic studies. Karyotyping was performed on metaphase preparations from the cultured UL samples. A normal karyotype was revealed in 20 out of the 32 ULs, of which 9 were selected for further study based on the good quality of the interphase preparations. Then, using interphase FISH with centromeric DNA probes, we analyzed the copy number of chromosomes 7 and 16 in 1,000 uncultured and 1,000 cultured cells of each selected UL. All of the ULs included both disomic cells representing a predominant subpopulation and heteroploid cells reaching a maximum frequency of 21.6% (mean 9.8%) in vivo and 11.5% (mean 6.1%) in vitro. The spectrum of heteroploid cells was similar in vivo and in vitro and mostly consisted of monosomic and tetrasomic cells. However, their frequencies in the cultured samples differed from those in the uncultured ones: while the monosomic cells decreased in number, the tetrasomic cells became more numerous. The frequency of either monosomic or tetrasomic cells both in vivo and in vitro was not associated with the presence of MED12 exon 2 mutations in the tumors. Our results suggest that ULs with an apparently normal karyotype consist of both karyotypically normal and heteroploid cells, implying that the occurrence of minor cell subpopulations with numerical chromosome abnormalities may be considered a characteristic of UL tumorigenesis. Different frequencies of heteroploid cells in vivo and in vitro suggest their dependence on microenvironmental conditions, thus providing a pathway for regulation of their propagation, which may be important for the UL pathogenesis.

Analysis of the Spectrum of ACE2 Variation Suggests a Possible Influence of Rare and Common Variants on Susceptibility to COVID-19 and Severity of Outcome.

OBJECTIVES: In March 2020, the World Health Organization declared that an infectious respiratory disease caused by a new severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2, causing coronavirus disease 2019 (COVID-19)] became a pandemic. In our study, we have analyzed a large publicly available dataset, the Genome Aggregation Database (gnomAD), as well as a cohort of 37 Russian patients with COVID-19 to assess the influence of different classes of genetic variants in the angiotensin-converting enzyme-2 (ACE2) gene on the susceptibility to COVID-19 and the severity of disease outcome. RESULTS: We demonstrate that the European populations slightly differ in alternative allele frequencies at the 2,754 variant sites in ACE2 identified in the gnomAD database. We find that the Southern European population has a lower frequency of missense variants and slightly higher frequency of regulatory variants. However, we found no statistical support for the significance of these differences. We also show that the Russian population is similar to other European populations when comparing the frequencies of the ACE2 variants. Evaluation of the effect of various classes of ACE2 variants on COVID-19 outcome in a cohort of Russian patients showed that common missense and regulatory variants do not explain the differences in disease severity. At the same time, we find several rare ACE2 variants (including rs146598386, rs73195521, rs755766792, and others) that are likely to affect the outcome of COVID-19. Our results demonstrate that the spectrum of genetic variants in ACE2 may partially explain the differences in severity of the COVID-19 outcome.

The spectrum of pathogenic variants of the ATP7B gene in Wilson disease in the Russian Federation.

BACKGROUND: Wilson's disease (WD) is a rare inherited disorder caused by mutations in the ATP7B gene resulting in copper accumulation in different organs. However, data on ATP7B mutation spectrum in Russia and worldwide are insufficient and contradictory. The objective of the present study was estimation of the frequency of ATP7B gene mutations in the Russian population of WD patients. MATERIALS AND METHODS: 75 WDpatients were examined by next-generation sequencing (NGS). A targeted panel NimbleGen SeqCap EZ Choice: 151012_HG38_CysFib_EZ_HX3 (ROCHE)was designed for analysis of ATP7B gene and possible modifier genes. Retrospective assessment of a diagnostic WD score (Leipzig, 2001) was also performed. RESULTS: 31 mutations in ATP7B gene were detected. Two most frequent mutations were c.3207C > A (51,85% of alleles) and c.3190 G > A (8,64% of alleles). Single rare mutations were detected in 29% of cases. In 96% cases mutations of both copies of the ATP7B were revealed. We also observed 3 novel potentially pathogenic variants which were not previously described (c.1870-8A > G, c.3655A > T (p.Ile1219Phe), c.3036dupC (p.Lys1013fs). For 25% of patients at the time of the manifestation the diagnosis WD could not be established using the earlier proposed diagnostic score. There was a remarkable delay in diagnosis for the majority of patients. Only 33% of patients WD was diagnosed in three months after the first symptoms, 29%patients - in 3-12 months, 30% - in 1-10 years, in 8% - more than 10 years. Generally, clinical appearance of WD may be rather variable at manifestation and genetic profiling at this step is the only way to confirm the presence of WD.

Reproductive History of a Woman With 8p and 18p Genetic Imbalance and Minor Phenotypic Abnormalities.

We report on the phenotype and the reproductive history of an adult female patient with an unbalanced karyotype: 8p23 and 18p11.3 terminal deletions and 8p22 duplication. The indication for karyotyping of the 28-year-old patient was a structural rearrangement in her miscarriage specimen: 45,ХХ,der(8;18)t(8;18)(p23;p11.3). Unexpectedly, the patient had the same karyotype with only one normal chromosome 8, one normal chromosome 18, and a derivative chromosome, which was a product of chromosomes 8 and 18 fusion with loss of their short arm terminal regions. Fluorescence in situ hybridization revealed that derivative chromosome was a pseudodicentric with an active centromere of chromosome 8. Array comparative genomic hybridization confirmed 8p and 18p terminal deletions and additionally revealed 8p22 duplication with a total of 43 OMIM annotated genes being affected by the rearrangement. The patient had minor facial and cranial dysmorphia and no pronounced physical or mental abnormalities. She was socially normal, had higher education and had been married since the age of 26 years. Considering genetic counseling, the patient had decided to conceive the next pregnancy through in vitro fertilization (IVF) with preimplantation genetic testing for structural chromosomal aberrations (PGT-SR). She underwent four IVF/PGT-SR cycles with a total of 25 oocytes obtained and a total of 10 embryos analyzed. Only one embryo was balanced regarding chromosomes 8 and 18, while the others were unbalanced and demonstrated different combinations of the normal chromosomes 8 and 18 and the derivative chromosome. The balanced embryo was transferred, but the pregnancy was not registered. After four unsuccessful IVF/PGT-SR cycles, the patient conceived naturally. Non-invasive prenatal testing showed additional chromosome 18. The prenatal cytogenetic analysis of chorionic villi revealed an abnormal karyotype: 46,ХХ,der(8;18)t(8;18)(p23;p11.3)mat,+18. The pregnancy was terminated for medical reasons. The patient has a strong intention to conceive a karyotypically normal fetus. However, genetic counseling regarding this issue is highly challenging. Taking into account a very low chance of balanced gametes, emotional stress caused by numerous unsuccessful attempts to conceive a balanced embryo and increasing age of the patient, an IVF cycle with a donor oocyte should probably be considered.

Pathogenomics of Uterine Fibroids Development.

We review recent studies dealing with the molecular genetics and basic results of omics analysis of uterine leiomyoma (LM)-a common benign muscle tumor of the uterus. Whole genome studies of LM resulted in the discovery of many new gene nets and biological pathways, including its origin, transcriptomic, and epigenetic profiles, as well as the impact of the inter-cell matrix in LM growth and involvement of microRNA in its regulation. New data on somatic cell mutations ultimately involved in the origin, distribution and growth of LM are reviewed. Putative identification of LM progenitor SC (stem cells) giving rise to maternal fibroid nodes and junctional zones provide a new clue for hypotheses on the pathogenomics of LM. The reviewed data are consistent with at least two different but probably intimately interacted molecular mechanisms of LM. One of them (the genetic hypothesis) is focused primarily on the MED12 gene mutations and suggests its onset in the side population of embryonic myoblasts of the female reproductive system, which later gave rise to multiple small and medium fibroids. The single and usually large-size fibroids are induced by predominantly epigenetic disorders in LM SC, provoked by enhanced expression of the HMGA2 gene caused by its hypomethylation and epigenetic deregulation enhanced by hypoxia, muscle tension, or chromosome instability/aberrations. The pathogenomics of both genetic and epigenetic programs of LM with many peculiarities at the beginning later became rather similar and partly overlapped due to the proximity of their gene nets and epigenetic landscape. Pathogenomic studies of LM open ways for elaboration of novel strategies of prevention and treatment of this common disease.

Whole­exome sequencing in Russian children with non­type 1 diabetes mellitus reveals a wide spectrum of genetic variants in MODY­related and unrelated genes.

The present study reports on the frequency and the spectrum of genetic variants causative of monogenic diabetes in Russian children with non­type 1 diabetes mellitus. The present study included 60 unrelated Russian children with non­type 1 diabetes mellitus diagnosed before the age of 18 years. Genetic variants were screened using whole­exome sequencing (WES) in a panel of 35 genes causative of maturity onset diabetes of the young (MODY) and transient or permanent neonatal diabetes. Verification of the WES results was performed using PCR­direct sequencing. A total of 38 genetic variants were identified in 33 out of 60 patients (55%). The majority of patients (27/33, 81.8%) had variants in MODY­related genes: GCK (n=19), HNF1A (n=2), PAX4 (n=1), ABCC8 (n=1), KCNJ11 (n=1), GCK+HNF1A (n=1), GCK+BLK (n=1) and GCK+BLK+WFS1 (n=1). A total of 6 patients (6/33, 18.2%) had variants in MODY­unrelated genes: GATA6 (n=1), WFS1 (n=3), EIF2AK3 (n=1) and SLC19A2 (n=1). A total of 15 out of 38 variants were novel, including GCK, HNF1A, BLK, WFS1, EIF2AK3 and SLC19A2. To summarize, the present study demonstrates a high frequency and a wide spectrum of genetic variants causative of monogenic diabetes in Russian children with non­type 1 diabetes mellitus. The spectrum includes previously known and novel variants in MODY­related and unrelated genes, with multiple variants in a number of patients. The prevalence of GCK variants indicates that diagnostics of monogenic diabetes in Russian children may begin with testing for MODY2. However, the remaining variants are present at low frequencies in 9 different genes, altogether amounting to ~50% of the cases and highlighting the efficiency of using WES in non­GCK­MODY cases.

Synergistic Anti-Angiogenic Effects Using Peptide-Based Combinatorial Delivery of siRNAs Targeting VEGFA, VEGFR1, and Endoglin Genes.

Angiogenesis is a process of new blood vessel formation, which plays a significant role in carcinogenesis and the development of diseases associated with pathological neovascularization. An important role in the regulation of angiogenesis belongs to several key pathways such as VEGF-pathways, TGF-ß-pathways, and some others. Introduction of small interfering RNA (siRNA) against genes of pro-angogenic factors is a promising strategy for the therapeutic suppression of angiogenesis. These siRNA molecules need to be specifically delivered into endothelial cells, and non-viral carriers modified with cellular receptor ligands can be proposed as perspective delivery systems for anti-angiogenic therapy purposes. Here we used modular peptide carrier L1, containing a ligand for the CXCR4 receptor, for the delivery of siRNAs targeting expression of VEGFA, VEGFR1 and endoglin genes. Transfection properties of siRNA/L1 polyplexes were studied in CXCR4-positive breast cancer cells MDA-MB-231 and endothelial cells EA.Hy926. We have demonstrated the efficient down-regulation of endothelial cells migration and proliferation by anti-VEGFA, anti-VEGFR1, and anti-endoglin siRNA-induced silencing. It was found that the efficiency of anti-angiogenic treatment can be synergistically improved via the combinatorial delivery of anti-VEGFA and anti-VEGFR1 siRNAs. Thus, this approach can be useful for the development of therapeutic angiogenesis inhibition.

On the Complexity of Mechanisms and Consequences of Chromothripsis: An Update.

In the present review, we focus on the phenomenon of chromothripsis, a new type of complex chromosomal rearrangements. We discuss the challenges of chromothripsis detection and its distinction from other chromoanagenesis events. Along with already known causes and mechanisms, we introduce aberrant epigenetic regulation as a possible pathway to chromothripsis. We address the issue of chromothripsis characteristics in cancers and benign tumours, as well as chromothripsis inheritance in cases of its occurrence in germ cells, zygotes and early embryos. Summarising the presented data on different phenotypic effect of chromothripsis, we assume that its consequences are most likely determined not by the chromosome shattering and reassembly themselves, but by the genome regions involved in the rearrangement.

Targeted sequencing analysis of ACVR2A gene identifies novel risk variants associated with preeclampsia.

Background: Preeclampsia (PE) is the most common complication of pregnancy that remains to be a major cause of maternal and fetal mortality. Prediction and early diagnosis of PE would allow for timely initiation of preventive therapy. According to recent studies of ACVR2A gene polymorphism is associated with PE, but it is still unclear whether these findings reflect specific pathogenetic mechanisms of this disease. Methods: We performed targeted next-generation sequencing (NGS) sequencing of ACVR2A gene by means of Ion Torrent Personal Genome machine (PGM) Sequencer. A genetic analysis of patients with PE and control group was performed. Bioinformatics analysis using Polyphen2 (Boston, MA), SIFT (La Jolla, CA), and SnpSift software were used. To select genetic markers in PE patients two additive models and score analysis were applied. Results: Based on the score analysis, we detected two substitutions (rs145399059 and rs17692648) and one insertion insAA at position 148642724 that were associated with PE in our cohorts. We also detected a variant rs17742573 that can be considered as protective against preeclampsia. Conclusions: Our data suggest that some variants in ACVR2A gene are associated with PE. But more studies are required to reveal the role of ACVR2A gene in the pathogenesis of this disease during pregnancy.

Inter-Cell and Inter-Chromosome Variability of 5-Hydroxymethylcytosine Patterns in Noncultured Human Embryonic and Extraembryonic Cells.

5-hydroxymethylcytosine (5hmC) is an oxidative derivative of 5-methylcytosine (5mC). Recent studies have revealed a sharp difference in the levels of 5hmC in 2 opposite DNA strands of a given chromosome and a chromosome-wide cell-to-cell variability in mammalian cells. This asymmetric 5hmC distribution was found in cultured cells, which may not fully mimic in vivo epigenetic processes. We have checked whether inter-chromosome and inter-cell variability of 5hmC patterns is typical for noncultured human cells. Using indirect immunofluorescence, we analyzed the localization of 5hmC and its co-distribution with 5mC on direct preparations of mitotically active cells from human embryonic lung and chorionic cytotrophoblast samples. We demonstrated 3 types of chromosomes according to the 5hmC accumulation pattern: hydroxymethylated (5hmC in both sister chromatids), hemihydroxymethylated (5hmC in only 1 sister chromatid), and nonhydroxymethylated ones. Each accumulation type was not specific to any particular chromosome, resulting in different 5hmC patterns between homologous chromosomes, among chromosomes within each metaphase plate, among metaphases in one tissue, and between the tissues. The 5mC distribution was stable: chromosomes were methylated in R-bands and, especially in embryonic lung cells, in the heterochromatic regions 1q12, 9q12, and 16q11.2. Our results provide the first evidence of inter-cell and inter-chromosome variability of 5hmC patterns in human noncultured embryonic and extraembryonic cells.

Molecular Factors Involved in Spinal Muscular Atrophy Pathways as Possible Disease-modifying Candidates.

Spinal Muscular Atrophy (SMA) is a neuromuscular disorder caused by mutations in the SMN1 gene. Being a monogenic disease, it is characterized by high clinical heterogeneity. Variations in penetrance and severity of symptoms, as well as clinical discrepancies between affected family members can result from modifier genes influence on disease manifestation. SMN2 gene copy number is known to be the main phenotype modifier and there is growing evidence of additional factors contributing to SMA severity. Potential modifiers of spinal muscular atrophy can be found among the wide variety of different factors, such as multiple proteins interacting with SMN or promoting motor neuron survival, epigenetic modifications, transcriptional or splicing factors influencing SMN2 expression. Study of these factors enables to reveal mechanisms underlying SMA pathology and can have pronounced clinical application.

Genome-wide 5-hydroxymethylcytosine patterns in human spermatogenesis are associated with semen quality.

We performed immunofluorescent analysis of DNA hydroxymethylation and methylation in human testicular spermatogenic cells from azoospermic patients and ejaculated spermatozoa from sperm donors and patients from infertile couples. In contrast to methylation which was present throughout spermatogenesis, hydroxymethylation was either high or almost undetectable in both spermatogenic cells and ejaculated spermatozoa. On testicular cytogenetic preparations, 5-hydroxymethylcytosine was undetectable in mitotic and meiotic chromosomes, and was present exclusively in interphase spermatogonia Ad and in a minor spermatid population. The proportions of hydroxymethylated and non-hydroxymethylated diploid and haploid nuclei were similar among samples, suggesting that the observed alterations of 5-hydroxymethylcytosine patterns in differentiating spermatogenic cells are programmed. In ejaculates, a few spermatozoa had high 5-hydroxymethylcytosine level, while in the other ones hydroxymethylation was almost undetectable. The percentage of highly hydroxymethylated (5-hydroxymethylcytosine-positive) spermatozoa varied strongly among individuals. In patients from infertile couples, it was higher than in sperm donors (P<0.0001) and varied in a wider range: 0.12-21.24% versus 0.02-0.46%. The percentage of highly hydroxymethylated spermatozoa correlated strongly negatively with the indicators of good semen quality - normal morphology (r=-0.567, P<0.0001) and normal head morphology (r=-0.609, P<0.0001) - and strongly positively with the indicator of poor semen quality: sperm DNA fragmentation (r=0.46, P=0.001). Thus, the immunocytochemically detected increase of 5hmC in individual spermatozoa is associated with infertility in a couple and with deterioration of sperm parameters. We hypothesize that this increase is not programmed, but represents an induced abnormality and, therefore, it can be potentially used as a novel indicator of semen quality.

Aging and Ambiguous ROS. System Genetics Analysis.

Famous Free Radical Theory (FRT) of aging, the 50th year anniversary of which is celebrated in 2015 postulates a crucial role of Reactive Oxygen Species (ROS) in aging. Still it is the most robust theory of aging as mitochondria ROS production (mtROSp) correlates well with four principal ''rules" of aging being universal, endogenous, progressive, and deleterious. Vast number of experiments in different species prove mutagenic effect of ROS and their carcinogenic properties. So far, FRT stimulates the search of new pharmaceuticals with antioxidant activity. However, some recent experimental data and clinical findings render doubt to ROS as a principal senescence drivers and come in conflict with original version of FRT. Growth stimulating effects of ROS and their modest antitumor properties support these objections. One should remember that FRT is only one of the numerous theories of aging. Molecular mechanisms of senescence involve all living systems and numerous metabolic pathways which are also variable owing to the unique properties of individual genome and unique epigenetic modulations operating throughout the lifetime thus making aging a unique private matter. Universal theory of aging that incorporates and explains all known and suggested mechanisms of aging, is illusive. However, knowledge of unique peculiarities of individual genome, its feasible editing and efficient epigenetic regulation of metabolic pathways give a chance to postpone aging and extend period of active longevity.