The autism-associated gene chromodomain helicase DNA-binding protein 8 (CHD8) regulates noncoding RNAs and autism-related genes.

Chromodomain helicase DNA-binding protein 8 (CHD8) was identified as a leading autism spectrum disorder (ASD) candidate gene by whole-exome sequencing and subsequent targeted-sequencing studies. De novo loss-of-function mutations were identified in 12 individuals with ASD and zero controls, accounting for a highly significant association. Small interfering RNA-mediated knockdown of CHD8 in human neural progenitor cells followed by RNA sequencing revealed that CHD8 insufficiency results in altered expression of 1715 genes, including both protein-coding and noncoding RNAs. Among the 10 most changed transcripts, 4 (40%) were noncoding RNAs. The transcriptional changes among protein-coding genes involved a highly interconnected network of genes that are enriched in neuronal development and in previously identified ASD candidate genes. These results suggest that CHD8 insufficiency may be a central hub in neuronal development and ASD risk.

Compound heterozygous deletion of the PROP-1 gene in children with combined pituitary hormone deficiency.

Mutations in the prophet of Pit-1 gene (PROP1) have been shown to be responsible for combined pituitary hormone deficiency (CPHD) with deficiencies of growth hormone (GH), Prolactin (Prl), thyroid-stimulating hormone (TSH) and gonadotropins. We previously reported that homozygosity for a 2bp deletion in exon 2 (296delGA) accounted for CPHD in three patients from two Russian families. Here we report a second mutational hot spot in exon 2. This 2bp 149delGA deletion results in a frame shift that leads to the same serine to stop codon change at codon 109 (S109X). The predicted proteins are each truncated at residue 108 but diverge from the wild type sequence at different points in the homeodomain. Compound heterozygosity for the two mutations (149delGA/296delGA) was detected in 5 of 14 CPHD children from 4 families (36%). This provides the first evidence of heterozygosity for two common deletions as a cause of CPHD in Russian children.

A new locus for autosomal dominant Charcot-Marie-Tooth disease type 2 (CMT2F) maps to chromosome 7q11-q21.

Charcot-Marie-Tooth disease (CMT) constitutes a genetically heterogeneous group of inherited motor and sensory peripheral neuropathies. The axonal type of CMT is designated CMT type 2 (CMT2). Four loci for autosomal dominant CMT2 have been reported so far. Only in CMT2E, linked to chromosome 8p21, disease-causing mutations in the gene for neurofilament light chain (NEFL) were identified. In this study we report a multigenerational Russian family with autosomal dominant CMT2 and assign the locus to chromosome 7q11-q21. The CMT2 neuropathy in this family represents a novel genetic entity designated CMT2F.

Bacterial beta-galactosidase and human dystrophin genes are expressed in mouse skeletal muscle fibers after ballistic transfection.

Ballistic transfection, based on cell and tissue bombardment by the tungsten and gold microparticles covered with the gene DNA, was used for the delivery of a bacterial beta-galactosidase and a full-length cDNA copy of the human dystrophin genes into mouse skeletal muscles. CMV-lacZ, SV40-lacZ, LTR-lacZneo and full-length cDNA dystrophin (pDMD-1, approximately 16 kb) in eukaryotic expression vector pJ OMEGA driven by mouse leukaemia virus promotor (pMLVDy) were used throughout the studies. Musculus glutaeus superficialis of C57BL/6J and quadriceps femoris of mdx male mice were opened surgically under anesthesia and bombarded by means of the gene-gun technique originally developed by us. Different mixtures of gold and tungsten particles at ratios of 4:1, 1:1, 1:4 were applied. X-gal assay revealed marked beta-gal activity, both in total muscles and whole muscle fibers on histological sections, up to three months after transfection. The most intensive staining was observed after SV40-lacZ delivery. No staining was detected with LTR-lacZneo DNA as well as in untreated muscles. The higher tungsten particle concentration in the bombardment mixture correlated with more intense X-gal staining. At the gold/tungsten ratio of 1:4 the microparticles penetrated the musculus glutaeus superficialis and transfected the underlying musculus glutaeus medius as well. Immuno-cytochemical assay for human dystrophin revealed dystrophin positive myofibers (DPM) in the bombarded area up to two months after transfection. The proportion of DMP varied from 2.5% on day 17 up two 5% on day 60 after bombardment compared to only 0.5% in the control mdx mice. These results suggest the applicability of particle bombardment for gene delivery into muscle fibers.

PCR detection of Y-specific sequences in patients with Ullrich-Turner syndrome: clinical implications and limitations.

Cytogenetic analysis of patients with Ullrich-Turner syndrome (UTS) may fail to detect low levels of Y chromosome mosaicism or Y-derived marker chromosomes. More sensitive polymerase chain reaction (PCR)-based tests have been developed; however, applicability of these data to prognosis of virilization and gonadoblastoma development has not been investigated adequately. We used a multiplex PCR-based method to detect two Y-specific sequences, SRY and AMGLY. Thirteen patients with UTS without cytogenetically detected Y chromosomes were studied. Y-specific sequences were detected in 5 patients by multiplex PCR. A cryptic translocation involving the Y chromosome was found in one patient with severe virilization of external genitalia and a male phenotype. Y chromosomal mosaicism was detected in peripheral blood and in both gonads of one patient, and only in the left gonad of another patient. Existence of a Y-derived marker was demonstrated in 2 patients, one of whom had no testicular tissue or virilization. Consistent with previous reports, we conclude that PCR is more sensitive than classical cytogenetic analysis and detects patients with Y-specific sequences in blood cells. However, the absence of Y-specific material in blood is not a sufficient reason to reject surgical treatment in case of virilization.

Rarity of PIT1 involvement in children from Russia with combined pituitary hormone deficiency.

To ascertain the molecular background of combined pituitary hormone deficiency, screening for mutations in the pituitary-specific transcription factor (Pit-1/GHF-1) gene (PIT1) was performed on a cohort of 15 children from Russia with combined growth hormone (GH)/prolactin (Prl)/thyroid-stimulating hormone (TSH) deficiency. The group of patients, suspected of PIT1 mutations, consisted of four familial cases (seven patients) and eight sporadic cases. All had complete GH deficiency and complete or partial Prl and TSH deficiency. Direct sequencing of all six exons of PIT1 and its promoter region showed a C to T transition mutation at codon 14 of exon 1 in a 3 8/12-year-old girl. This novel PIT1 mutation results in a proline to leucine substitution (P14L). The patient was heterozygous for mutant and normal alleles. The heterozygous P14L mutation was also present in her mother as well as in her maternal aunt and grandmother, all of whom were phenotypically normal. There was no mutation in the father's DNA, suggesting the need for reevaluation of genomic imprinting. In other children of our series, no mutation in PIT1 or in its promotor region was identified. This is the first report on the analysis of PIT1 and its promoter region in Russian children with GH/Prl/TSH deficiency. However, as the involvement of PIT1 mutation is rare in Russia, the other negative cases need to be analyzed for another candidate gene responsible for combined GH/Pr/TSH deficiency.

[Use of nonradioactively labelled DNA-probes for DNA-diagnosis of Duchenne's muscular dystrophy]. / Ispol'zovanie neradioaktivno mechennykh DNK-zondov dlia DNA-diagnostiki miodistrofii Diushenna.

Routinely, we detect 0,1 pg of plasmid DNA using the nonradioactive DNA labeling and detection kit produced by Boehringer Mannheim (FRG). Using the kit we have determined the carrier status of a woman in a family with a case of Duchenne muscular dystrophy by the blot hybridization technique.

[The effective method of gene transfer into mammalian cells cultured in vitro]. / Effektivnyi metod perenosa genov v kletki mlekopitaiushchikh, kul'tiviruemye in vitro.

Efficiency of transformation by a number of vectors with the different selective markers of a set of cell lines has been studied for three different methods based on using calcium phosphate, polybrene, or electroporation. Electroporation is shown to be the most efficient one. Using this method with the system rat2k-cells-pAGO vector we have obtained the frequencies of transformation up to 2-3.10(-3). We suggest to use this system as a model for investigation of homologous recombination in the framework of the gene therapy project.

[DNA-diagnosis of carriers of the Duchenne muscular dystrophy gene]. / DNK-diagnostika nositel'stva gena miodistrofii diushenna.

Duchenne muscular dystrophy carrier detection has been performed by using probes XJ1.1 (intragenic probe) and probe 754 for a girl. The carrier probability was estimated by means of a computer program GenRisk combining pedigree and DNA-probe data and turned out to be 95%.

[Analysis of deletions in the dystrophin gene by the multiplex amplification method in patients suffering from Duchenne's muscular dystrophy]. / Analiz deletsii v gene distrofina metodom mul'tipleksnoi amplifikatsii u patsientov, stradaiushchikh miodistrofiei diushenna.

Multiplex polymerase chain reaction was carried out with the material from 68 patients suffering from Duchenne muscular dystrophy in Moscow and Leningrad clinics. Six pairs of oligoprimers were used. Deletions were detected in the material from 22 patients. A new type of deletion was found. Data on deletion frequencies and spectrum were compared with the results published by other authors.

[Prenatal DNA-diagnosis of Duchenne muscular dystrophy]. / Prenatal'naia DNK-diagnostika miodistrofii Diushenna.

Two prenatal diagnoses were carried out by the technique of intragenic polymorphous marker detecting heterozygosity in pregnant women in the families with cases of Duchenne muscular dystrophy. In both cases the DNA fragment from pERT87-15 region was amplified. This fragment includes a polymorphous site in BamHI region of recognition. DNA analyses of the families members have been made and the genetical risk has been calculated by the Bayes method. The prognoses for both fetuses are good.

[Determination of the effectiveness of Chinese hamster cell fusion in monolayers]. / Opredelenie éffektivnosti sliianiia kletok kitaiskogo khomiachka v monosloe.

Using polyethylene glycol 6000 (PEG), cell fusion was obtained for the Chinese hamster transplantable culture. A most rational character of cell fusion efficiency was the amount of nuclei found in multinuclear cells (polykaryocytar index) determined within 30-60 minutes after PEG treatment of the culture. After a 1-6 hour treatment, a mass death of polykaryocytes was more than 8 hours; in 3 hours and up to the end of a 24 hour's period of observation an intensive cell division is observed.

[Analysis of mutations and haplotypes of polymorphic markers in patients with Wilson-Konovalov disease from Bashkir]. / Analiz mutatsii i gaplotipov polimorfnykh markerov u patsientov s bolezn'iu Vil'sona-Konovalova iz Bashkirii.

Mutations of the Wilson disease (WD) gene were studied in patients from Bashkortostan. Four mutations were identified: His1069Gln, 3402delC, Glu1064Lys, and 3559 + 1G-->T. The latter mutation was described for the first time. Mutation His1069Gln was found to be the most prevalent in Bashkortostan; its frequency was 43.5%. The associations of the mutations found with the haplotypes for polymorphic loci D13S316, D13S133, and D13S228 were studied. The mutations were found to be linked with specific haplotypes, and the study of polymorphic haplotypes can therefore facilitate the search for mutations in the gene for WD. The results of the molecular genetic study of WD can be used for direct and indirect DNA diagnostics of this disease in Bashkortostan.

[Polymorphism of microsatellite loci DYS19, DYS393, and frequency of the T-C transition of the RBF5 locus on the Y-chromosome in inhabitants of the Volga-Ural region]. / Polimorfizm mikrosatellitnykh lokusov DYS19, DYS393 i chastota T-C-tranzitsii lokusa RBF5 Y-khromosomy u narodov Volgo-Ural'skogo regiona.

Polymorphism of the DYS19 and DYS393 microsatellite loci and T-C transition at the RBF5 locus of the Y chromosome were analyzed in Volga-Ural populations of Bashkirs, Tatars, Chuvashes, Maris, Mordovians, Udmurts, and Komis. For the DYS19 locus, statistically significant differences were observed between Trans-Ural and Northeastern Bashkirs; between Trans-Ural Bashkirs and Tatars; and between Udmurts and other populations of the Volga-Ural region, excluding Trans-Ural Bashkirs. The DYS393 locus allele frequency distribution patterns were similar in all populations studied. The highest and the lowest frequencies of T-C transition at the RBF5 locus was detected in Udmurts (0.68) and in Mordovians (0.09), respectively. Association of C-alleles with the DYS19/DYS393 microsatellite haplotypes was investigated. The major haplotypes specific to the Turkic- and Finno-Ugric populations were revealed.

[Distribution of spontaneous and 8-methoxypsoralen-induced sister chromatid exchanges along the length of the 1st chromosome of Chinese hamster cells]. / Raspredelenie spontannykh i indutsirovannykh 8-metoksipsoralenom sestrinskikh khromatidnykh obmenov po dline pervoi khromosomy kletok kitaiskogo khomiachka.

Distributions of spontaneous, induced by monoadducts and induced by crosslinks sister chromatid exchanges (SCE) along the first chromosome of Chinese hamster cells are nonrandom. All experimental distributions have low frequency of SCE in centromeric and telomeric regions. It can be explained by specific structural organization of the chromosome. However, there are some differences between experimental distributions. Distribution of SCE induced by crosslinks differs from that of spontaneous SCE. Distribution of SCE induced by monoadducts, unlike other distributions, has an increased frequency of exchanges in the q11 region. This region contains several narrow closely disposed G+ bands. It is possible that monoadducts lead to increasing SCE frequency on G+-G- junctions. Distribution of SCE induced by crosslinks resembles random distribution, except centromeric and telomeric regions. These results lead to conclusion that the mechanisms of formation of spontaneous, induced by monoadducts and induced by crosslinks SCE differ from each other.

[Expression of the human dystrophin gene in mdx mouse muscle fibers after transfection using liposomes and synthetic oligopeptides]. / Ekspressiia gena distrofina cheloveka v myshechnykh voloknakh myshei mdx posle transfektsii s pomoshch'iu liposom i sinteticheskikh oligopeptidov.

The number of dysrophin-positive fibers appearing in the femoral quadriceps muscle of mdx mice after injection of the full-length human dystrophin cDNA within the pHSADy plasmid was examined by means of immunohystochemical techniques. Transfection was carried out using lipofectamine (LFA), or synthetic oligopeptide complexes that provided the condensation of plasmid DNA (K8) and its release from endosomes gopeptide complexes that provided the condensation of plasmid DNA (K8) and its release from endosomes (JTS1). The LFA + pHSADy at a dose of 10 micrograms DNA did not affect the number of dystrophin-positive fibers at the site of injection (0.6-0.8%), whereas it caused a statistically significant increase in the number of these fibers in the same muscle of the contralateral leg (up to 2.3%). Injection of the SO + pHSADy complex resulted in the occurrence of dystrophin-positive muscle fibers characterized by a heterogeneous content and the distribution of dystrophin. The greatest number of dystrophin-positive fibers (about 16%) was observed under a ratio of pHSADy to K8 of 1:3 or 1:4. The observed maximal number of dystrophin-positive fibers after a single injection of SO + pHSADy was 3.8%, and it was 17.7% after three injections. These values were statistically significantly higher compared to intact mice (0.6%), the injection of pure plasmid (2.2%), or the intramuscular injection of sucrose (from 0.7 to 1.3%). A relatively high level of transfection (about 5%) was observed after an intracardiac injection of a large dose of the pHSADy (70 micrograms DNA). The perspectives of the targeted delivery of the dystrophin gene into muscles under conditions of parenteral administration are discussed.

[Expression of the human dystrophin gene in mdx mouse skeletal muscles after ballistic transfection]. / Ekspressiia gena distrofina cheloveka v skeletnykh myshtsakh myshei mdx posle ballisticheskoi transfektsii.

"Gene-gun" ballistic transfection (BT) was used to deliver genetic constructs pMLVDy and pHSADy containing full-length cDNA of the dystrophin gene to musculus quadriceps remoris and musculus gluteus of mdx mice, which represent a natural model of Duchenne muscular dystrophy. Clusters of dystrophin-positive muscular fibers (DPMF) were immunocytochemically detected in sites exposed to BT. The average number of DPMF was 2% by the 17th day and 3% by the 60th day after BT with pMLVDy, whereas the number of revertant DPMF was 0.2% in control mice (without BT). When pHSADy was used, the average number of DPMF was 3% 20 days after BT. In this case, dystrophin was uniformly spread though the myoplasm in 3% of cells and produced a slight signal in separate regions under the sarcolemma in 10% of muscle fibers. The number of revertant DPMF increased to 0.6% after BT with naked particles and to 2.8% after BT with the marker lacZ gene, in both bombarded and contralateral legs. The number of DPMF in the corresponding muscles of the contralateral leg significantly increased and reached 2.8% by the 60th day after BT with pMLVDy and 6.7% by the 20th day after BT with pHSADy. Human dystrophin gene cDNA was detected in all skeletal muscles, heart, intestine, tongue, and brain by polymerase chain reaction (PCR) three weeks after BT. Immunoblot analysis showed that normal 427-kDa human dystrophin was synthesized in muscles of mdx mice. The results suggest applicability of BT for delivery of dystrophin constructs into muscles.

[Congenital dysfunction of adrenal cortex - detection of new mutant gene of 21-hydroxylase]. / Vrozhdennaia disfunktsiia kory nadpochechnikov - obnaruzhenie novoi mutatsii gena 21-gidroksilazy.

The paper presents the results of investigations of 30 Slavic families with different types of congenial adrenal hyperplasia (CAH). The classic types of CAH were established to be associated with HLA B14 in most cases. This fact proves the presence of new mutation of 21-hydroxylase (21-OH) gene. The nature of this mutation was studied by polymerase chain reactions in two points: 3rd and 8th exons. The mutation in the third exon was recorded as deletion of 8 nucleotide pairs. The 8th exon appeared to be unchanged. The mutation in the homozygotic state causes a salt-losing type of disease with marked decreases in 21-OH activity. A significant decreases of 21-OH activity were also detected in the heterozygotic carriers during ACTH stimulation. This mutation was discovered in 28% of chromosomes of patients with salt-losing type of CAH.

[The preclinical DNA diagnosis of Huntington's chorea]. / Preklinicheskaia DNK-diagnostika khorei Gentingtona.

Presymptomatic DNA diagnosis of Huntington's chorea (HC) was made for two sons of a patient affected with the disease using amplification of the DNA fragment in the area of locus G 8 linked with HC gene. That fragment contains a polymorphous site in the area of restrictase recognition Hind III, being of information value as regards the family under examination. The familial analysis with the use of the DNA diagnosis data makes it possible to exclude the inheritance of HC gene for both the sons of the patient with a probability of 96%.

[Carrier detection and prenatal diagnosis of Duchenne's muscular dystrophy based on DNA analysis]. / Ustanovlenie nositel'stva i prenatal'naia diagnostika miodistrofii Diushenna na osnove analiza DNK.

Seven families with histories of Duchenne's muscular dystrophy underwent DNA diagnosis. The daughters of those consulted were examined for the carriage in 4 families. Their carriage was rejected or confirmed. Prenatal diagnosis was made in 2 families. In another family an abortion preceded obtaining molecular-genetic evidence. Probes 754, p20, XJI.I and primers for amplification of the site pERI87-15 containing a polymorphic locus were employed. The genetic risk was assessed using the computer program GenRisk adjusted for family history and DNA test allowances.