Analysis of copy number variation in men with non-obstructive azoospermia.

BACKGROUND: Recent findings demonstrate that single nucleotide variants can cause non-obstructive azoospermia (NOA). In contrast, copy number variants (CNVs) were only analysed in few studies in infertile men. Some have reported a higher prevalence of CNVs in infertile versus fertile men. OBJECTIVES: This study aimed to elucidate if CNVs are associated with NOA. MATERIALS AND METHODS: We performed array-based comparative genomic hybridisation (aCGH) in 37 men with meiotic arrest, 194 men with Sertoli cell-only phenotype, and 21 control men. We filtered our data for deletions affecting genes and prioritised the affected genes according to the literature search. Prevalence of CNVs was compared between all groups. Exome data of 2,030 men were screened to detect further genetic variants in prioritised genes. Modelling was performed for the protein encoded by the novel candidate gene TEKT5 and we stained for TEKT5 in human testicular tissue. RESULTS: We determined the cause of infertility in two individuals with homozygous deletions of SYCE1 and in one individual with a heterozygous deletion of SYCE1 combined with a likely pathogenic missense variant on the second allele. We detected heterozygous deletions affecting MLH3, EIF2B2, SLX4, CLPP and TEKT5, in one subject each. CNVs were not detected more frequently in infertile men compared with controls. DISCUSSION: While SYCE1 and MLH3 encode known meiosis-specific proteins, much less is known about the proteins encoded by the other identified candidate genes, warranting further analyses. We were able to identify the cause of infertility in one out of the 231 infertile men by aCGH and in two men by using exome sequencing data. CONCLUSION: As aCGH and exome sequencing are both expensive methods, combining both in a clinical routine is not an effective strategy. Instead, using CNV calling from exome data has recently become more precise, potentially making aCGH dispensable.

Genomic testing for copy number and single nucleotide variants in spermatogenic failure.

PURPOSE: To identify clinically significant genomic copy number (CNV) and single nucleotide variants (SNV) in males with unexplained spermatogenic failure (SPGF). MATERIALS AND METHODS: Peripheral blood DNA from 97/102 study participants diagnosed with oligozoospermia, severe oligozoospermia, or non-obstructive azoospermia (NOA) was analyzed for CNVs via array comparative genomic hybridization (aCGH) and SNVs using whole-exome sequencing (WES). RESULTS: Of the 2544 CNVs identified in individuals with SPGF, > 90% were small, ranging from 0.6 to 75 kb. Thirty, clinically relevant genomic aberrations, were detected in 28 patients (~ 29%). These included likely diagnostic CNVs in 3/41 NOA patients (~ 7%): 1 hemizygous, intragenic TEX11 deletion, 1 hemizygous DDX53 full gene deletion, and 1 homozygous, intragenic STK11 deletion. High-level mosaicism for X chromosome disomy (~ 10% 46,XY and ~ 90% 47,XXY) was also identified in 3 of 41 NOA patients who previously tested normal with conventional karyotyping. The remaining 24 CNVs detected were heterozygous, autosomal recessive carrier variants. Follow-up WES analysis confirmed 8 of 27 (30%) CNVs (X chromosome disomy excluded). WES analysis additionally identified 13 significant SNVs and/or indels in 9 patients (~ 9%) including X-linked AR, KAL1, and NR0B1 variants. CONCLUSION: Using a combined genome-wide aCGH/WES approach, we identified pathogenic and likely pathogenic SNVs and CNVs in 15 patients (15%) with unexplained SPGF. This value equals the detection rate of conventional testing for aneuploidies and is considerably higher than the prevalence of Y chromosome microdeletions. Our results underscore the importance of comprehensive genomic analysis in emerging diagnostic testing of complex conditions like male infertility.

Structural Insight into Complexation Ability and Coordination of Uranyl Nitrate by 1,10-Phenanthroline-2,9-diamides.

Reprocessing of spent nuclear fuel (SNF) is an important task in a frame of ecology and rational use of natural resources. Uranium, as the main component of SNF (>95%), can be recovered for further use as fresh nuclear fuel. To minimize an amount of solid radioactive waste generated during SNF reprocessing, new extractants are under investigation. Diamides of 1,10-phenanthroline-2,9-dicarboxylic acid are perspective tetradentate N-donor ligands that form strong complexes with f-elements, which are soluble in polar organic solvents. As an example of three ligands of this class, we conducted a comparative study and showed how the substituent in the amide functional group affects the extraction ability toward uranyl nitrate from nitric acid media. We have performed a careful study (NMR, FT-IR, XRD, RMC-EXAFS) of the structures of synthesized complexes of new ligands with uranyl nitrate and used quantum mechanical calculations to explain the discovered regularities through.

High-resolution microarray analysis unravels complex Xq28 aberrations in patients and carriers affected by X-linked blue cone monochromacy.

The human X chromosome contains ∼ 1600 genes, about 15% of which have been associated with a specific genetic condition, mainly affecting males. Blue cone monochromacy (BCM) is an X-linked condition caused by a loss-of-function of both the OPN1LW and OPN1MW opsin genes. The cone opsin gene cluster is composed of 2-9 paralogs with 99.8% sequence homology and is susceptible to deletions, duplications, and mutations. Current diagnostic tests employ polymerase chain reaction (PCR)-based technologies; however, alterations remain undetermined in 10% of patients. Furthermore, carrier testing in females is limited or unavailable. High-resolution X chromosome-targeted CGH microarray was applied to test for rearrangements in males with BCM and female carriers from three unrelated families. Pathogenic alterations were revealed in all probands, characterized by sequencing of the breakpoint junctions and quantitative real-time PCR. In two families, we identified a novel founder mutation that consisted of a complex 3-kb deletion that embraced the cis-regulatory locus control region and insertion of an additional aberrant OPN1MW gene. The application of high-resolution X-chromosome microarray in clinical diagnosis brings significant advantages in detection of small aberrations that are beyond the resolution of clinically available aCGH analysis and which can improve molecular diagnosis of the known conditions and unravel previously unrecognized X-linked diseases.

[[Impact of plestazol preparation on hyperplasia of inter vascular layer in the patients, suffering obliterating atherosclerosis of the lower extremities arteries after reconstructive operations].]

In the clinic 60 patients were examined, in whom reconstructive operations on the main arteries were performed for obliterating atherosclerosis (OA) of the lower extremities vessels. Efficacy of impact of Plestazol (in a 200 mg/day dose) on neointima hyperpla- sia was studied. Clopidogrel (75 mg/day) was administered to patients of comparative group. Effective criteria for estimation of the migration-proliferation disorders state in endothelial dysfunction are concentration of the intercellular adhesion molecules (ICAM) and the basic factor of the fibroblasts growth (FGFb); morphological disorders in hyperplastic reactions of neointima - determination of thickness of "intima-media" complex in accordance to the ultrasound duplex scanning data. There was established, that Plestazol constitutes an effective disaggregate preparation, positively impacting decelerating reaction of neointima hyperplasia, including, deceleration of the smooth- muscle cells migration into subendotelial layer in the formation zone of the femoro- popliteal shunt distal anastomosis.

[MODERN VIEW ON CHILDREN'S BIOLOGICAL DEVELOPMENT].

The present review is devoted to the modern analysis of understanding of biological development, as a health index. There are considered works about tendencies of biological development of the child's organism in the conditions of environment, training and education. There are presented results of the study of indices of biological age of children's and teenagers' residing in various regions of the Russian Federation, and also in the near-abroad and far-abroad countries. The scientific materials devoted to the study of the biological development of the children's population were analyzed in the context of epochal variability. Modern approaches to the identification of the causes and risk factors for the formation of deviations in biological development of children and teenagers are presented.

The power of mouse genetics to study spermatogenesis.

Approximately 80 million people worldwide are infertile, and nearly half of all infertility cases are attributed to a male factor. Therefore, progress in reproductive genetics becomes crucial for future diagnosis and treatment of infertility. In recent years, enormous progress has been made in this field. More than 400 mutant mouse models with specific reproductive abnormalities have been produced, and numerous human association studies have been discovered. However, the translation of basic science findings to clinical practice remains protracted, with only modest progress in the application of novel findings to clinical genetic testing and cures. To date, the most significant findings in male infertility remain numeric and structural chromosomal abnormalities and Y-chromosome microdeletions in infertile men. Thus, we anticipate that future genetic investigations will focus on infertile men with a normal somatic karyotype but with various spermatozoal defects, like insufficient production of spermatozoa (oligozoospermia), inadequate motility (asthenozoospermia), abnormal morphology (teratozoospermia), or combinations of these defects. Ultimately, basic advances in mammalian nonhuman reproduction will translate to clinical advances in human reproduction and testing for infertile humans, thereby helping to improve diagnostics and health care for infertile patients.

The conserved WW-domain binding sites in Dystroglycan C-terminus are essential but partially redundant for Dystroglycan function.

BACKGROUND: Dystroglycan (Dg) is a transmembrane protein that is a part of the Dystrophin Glycoprotein Complex (DGC) which connects the extracellular matrix to the actin cytoskeleton. The C-terminal end of Dg contains a number of putative SH3, SH2 and WW domain binding sites. The most C-terminal PPXY motif has been established as a binding site for Dystrophin (Dys) WW-domain. However, our previous studies indicate that both Dystroglycan PPXY motives, WWbsI and WWbsII can bind Dystrophin protein in vitro. RESULTS: We now find that both WW binding sites are important for maintaining full Dg function in the establishment of oocyte polarity in Drosophila. If either WW binding site is mutated, the Dg protein can still be active. However, simultaneous mutations in both WW binding sites abolish the Dg activities in both overexpression and loss-of-function oocyte polarity assays in vivo. Additionally, sequence comparisons of WW binding sites in 12 species of Drosophila, as well as in humans, reveal a high level of conservation. This preservation throughout evolution supports the idea that both WW binding sites are functionally required. CONCLUSION: Based on the obtained results we propose that the presence of the two WW binding sites in Dystroglycan secures the essential interaction between Dg and Dys and might further provide additional regulation for the cytoskeletal interactions of this complex.

Interstitial deletion of 10p and atrial septal defect in DiGeorge 2 syndrome.

We present molecular genetic investigations of a 4-year-old boy with craniofacial dysmorphism and developmental delay. Trivial mitral and tricuspid regurgitation without gross structural abnormality was diagnosed by echocardiography. High-resolution chromosome analysis revealed an interstitial deletion, del(10)(p12.1p12.32). To characterize the deletion size and breakpoints, we performed fluorescence in situ hybridization analysis using 27 BAC clones. Our data demonstrate an approximately 5.5 Mb deletion del(10)(p12.1p12.31). Surprisingly, the BAC clone RP11-56H7 that contains NEBL, an apparent downstream gene of the cardiogenic transcription factor HAND2 previously shown to be deleted in the patients with DiGeorge 2 syndrome and 10p13 deletion, was deleted in our patient with 10p12.1-p12.31 deletion. In addition, we provide clinical data and results of molecular analysis for a patient with multiple congenital anomalies including Ebstein's anomaly, kidney malformations, and 10p13-p14 deletion. We also reviewed 19 patients with congenital heart defects and deletions involving 10p and propose that atrial septal defect (ASD) is a common cardiac anomaly associated with DiGeorge 2 syndrome. Based on genotype-phenotype analysis of published patients and those reported herein, we propose an approximately 1.0 Mb critical region between loci D10S547 and D10S2176 in 10p14 to be associated with ASD. Considering that septal defects are the most frequent congenital heart anomaly, we suggest that further investigations in the 10p critical region are important to identify gene(s) responsible for this common birth defect.

Intermolecular -CH(3)...O(2)N-- contacts in two polymorphic modifications of (1E)-N'-[(E)-2-cyano-1-(dimethylamino)-2-nitrovinyl]-N,N-dimethylethanimidamide.

The title compound was synthesized and isolated in two crystal modifications. The structure of the orthorhombic modification was determined by the X-ray powder diffraction method and the structure of the monoclinic modification was determined using the X-ray single-crystal diffraction technique. The molecules in both polymorphs are E,E isomers. Intermolecular H(3)C....NO(2) contacts and their role in the formation of the polymorphic modifications are analyzed.

Powder diffraction study of the hydrogen bonds in nitroxoline and its hydrochloride.

The crystal structures of 8-hydroxy-5-nitroquinoline, C9H6N2O3, (I), and 8-hydroxy-5-nitroquinolinium chloride, C9H7N2O3+*Cl-, (II), have been determined from X-ray powder data. In (I), the molecules are linked via moderately strong hydrogen bonds to form dimers. Such a packing motif is likely to be responsible for the low solubility of (I) in water. In (II), the inversion-related cations form stacks, and anions fill the interstack channels.

Cosegregation and functional analysis of mutant ABCR (ABCA4) alleles in families that manifest both Stargardt disease and age-related macular degeneration.

Mutations in ABCR (ABCA4) have been reported to cause a spectrum of autosomal recessively inherited retinopathies, including Stargardt disease (STGD), cone-rod dystrophy and retinitis pigmentosa. Individuals heterozygous for ABCR mutations may be predisposed to develop the multifactorial disorder age-related macular degeneration (AMD). We hypothesized that some carriers of STGD alleles have an increased risk to develop AMD. We tested this hypothesis in a cohort of families that manifest both STGD and AMD. With a direct-sequencing mutation detection strategy, we found that AMD-affected relatives of STGD patients are more likely to be carriers of pathogenic STGD alleles than predicted based on chance alone. We further investigated the role of AMD-associated ABCR mutations by testing for expression and ATP-binding defects in an in vitro biochemical assay. We found that mutations associated with AMD have a range of assayable defects ranging from no detectable defect to apparent null alleles. Of the 21 missense ABCR mutations reported in patients with AMD, 16 (76%) show abnormalities in protein expression, ATP-binding or ATPase activity. We infer that carrier relatives of STGD patients are predisposed to develop AMD.

Null missense ABCR (ABCA4) mutations in a family with stargardt disease and retinitis pigmentosa.

PURPOSE: To determine the type of ABCR mutations that segregate in a family that manifests both Stargardt disease (STGD) and retinitis pigmentosa (RP), and the functional consequences of the underlying mutations. METHODS: Direct sequencing of all 50 exons and flanking intronic regions of ABCR was performed for the STGD- and RP-affected relatives. RNA hybridization, Western blot analysis, and azido-adenosine triphosphate (ATP) labeling was used to determine the effect of disease-associated ABCR mutations in an in vitro assay system. RESULTS: Compound heterozygous missense mutations were identified in patients with STGD and RP. STGD-affected individual AR682-03 was compound heterozygous for the mutation 2588G-->C and a complex allele, [W1408R; R1640W]. RP-affected individuals AR682-04 and-05 were compound heterozygous for the complex allele [W1408R; R1640W] and the missense mutation V767D. Functional analysis of the mutation V767D by Western blot and ATP binding revealed a severe reduction in protein expression. In vitro analysis of ABCR protein with the mutations W1408R and R1640W showed a moderate effect of these individual mutations on expression and ATP-binding; the complex allele [W1408R; R1640W] caused a severe reduction in protein expression. CONCLUSIONS: These data reveal that missense ABCR mutations may be associated with RP. Functional analysis reveals that the RP-associated missense ABCR mutations are likely to be functionally null. These studies of the complex allele W1408R; R1640W suggest a synergistic effect of the individual mutations. These data are congruent with a model in which RP is associated with homozygous null mutations and with the notion that severity of retinal disease is inversely related to residual ABCR activity.

Differently coloured polymorphs of 4-[(2-nitrophenyl)azo]phenol.

The crystal structures of the brown-yellow and orange polymorphs of the title compound, 4-[(2-nitrophenyl)diazenyl]phenol, C(12)H(9)N(3)O(3), have been determined and their visible reflection spectra recorded. Both structures adopt a stacking arrangement with interstack hydrogen bonds. Ab initio and semi-empirical (AM1 and INDO-CISD) calculations were performed in order to rationalize the difference in colour. It can be attributed neither to the subtle distinctions in molecular geometry nor to the effect of intermolecular electrostatic interactions. The most probable origin of this difference is the mixing of intramolecular n --> pi* and intermolecular charge-transfer excitations.

Late-onset Stargardt disease is associated with missense mutations that map outside known functional regions of ABCR (ABCA4).

Based on recent studies of the photoreceptor-specific ABC transporter gene ABCR (ABCA4) in Stargardt disease (STGD1) and other retinal dystrophies, we and others have developed a model in which the severity of retinal disease correlates inversely with residual ABCR activity. This model predicts that patients with late-onset STGDI may retain partial ABCR activity attributable to mild missense alleles. To test this hypothesis, we used late-onset STGDI patients (onset: > or =35 years) to provide an in vivo functional analysis of various combinations of mutant alleles. We sequenced directly the entire coding region of ABCR and detected mutations in 33/50 (66%) disease chromosomes, but surprisingly, 11/33 (33%) were truncating alleles. Importantly, all 22 missense mutations were located outside the known functional domains of ABCR (ATP-binding or transmembrane), whereas in our general cohort of STGDI subjects, alterations occurred with equal frequency across the entire protein. We suggest that these missense mutations in regions of unknown function are milder alleles and more susceptible to modifier effects. Thus, we have corroborated a prediction from the model of ABCR pathogenicity that (1) one mutant ABCR allele is always missense in late-onset STGD1 patients, and (2) the age-of-onset is correlated with the amount of ABCR activity of this allele. In addition, we report three new pseudodominant families that now comprise eight of 178 outbred STGD1 families and suggest a carrier frequency of STGD1-associated ABCR mutations of about 4.5% (approximately 1/22).

Sodium 4-(2-pyridinyldiazenyl)resorcinolate monohydrate and ammonium 2,4-dinitro-1-naphthalenolate from powder diffraction data.

The crystal structures of two dyestuffs, Na(+).C(11)H(8)N(3)O(2)(-).H(2)O, (I), and NH(4)(+).C(10)H(5)N(2)O(5)(-), (II), were determined from X-ray powder diffraction data. In both structures, translationally related anions form stacks, and cations fill interstack channels. A comparison of the diffuse reflectance spectra of crystalline (I) and (II) with the absorption spectra of their aqueous solutions demonstrates that the geometry of their anions does not change significantly upon transfer from the crystalline to the solution state.

4-(Phenyldiazenyl)naphthalen-1-amine and its hydrochloride.

The crystal structures of 4-(phenyldiazenyl)naphthalen-1-amine, C(16)H(13)N(3), (I), and its hydrochloride, (4-aminonaphthalen-1-yl)phenyldiazenium chloride, C(16)H(14)N(3)(+) x Cl(-), (II), have been determined from X-ray single-crystal and powder data, respectively. The effect of the crystal environment on the molecular electronic structure was analysed on the AM1 level. One of the two symmetry-independent molecules in (I) is involved in intermolecular hydrogen bonding, so that its dipole moment is twice as large as that of the other molecule. The cations in (II) form stacks along [100], with the Cl(-) anions forming hydrogen bonds to all three H atoms attached to N atoms.

Nuclear magnetic resonance of 93Nb in LiNbO3: effect of structural distortions on lineshapes.

The expression for the angular dependence of the first moment of the nuclear magnetic resonance central transition (+/-1/2<-->-/+1/2) lineshapes of a quadrupolar nuclei with half integer spins in a disordered solid is derived and used for investigation of the structural distortions in LiNbO3.