MBOAT1 homozygous missense variant causes nonobstructive azoospermia / 亚洲男科学杂志(英文版)

Nonobstructive azoospermia (NOA) is a common cause of infertility and is defined as the complete absence of sperm in ejaculation due to defective spermatogenesis. The aim of this study was to identify the genetic etiology of NOA in an infertile male from a Chinese consanguineous family. A homozygous missense variant of the membrane-bound O-acyltransferase domain-containing 1 (MBOAT1) gene (c.770C>T, p.Thr257Met) was found by whole-exome sequencing (WES). Bioinformatic analysis also showed that this variant was a pathogenic variant and that the amino acid residue in MBOAT1 was highly conserved in mammals. Quantitative polymerase chain reaction (Q-PCR) analysis showed that the mRNA level of MBOAT1 in the patient was 22.0% lower than that in his father. Furthermore, we screened variants of MBOAT1 in a broader population and found an additional homozygous variant of the MBOAT1 gene in 123 infertile men. Our data identified homozygous variants of the MBOAT1 gene associated with male infertility. This study will provide new insights for researchers to understand the molecular mechanisms of male infertility and will help clinicians make accurate diagnoses.

Transgenesis of Drosophila melanogaster with an Elovl5 gene enables the production of longer-chain fatty acids / 生物工程学报

In most insects, polyunsaturated fatty acids (PUFAs) are mainly polyunsaturated fatty acids with a carbon-chain length less than 18 carbon atoms, hardly any long-chain polyunsaturated fatty acids such as C20 and C22 that are more valuable and bioactive. This study, by using Drosophila melanogaster (Fruit fly) as a model organism, optimized the Δ6-fatty acid elongase enzyme Elovl5 gene from mice and transferred it to fruit flies for expression. Vectors containing Elovl5 gene were successfully injected into drosophila embryo through the microscopic injection. There were enhanced green fluorescent proteins expressed in the whole developmental stage of Drosophila be means of fluorescence microscope. At the same time, expression of Elovl5 gene significantly contributed to the transformation of fruit flies C18-polyunsaturated fatty acids in the body towards the biosynthesis of longer-chain polyunsaturated fatty acids. The transgenic fruit fly model rich in long-chain polyunsaturated fatty acids such as C20 and C22 were obtained, providing a basis for further research on biosynthesis of polyunsaturated fatty acids in fruit flies.

Quinolonas: Perspectivas actuales y mecanismos de resistencia / Quinolones: Nowadays perspectives and mechanisms of resistance

Quinolones are a family of synthetic broad-spectrum antimicrobial drugs whose target is the synthesis of DNA. They directly inhibit DNA replication by interacting with two enzymes; DNA gyrase and topoisomerase IV. They have been widely used for the treatment of several community and hospital acquired infections, in the food processing industry and in the agricultural field, making the increasing incidence of quinolone resistance a frequent problem associated with constant exposition to diverse microorganisms. Resistance may be achieved by three non-exclusive mechanisms; through chromosomic mutations in the Quinolone Resistance-Determining Regions of DNA gyrase and topoisomerase IV, by reducing the intracytoplasmic concentrations of quinolones actively or passively and by Plasmid-Mediated Quinolones-Resistance genes, [Qnr determinant genes of resistance to quinolones, variant gene of the aminoglycoside acetyltransferase (AAC(6')-Ib-c)] and encoding genes of efflux pumps (qepA and oqxAB)]. The future of quinolones is uncertain, however, meanwhile they continue to be used in an irrational way, increasing resistance to quinolones should remain as an area of primary priority for research.

Las quinolonas son un grupo de antimicrobianos sintéticos de amplio espectro, cuyo objetivo es la síntesis del ADN. Inhiben directamente su replicación al interactuar con dos enzimas; ADN girasa y topoisomerasa IV. Se han utilizado ampliamente para el tratamiento de infecciones intra y extra-hospitalarias, en el campo de la agricultura y en el procesamiento de alimentos, lo que hace que el incremento de resistencia a quinolonas sea un problema cada vez más frecuente, asociado a la constante exposición de diversos microorganismos. La resistencia puede alcanzarse mediante tres mecanismos no excluyentes entre sí; a través de mutaciones cromosómicas en genes codificantes que afectan las regiones determinantes de resistencia a quinolonas de ADN girasa y topoisomerasa IV, al reducir las concentraciones intracitoplásmicas de quinolonas de manera activa o pasiva y por genes de resistencia a quinolonas mediados por plásmidos [genes de resistencia a quinolonas determinates de qnr, gen variante de la aminoglucósido acetil transferasa (AAC(6’)-lb-cr) y genes codificadores de bombas de eflujo (qepAy oqxAB)]. El futuro de las quinolonas es incierto; sin embargo, mientras continúen empleándose para el manejo de infecciones en el ser humano, el incremento de resistencia a quinolonas debe permanecer como un área de importancia primaria para la investigación.

Genetic analysis of the ELOVL6 gene polymorphism associated with type 2 diabetes mellitus

Recent animal studies have indicated that overexpression of the elongation of long-chain fatty acids family member 6 (Elovl6) gene can cause insulin resistance and β-cell dysfunction. These are the major factors involved in the development of type 2 diabetes mellitus (T2DM). To identify the relationship between single nucleotide polymorphisms (SNP) of ELOVL6 and T2DM pathogenesis, we conducted a case-control study of 610 Han Chinese individuals (328 newly diagnosed T2DM and 282 healthy subjects). Insulin resistance and islet first-phase secretion function were evaluated by assessment of insulin resistance in a homeostasis model (HOMA-IR) and an arginine stimulation test. Three SNPs of the ELOVL6 gene were genotyped with polymerase chain reaction-restriction fragment length polymorphism, with DNA sequencing used to confirm the results. Only genotypes TT and CT of the ELOVL6 SNP rs12504538 were detected in the samples. Genotype CC was not observed. The T2DM group had a higher frequency of the C allele and the CT genotype than the control group. Subjects with the CT genotype had higher HOMA-IR values than those with the TT genotype. In addition, no statistical significance was observed between the genotype and allele frequencies of the control and T2DM groups for SNPs rs17041272 and rs6824447. The study indicated that the ELOVL6 gene polymorphism rs12504538 is associated with an increased risk of T2DM, because it causes an increase in insulin resistance.

The First Korean Case of Mucopolysaccharidosis IIIC (Sanfilippo Syndrome Type C) Confirmed by Biochemical and Molecular Investigation

Mucopolysaccharidosis (MPS) III has 4 enzymatically distinct forms (A, B, C, and D), and MPS IIIC, also known as Sanfilippo C syndrome, is an autosomal recessive lysosomal storage disease caused by a deficiency of heparan acetyl-CoA:alpha-glucosaminide N-acetyltransferase (HGSNAT). Here, we report a case of MPS IIIC that was confirmed by molecular genetic analysis. The patient was a 2-yr-old girl presenting with skeletal deformity, hepatomegaly, and delayed motor development. Urinary excretion of glycosaminoglycan (GAG) was markedly elevated (984.4 mg GAG/g creatinine) compared with the age-specific reference range (A (IVS2+1G>A) and c.1150C>T (p.Arg384*). To the best of our knowledge, this is the first case of MPS IIIC to be confirmed by clinical, biochemical, and molecular genetic findings in Korea.

The first report of the qnrB19, qnrS1 and aac(6´)-Ib-cr genes in urinary isolates of ciprofloxacin-resistant Escherichia coli in Brazil

In this study, we investigated the presence of plasmid-mediated quinolone resistance (PMQR) genes among 101 ciprofloxacin-resistant urinary Escherichia coli isolates and searched for mutations in the quinolone-resistance-determining regions (QRDRs) of the DNA gyrase and topoisomerase IV genes in PMQR-carrying isolates. Eight isolates harboured the qnr and aac(6')-Ib-cr genes (3 qnrS1, 1 qnrB19 and 4 aac(6')-Ib-cr). A mutational analysis of the QRDRs in qnr and aac(6')-Ib-cr-positive isolates revealed mutations in gyrA, parC and parE that might be associated with high levels of resistance to quinolones. No mutation was detected in gyrB. Rare gyrA, parC and parE mutations were detected outside of the QRDRs. This is the first report of qnrB19, qnrS1 and aac(6')-Ib-cr -carrying E. coli isolates in Brazil.

Aminoglycoside Susceptibility Profiles of Enterobacter cloacae Isolates Harboring the aac(6')-Ib Gene / 대한진단검사의학회지

The aminoglycoside 6'-N-acetyltransferases of type Ib (aac(6')-Ib) gene confers resistance to amikacin, tobramycin, kanamycin, and netilmicin but not gentamicin. However, some isolates harboring this gene show reduced susceptibility to amikacin. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommends a revision of the phenotypic description for isolates harboring the aac(6')-Ib gene. In this study, we determined the aminoglycoside susceptibility profiles of 58 AAC(6')-Ib-producing Enterobacter cloacae isolates. On the basis of the CLSI and EUCAST breakpoints, a large proportion (84.5% and 55.2%, respectively) of these 58 isolates were found to be susceptible to amikacin. However, among the isolates that were shown to be anikacin-susceptible according to the CLSI and EUCAST breakpoints, only 30.6% and 18.8% isolates, respectively, could be considered to have intermediate resistance on the basis of the EUCAST expert rules. Further studies should be conducted to determine the aminoglycoside susceptibility profiles of aac(6')-Ib-harboring isolates from various geographic regions and to monitor the therapeutic efficacy of amikacin in infections caused by these isolates.

The prevalence of aminoglycoside-modifying enzyme genes (aac (6')-I, aac (6')-II, ant (2")-I, aph (3')-VI) in Pseudomonas aeruginosa

INTRODUCTION: Pseudomonas aeruginosa (P. aeruginosa) is one of the primary opportunistic pathogens responsible for nosocomial infections. Aminoglycosides are an import ant component of antipseudomonal chemotherapy. The inactivation of drugs by modifying enzymes is the most common mechanism of aminoglycoside resistance. OBJECTIVES: The inactivation of aminoglycosides by modifying enzymes is the primary resistance mechanism employed by P. aeruginosa. The aim of the present study was to investigate the occurrence of aminoglycoside resistance and the prevalence of four import ant modifying enzyme genes (aac (6')-I, aac (6')-II, ant (2")-I, aph (3')-VI) in P. aeruginosa in Iran. METHODS: A total of 250 clinical isolates of P. aeruginosa were collected from several hospitals in seven cities in Iran. Antimicrobial susceptibility tests (using the disk diffusion method and E-tests) were performed for all 250 isolates. In addition, all isolates were screened for the presence of modifying enzyme genes by polymerase chain reaction. RESULTS: The resistance rates, as determined by the disk diffusion method, were as follows: gentamicin 43 percent, tobramycin 38 percent, and amikacin 24 percent. Of the genes examined, aac (6')-II (36 percent) was the most frequently identified gene in phenotypic resist ant isolates, followed by ant (2")-I, aph (3')-VI, and aac (6')-I. CONCLUSIONS: Aminoglycoside resistance in P. aeruginosa remains a signific ant problem in Iran. Therefore, there is considerable local surveillance of aminoglycoside resistance.

child with roberts syndrome

Roberts syndrome Is a genetically determined rare birth defect causing, skeletal deformities, particularly symmetrical limb reduction and craniofacial anomalies. For any child with limb and craniofacial bony malformations, this syndrome should be considered in the differentials. Although this syndrome represents only a small proportion of the total number of individuals with limb deficiency, it is important to be identified in order to give accurate genetic counselling including recurrence risk in siblings and possible prenatal diagnosis. This is the case report of a 22 days old male infant who presented with defective development of all four extremities and craniofacial abnormalities. The overall clinical and radiological features were suggestive of Roberts syndrome

Characteristics of aac(6')-Ib-cr Gene in Extended-Spectrum beta-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae Isolated from Chungnam Area / 대한진단검사의학회지

BACKGROUND: Concomitant quinolone resistance in extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae is a crucial problem in the clinical management of infections. In foreign countries, the fluoroquinolone acetylating aminoglycoside-(6)-N-acetyltransferase (aac[6']-Ib-cr) gene, a novel plasmid-mediated quinolone resistance determinant has been reported to occur in conjunction with qnr. We aim to investigate the prevalence and characteristics of concomitant aac(6')-Ib-cr and qnr expression in ESBL-producing Escherichia coli and Klebsiella pneumoniae in Korea. METHODS: Between December 2007 and April 2008, we collected 60 and 69 clonally unrelated non-repetitive clinical isolates of ESBL-producing E. coli and K. pneumoniae, respectively. We studied the expressions of 11 types of ESBL-encoding genes, 4 types of 16s rRNA methylase genes; rmtA, rmtB, rmtC and armA, 3 types of qnr genes; qnrA, qnrB, qnrS and aac(6')-Ib. The presence of aac(6')-Ib-cr variants was detected by sequencing. The involvement of integrons was studied using multiplex PCR and sequencing of gene-cassette arrays. Conjugation experiments were performed to confirm plasmid-mediated resistance and the relationships among coharbored genes. RESULTS: We observed a high prevalence of the cr variant (61.1%) of aac(6')-Ib, and the prevalence of this variant in qnr and aac(6')-Ib-coharboring isolates (67.4%) was higher than in qnr-negative isolates (51.7%). The high prevalence of the cr variant was significantly related to the high minimum inhibitory concentrations (MICs) of ciprofloxacin, tobramycin, and amikacin and indicated the statistically significant roles of qnrB, qnrS, rmtA, and rmtB in quinolone and aminoglycoside resistance. CONCLUSIONS: The aac(6')-Ib-cr variants were widespread and showed significant relation to the high-level quinolone and aminoglycoside resistance in ESBL-producing E. coli and K. pneumoniae.

Identificación molecular de enzimas modificantes de aminoglucósidos en cepas de Enterococcus spp. aisladas en hospitales de la Octava Región de Chile / Molecular identification of aminoglycoside-modifying enzymes among strains of Enterococcus spp. isolated in hospitals of the VIII Region of Chile

Background: Infectious diseases produced by Enterococcus spp, must be treated with a synergistic combination between a penicillin and an aminoglycoside. High level resistance to aminoglycosides is a serious therapeutic problem, since it predicts the loss of synergistic activity of this antimicrobial combination. Aim: To investigate the presence of genes encoding aminoglycoside-modifying enzymes (AMEs) among strains of Enterococcus spp with high level of resistance to aminoglycosides. Material and methods: The genes encoding some of the AMEs were investigated among 305 aminoglycoside-resistant strains of Enterococcus spp isolated in hospitals of the VIII region of Chile, by dot blot hybridization and Polymerase Chain Reaction (PCS). Results: High level resistance to some aminoglycosides was observed in 104 strains (34.1 percent) and 93 of these harbored at íeast one of the genes encoding the investigated AMEs. Three genes were detected: aac(6)Ie-aph(2")Ia (14.8 percent) encoding for the enzyme AAC(6)Ie-APH(2")Ia (resistance to all aminoglycosides, except streptomycin); aph(3)IIIa (26 percent), and ant(6)la (28.5 percent) encoding for the phosphorylating enzymes APH(3)Ilia (resistance to kanamycin, amikacin and neomycin), and ANT(6)-la (resistance only to streptomycin), respectively. None of the strains harbored the gene ant (4) which encode for the enzyme ANT (4). Conclusion: The low frequency of strains harbouring the bifunctional enzyme (<15 percent), conferring an extended resistance profile to aminoglycosides, allows us to propose the empirical use of aminoglycoside-aminocyclitols, associated to a penicillin, in the treatment of serious infections produced by species of enterococci.

Expression of a recombinant branched chain alpha-oxo acid dehydrogenase complex E2 (BCOADC-E2) in insect cells and its immunoreactivity to autoimmune sera

Preparation of a pure autoantigen by way of recombinant DNA technology has an important value in an accurate diagnosis or prognosis of an autoimmune disease. BCOADC-E2 subunit, a mitochondrial protein, has been known to be the autoantigen of primary biliary cirrhosis (PBC), a chronic autoimmune liver disease, as well as idiopathic dilated cardiomypathy (IDCM), a chronic autoimmune heart disease. Recombinant form of this molecule had been expressed in E. coli but with low yield and severe degradation. Furthermore, sera from IDCM patients failed to recognized BCOADC-E2 molecule produced in prokaryotic expression system. In this study, a recombinant bovine BCOADC-E2 fusion protein has been expressed in insect cells using baculovirus expression system and analyzed anti-BCOADC-E2 reactivity in sera from patients with PBC or with IDCM. Optimal production of the recombinant fusion protein has been achieved at 20 multiplicity of infection (MOI), and the protein was affinity-purified using metal-binding resins. The affinity-purified BCOADC-E2 protein was successfully recognized by sera from PBC patients, but not by sera from IDCM patients suggesting that the different auto-immune response against BCOADC-E2 is needed to be elucidated in terms of epitope recognition.

La biodiversidad humana y sus efectos sobre la variabilidad farmacologica: enzimas CYP2D6 y NAT2 en poblaciones amerindias de Panama, Colombia y Costa Rica / Human biodiversity and its effects on the pharmacological variability: CYP2D6 and NAT2 enzymes in Amerind populations of Panama, Colombia and Costa Rica

Human biodiversity originates partially from human microevolution, which have produced different populations. This biodiversity is responsible for most of the variability in drug response. We present the methodology employed in population pharmacology studies and general information about the CYP2D6 and NAT2 systems. We report results obtained in Embera and Ngawbe Amerindians, who are characterized by a low phenotypic and genotypic CYP2D6 diversity. In regard to NAT2, Amerindians are distinguished by a high allelic frequency of S3 and low ones of S1 and S2, situation which is reversed in Caucasians

Classification of children as slow or rapid acetylators based on concentrations of isoniazid in saliva following oral administration of body-weight and surface-area-related dosages of the drug.

The acetylator phenotype of 180 children aged 3-11 years was determined on the basis of isoniazid concentrations in saliva collected at 5 hours after oral administration of body-weight and surface-area-related dosages of the drug in a syrup form. Isoniazid 2.5 mg/kg was administered on one occasion and 75 mg/m2 surface-area on another, with an interval of 3 days between the occasions. A cross-over design was employed and the sequence was determined by random allocation. The distribution of the concentrations was bimodal with both procedures, indicating the presence of two groups namely, the slow and rapid acetylators. The criterion for a rapid acetylator was a concentration of 0.3 micrograms/ml or less by body-weight-related dosage and 0.4 micrograms/ml or less by that based on surface-area. Based on these criteria, 62% of the children were classified as slow acetylators and 38% as rapid acetylators by body-weight, and 59 and 41%, respectively by surface-area, and the findings were similar in children in the different age-groups. The agreement between the two procedures was 98%.