CYP21A2 Mutation Analysis in Korean Patients With Congenital Adrenal Hyperplasia Using Complementary Methods: Sequencing After Long-Range PCR and Restriction Fragment Length Polymorphism Analysis With Multiple Ligation-Dependent Probe Amplification Assay

CYP21A2 mutation analysis of congenital adrenal hyperplasia (CAH) is challenging because of the genomic presence of a homologous CYP21A2 pseudogene and the significant incidence of pseudogene conversion and large deletions. The objective of this study was to accurately analyze the CYP21A2 genotype in Korean CAH patients using a combination of complementary methods. Long-range PCR and restriction fragment length polymorphism analyses were performed to confirm valid amplification of CYP21A2 and to detect large gene conversions and deletions before direct sequencing. Multiple ligation-dependent probe amplification (MLPA) analysis was conducted concurrently in 14 CAH-suspected patients and six family members of three patients. We identified 27 CYP21A2 mutant alleles in 14 CAH-suspected patients. The c.293-13A>G (or c.293-13C>G) was the most common mutation, and p.Ile173Asn was the second, identified in 25% and 17.9% of alleles, respectively. A novel frame-shift mutation of c.492delA (p.Glu 164Aspfs*24) was detected. Large deletions were detected by MLPA in 10.7% of the alleles. Mutation studies of the six familial members for three of the patients aided in the identification of haplotypes. In summary, we successfully identified CYP21A2 mutations using both long-range PCR and sequencing and dosage analyses. Our data correspond relatively well with the previously reported mutation spectrum analysis.

Obtención de suspensiones celulares y embriones somáticos de cafeto (Coffea canephora P) con el empleo de metabolitos bacterianos / Obtaining of cell suspensions and somatic embryos of coffee (Coffea canephora P) using of bacterial metabolites

La embriogénesis somática es importante como sistema modelo para estudiar el desarrollo de eventos fisiológicos, citológicos y moleculares que sustentan la embriogénesis en plantas, por ser un sistema adecuado para la propagación masiva de especies vegetales y servir de herramienta para el mejoramiento genético, la conservación de germoplasma y la validación de nuevos productos biológicos, y facilitar la producción a gran escala a través del cultivo en medio líquido y su aplicación en biorreactores, proporcionando alta frecuencia de multiplicación, rápido crecimiento del embrión, facilidad de absorción de nutrientes y reducción de la labor de subcultivo. En este trabajo se empleó la embriogénesis somática como vía de multiplicación para evaluar el efecto de metabolitos bacterianos en la inducción de suspensiones celulares y embriones somáticos en tres genotipos de cafeto pertenecientes a Coffea canephora P. variedad Robusta. Para ello se estudiaron densidades de inóculo entre 0,2, 0,5, 1,0 y 3,0 gMF/L-1, y se evaluó el efecto de diferentes medios de cultivo en el desarrollo del proceso. Los resultados mostraron un comportamiento diferenciado en el genotipo M-28, en medios de cultivo suplementados con reguladores de crecimiento convencionales y en los alternativos. Se evidenció una fuerte relación entre la viabilidad celular y el número de células, ante las diferentes condiciones de cultivo y según la densidad de inóculo, se observó un amplio rango de tamaño y forma en las poblaciones de embriones somáticos. Los porcentajes de conversión de ES con el medio MDE-2 evidenciaron mejoras de este indicador para el cultivo del cafeto.

The somatic embryogenesis is important as model system to study the development of physiologic and molecular events that sustain the embryogenesis in plants, is an appropriate system for the massive propagation of vegetable species and as tool for the genetic improvement, the germplasm conservation and the validation of new biological products and to facilitate the multiplication to great scale through the culture in liquid medium, as well as application in bioreactores, providing high multiplication frequency, quick growth of the embryo, easiness of absorption of nutritious and reduction of the subculturing. In this paper the somatic embryogenesis was used to evaluate the effect of bacterial compounds in the induction of cellular suspensions and somatic embryos in three coffee genotypes of Coffea canephora P. var. Robusta. Were studied inoculo densities among 0.2, 0.5, 1.0 and 3.0 gMF/L-1 and the effect of different culture medium in the development of the process. The results showed a behavior differed in the genotype M-28, in medium culture with conventional regulators of growth and the alternatives. Strong relationship was evidenced between the cellular viability and the number of cells, in the different cultivation conditions and according to the inoculo density, a wide range of size and forms as observed in the populations of somatic embryos. The conversion percentages with the medium MDE-2, evidenced improvements of this indicator for the coffee.

Analysis of survival motor neuron gene conversion in patients with spinal muscular atrophy / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the type and frequency of gene conversion from SMN1 to SMN2 in Chinese patients affected with spinal muscular atrophy (SMA), and to explore the relationship between gene conversion and clinical phenotype.</p><p><b>METHODS</b>Non-homozygous deletion of SMN1 gene exon 8 was screened among 417 patients with SMN1 exon 7 homozygous deletions. To analyze and verify the types of gene conversion, genomic DNA sequencing, multiplex ligation-dependent probe amplification (MLPA), and gene subcloning and sequencing were carried out.</p><p><b>RESULTS</b>Thirty-one patients (7.4% of all) with non-homozygous deletions of SMN1 exon 8 were detected. Through series of experiments, the fusion genes SMN1/SMN2 in all cases were delineated. Five types of gene conversions were identified, which included SMN2-I7b/SMN1 E8, SMN2-I7a/SMN1 I7b, SMN2-E7/SMN1 I7a, SMN1 I6/SMN2 E7/SMN1 I7a and SMN2-E7/SMN1 I7a/SMN2 I7b. Such conversions were found in the type I-III patients. For 10 patients with type I-III SMA and 3 copies of SMN2 gene produced by conversion, the average survival age was 5 year and 4 months.</p><p><b>CONCLUSION</b>Partial conversions of SMN1 gene have been found among Chinese SMA patients. The type of conversion and frequency seem to be different from those of other races. Gene conversion to some extent may impact on survival time and rate of SMA patients, especially type I SMA.</p>

Study of SMN gene in Chinese children with spinal muscular atrophy / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the incidence of homozygous absence of SMN1 exons 7 and 8, SMN gene conversion frequency and SMN subtle mutations in children with spinal muscular atrophy (SMA).</p><p><b>METHODS</b>The homozygous deletion was detected by PCR-RFLP in 106 Chinese children with SMA, gene conversion by RFLP and subtle mutations by sequencing.</p><p><b>RESULTS</b>The rate of deletion of SMN1 exons 7 and/or 8 was 91.5%. Deletion of SMN1 exon 8 but existence of exon 7 was noted in one child with SMA. There were no significant differences in the gene conversion frequency among children with different types of SMA and who had homozygous deletion of SMN1 exon 7 but existence of exon 8. The gene conversion frequency was 8.3% in children with homozygous deletion of SMN1 exon 7. No subtle mutations were found around SMN1 exon 7.</p><p><b>CONCLUSIONS</b>Deletion of SMN1 exons 7 and/or 8 is the main cause of SMA in Chinese children. There exists a SMN gene conversion phenomenon in SMA. Deletion of exon 8 might lead to SMA. The hot area of subtle mutations of this disease might not be around SMN1 exon 7.</p>

Congenital adrenal hyperplasia clinical: characteristics and genotype in newborn, childhood and adolescence / Hiperplasia suprarrenal congenital: características clínicas, seguimiento y genotipo en la etapa perinatal, la niñez y la adolescencia

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is a disorder which can adopt three clinical expressions: two classical forms –salt-wasting (SW), with residual enzymatic activity (EA) < 1% and simple virilizing (SV), with EA 1-2%– and a mild late onset or nonclassical (NC) form, with EA 10-60%. Our objective is to describe clinical characteristics, growth, and bone mass in a group of patients affected by 21-hydroxylase deficiency. Besides, molecular genetics studies were performed in patients, and also when available in their parents and siblings. Nine patients with neonatal diagnosis and 8 with pre or postpubertal diagnosis were studied. Analyses of 10-point mutations in the CYP21A2 gene were performed. We found that all the patients with the classical expression, except one with a de novo mutation R356W in one allele, were fully genotyped with predictive < 2% EA mutations. Signs of hyperandrogenism were present in 5/6 NC patients; one was diagnosed by searching for mutations in asymptomatic siblings. All the NC patients were compound heterozygotes carrying V281L mutation in one allele and a predictive low EA in the other, except for one not yet determined. In patients with neonatal diagnosis, mean height was low at one year of age, though it showed a significant increase before the onset of puberty. We conclude that neonatal diagnosis of classical CAH allows an adequate follow up enhancing growth. Molecular analyses of all members of an affected family may disclose asymptomatic patients. The presence of de novo mutations, as well as, the presence of mutations with low predicted EA in NC patients reinforces the importance of genotyping for appropriate genetic counseling. In fully genotyped NC patients, the lowest value of ACTH-stimulated 17OHP was 14 ng/ml. Lower cut-off values might overestimate the diagnosis of the NC form.

La hiperplasia suprarrenal congénita por déficit de 21-hidroxilasa presenta tres formas clínicas: dos clásicas, perdedora de sal, con actividad enzimática (AE) < 1% y virilizante simple, con AE 1-2% y una no clásica, con AE 10-60%. Nuestro objetivo es describir las características clínicas y el genotipo de un grupo de pacientes con hiperplasia suprarrenal congénita; este último también sedeterminó en todos los miembros de la familia. Se estudiaron 9 pacientes diagnosticados en la etapa perinatal y 8 durante la etapa pre y postpuberal. Se analizaron diez mutaciones en el gen CYP21A2 y se evaluó crecimiento y densidad mineral ósea. Once pacientes presentaron la forma clásica: 9 con diagnóstico perinatal y 2 diagnosticados más tardíamente, uno de ellos con agrandamiento testicular por restos adrenales. Todos los pacientes, salvo 1 con una mutación de novo R356W en un alelo, presentaron ambos alelos mutados con un genotipo que predice AE < 2%. Seis pacientes presentaron la forma no clásica, todos con signos clínicos de hiperandrogenismo salvo un familiar asintomático que se diagnosticó por el estudio molecular. Todos, a excepción de uno con un alelo aún no determinado, presentaron la mutación V281L acompañada de otra que predice AE < 2%. Durante la evolución de los pacientes de diagnóstico perinatal se observó talla baja al año con recuperación de la misma en la etapa prepuberal. La densidad mineral ósea fue normal. Podemos concluir que el diagnóstico en la etapa perinatal en pacientes con la forma clásica posibilita un mejor seguimiento y crecimiento. La genotipificación de todos los miembros de una familia permite el diagnóstico de formas asintomáticas. La presencia de mutaciones de novo y de un alelo con una mutación que predice baja AE en los pacientes con forma no clásica, refuerza la importancia de la genotipificación para un adecuado asesoramiento genético.

Protection the flavonoids, rutin and proto chatechuic acid, against mitotic crossing over, gene conversion and reverse mutation induced by [chlorpyritos] in saccharomyces cerevisia D7

Protection by the flavonoids, rutin and protochatechuic acid, against insecticide chlorpyrifos induced mitotic crossing over, gene conversion and reverse mutation were investigated in Saccharomyces cerevisia D7. The results indicate that Rutin and Protochatechuic acid have some antimutagenic potential against mutagenicity of chloropyrifos. There for, the flavonoids contained in Senna seem to be important as antimutagenic and antioxidants

Conversión y reversión génica en Saccharomyces cerevisiae. Un modelo para el estudio del daño producido por radiación gamma / Gen conversion and reversion events in Saccharomyces cerevisiae. A model for the study of gamma radiation damage

Se estudió la radiosensibilidad y la cinética de inducción de eventos de conversión y reversión génica en la cepa D7 de Sacharomyces cerevisiae frente a radiación gamma, en rangos de dosis entre 100-800 Gy y entre 50-300 Gy, respectivamente. Se utilizó una fuente de 60Co PX-g-30 con una tasa de dosis 49,43 Gy/min. La curva de supervivencia celular mostró un DL50 de 150 Gy. La cinética de muerte celular fue lineal con un ajuste superior a 98 por ciento. La inducción de eventos de conversión génica fue significativa respecto al control a partir de 50 Gy. Por el contrario, la reversión génica fue significativa solo a partir de 200 Gy. En general, las frecuencias de eventos de conversión génica fueron superiores a las de reversión, esto sugiere que la radiación gamma induce preferentemente eventos recombinogénicos. Tanto para los eventos de conversión como de reversión génica se obtuvo una dependencia exponencial de la dosis de radiación gamma. Se discutió sobre la utilidad relativa del ensayo para estudios de mutagénesis y antimutagénesis

The value of code conversion of human genome in medicine

The Human Genome Project is an international cooperative project that launched in 1990 and completely finished in April 14, 2003. The discovery of pathogenic genes is promising that light up the pathogenesis of many diseases in human. In the future, the understanding of these genes is helpful for patients and physicians, but the first advantages are in diagnosis and prognosis. It’s hoped to bring about new more effective therapies tailored for each patient according to his genome in the next 5 years

Phenotype and genotype correlation of the microconversion from the CYP21A1P to the CYP21A2 gene in congenital adrenal hyperplasia

Deficiency of 21-hydroxylase is the most common form of congenital adrenal hyperplasia (CAH-21OH). We determined by allele-specific PCR the frequency of microconversion in the CYP21A2 gene in 50 Brazilian patients with the classical (salt wasting: SW and simple virilizing: SV) forms and nonclassical (NC) form of CAH-21OH and correlated genotype with phenotype. Genotypes were classified into three mutation groups (A, B, and C) based on the amount of enzymatic activity in in vitro studies using adrenal cells. In 94 unrelated alleles, we diagnosed 76 percent of the affected alleles after screening for 7 microconversions. The most frequent point mutations observed in this series were I172N (19 percent), V281L (18 percent), and IVS2,A/C>G,-12 (15 percent). In the SW form, the most frequent mutation was IVS2,A/C>G,-12 (38 percent), in the SV form it was I172N (53 percent), and in the NC form it was V281L (57.7 percent). We observed a good correlation between genotype and phenotype. Discordance between genotype and phenotype was found in one SV patient with a mild mutation in one of the alleles (R356W/V281L). However, we cannot rule out the presence of an additional mutation in these alleles. We also observed a good correlation of genotype with 17alpha-hydroxyprogesterone, testosterone, and androstenedione levels. The severity of external genitalia virilization correlated with the severity of mutation. In conclusion, the frequencies described in the present study did not differ from worldwide studies, including the Brazilian population. The few differences observed may reflect individual sample variations. This new Brazilian cohort study suggests the presence of new mutations in Brazilian patients with different forms of CAH-21OH

Molecular Diagnosis of 21-hydroxylase (CYP21) Gene mutations in Congenital Adrenal Hyperplasia / 대한산부인과학회잡지

OBJECTIVES: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease which is most often caused by a deficiency in steroid 21-hydroxylase (21-OH), a microsomal enzyme encoded by the CYP21 gene. Although several CAH causing mutations have been identified in the CYP21 gene of patients with 21-OH deficiency, genotyping of the 21-OH locus is quite complex because of the high frequency of gene conversion and the presence of multiple mutations on single CAH alleles. This study was aimed to analyze the complete characterization of the CYP21 gene coding region in a Korean CAH patient and to conform the PCR-based single strand conformation polymorphism (SSCP) and heteroduplex analysis as a diagnostic tool. METHODS: We used a highly sensitive, non-radioactive method allowing PCR-based single strand conformation polymorphism (SSCP) analysis. This method was applied to the characterization of all the exons and intron-exon junctions of the CYP21 gene in one patients affected by the salt wasting form and 4 normal controls. RESULTS: In all samples showing SSCP abnormal band patterns, sequence analysis showed the presence of sequence variants. In particular, one mutation (I172N) which is already known to cause the disease and 3 silent mutations were detected. CONCLUSION: PCR-based single strand conformation polymorphism (SSCP) and heteroduplex analysis should be useful for the clinical application as a diagnostic tool for the detection of 21-hydroxylase gene mutations.

Correlation between Genotype and Phenotype in Korean patients with Spinal Muscular Atrophy

BACKGROUND: Spinal muscular atrophy (SMA) is the second most common disease with autosomal recessive mode of inheritance in children and characterized by degeneration of anterior horn cells of the spinal cord resulting in weakness and wasting of voluntary muscles. This disease is caused by deletion of many candidate genes including SMN, p44, NAIP on chromosome 5q11.2-13.3. Although molecular characteristics of candidate genes were identified, genotype-phenotype correlation has not been clearly elucidated yet. Nevertheless, gene conversion, previously described as simply as gene deletion, appears to be very important mechanism as a molecular pathogenesis, and even makes more difficult to pursue the correlation. PURPOSE: This study was aimed to define the correlation between genotype and phenotype of SMA in Korean patients. The significance of SMN gene as well as NAIP gene, p44 gene in the progress of disease process and phenotypic correlation with gene conversion was evaluated. This study was also undertaken to determine the frequency of gene rearrangements in normal population. METHOD: Eight type I SMA patients and two type II SMA patients were studied. SMN, NAIP, and p44 gene deletion were analyzed by PCR amplification and restriction enzyme digestion with DraI, DdeI and AluI, respectively. p44 gene was also analyzed by SSCP. Gene conversion was defined by centromeric and telomeric SMN gene exon 7 to exon 8 PCR amplification followed by DdeI restiction enzyme digestion. RESULT: 1) Five of eight type I patients showed deletion of SMN, NAIP and p44 gene, while the rest of type 1 and all type II patients showed deletion of SMN gene only. 2) We examined SMN and NAIP gene deletion on 100 normal newborns, which showed the deletion of centromeric SMN gene in two newborns, the relative frequency of 2% in gene rearrangement. 3) There was one case of type I SMA showing deletion of telomeric SMN exon 7 but not SMN exon 8 suggestive of gene conversion occurred during the recombination as a molecular pathogenesis. CONCLUSION: The major deletion of SMA candidate genes, SMN, NAIP, and p44 gene appear to be involved in severe phenotype since these three candidate genes deletion were noted only in type 1 cases. However, SMN gene deletion only identified both in type 1 and type 2 explains that SMN gene may plan an major role in the pathogenesis of SMA and also suggests that other factors may be affecting the severity in spinal muscular atrophy. One patient with type I which showed the conversion of the centromeric SMN gene to the teleomeric gene strongly supports that SMN gene copy number may not be correlated with the severity in SMA. Our molecular findings suggest that phenotype is not clearly correlated with genotype. Prenatal screening should be carefully undertaken to interpretate because of high frequency of gene rearrangements in normal populations.

2-Deoxy-D-glucose induced modulation of DNA damage repair, survival, mutagenesis and recombinogenesis in 8-MOP+UVA treated yeast.

Cellular and genomic effects of post-treatment repair modulation by 2-deoxy-D-glucose (2-DG) and yeast extract were studied in 8-MOP + UVA treated cells of Saccharomyces cerevisiae. The type of lesions and their repair in phosphate buffer glucose (PBG) differed with UVA dose. At low UVA dose (1.4 kJ/m2), lesions were sublethal and mutagenic and did not repair by recombinogensis. The fraction of potentially lethal lesions and lesions repaired by recombinogenesis increased with UVA dose. Cellular repair in PBG was largely error-free and was inhibited by 2-DG. Yeast extract enhanced cellular repair and also recombinogensis; 2-DG in presence of yeast extract promoted error-prone repair. Pulsed-field gel electrophoresed chromosomal DNA bands did not show observable alterations immediately after 8-MOP + UVA treatment. On post-treatment incubation in PBG, the intensity ratio (rho n), of each band altered in a biphasic manner showing decrease first, followed by either increase or no change upto 24 hr depending upon UVA exposure dose. Presence of 2-DG in PBG inhibited decrease in rho n in a concentration dependent manner. Yeast extract reduced the time of first phase of DNA repair. 2-DG and yeast extract together reduced the time of first phase of repair and also inhibited the subsequent increase in rho n, which was observed in the case of yeast extract in PBG. It is proposed that (i) 2-DG in PBG inhibits excision of DNA damage and error-free repair; (ii) yeast extract stimulates the error-prone repair associated with cell cycle and recombinogenesis; (iii) 2-DG in presence of yeast extract allows excision of damage but inhibits build up through recombinogenesis inducing instead, cell cycle associated error-prone repair. A simple schematic model has been proposed to explain these events.

Duplication and deletion of 21 hydroxylase gene among the normal Korean subjects and in adrenogenital syndrome patients

Steroid 21 hydroxylase deficiency is a major cause of congenital adrenal hyperplasia(CAH) and is caused by genetic impairment (CYP21B) of this enzyme. In the human genome, CYP21B is located within MHC class III region on the short arm of chromosome 6. Most of the genes in this region are highly polymorphic and crowded. Also the CYP21B gene is accompanied by its pseudogene (CYP21A) and tandemly arranged with two genes of fourth component of complement. This highly complex gene arrangement in this area may predispose genetic unstability of CYP21 genes,i.e. mutations. In the current study, we tried to investigate the frequency of duplication and deletion of CYP21 genes and pattern of the genetic alteration of these genes by RFLPs. We also compared the genetic alteration of CYP21 in normal subjects with those of the CAH patients. According to our study, 15% of the normal Korean population have duplication or deletion of CYP21. There was one normal subject with heterozygous deletion of CYP21B gene. Of the 5 CAH patients examined, we found abnormal patterns in 2 patients. One was a large scale gene conversion and the other was a deletion of CYP21B and C4 locus II genes with gene conversion. These results suggest that high frequency of duplication and deletion of CYP21 and C4 in the general population may provide the genetic pool of instable CYP21 genes and these duplicated or deleted genes may result in gene conversions between CYP21A(pseudogene) and CYP21B(true gene) by preventing the normal recombination event.

De cómo la conciencia es un proceso que comienza con una conversión analógica-digital y termina, probablemente, con una digital-analógica, integrada por los genes, la percepción y la memoria / Conscience as a process starting with an analogic-digital conversion and probably ending with a digital-analogic one integrated by gens, perception and memory

Se revisan los datos empíricos de las neurociencias que puedan ser útiles para una caracterización biológica de la conciencia. Se propone que la conversión analógica-digital que tiene lugar en los receptores sensoriales es seguida de una conversión digital-analógica que sería el contenido de la conciencia. Existen pruebas a nivel celular de que la información se transmite de este modo (potencial del receptor, analógico-impulso nervioso, digital-potencial postsináptico local, analógico). Se revisan datos de la neurología y de la psicología (trasplantes de córnea, miembro fantasma y Síndrome de Antón-Babinski), que apoyan la idea de una localización cerebral de la conciencia, junto con datos de las técnicas no invasivas

2-DG induced modulation of chromosomal DNA profile, cell survival, mutagenesis and gene conversion in X-irradiated yeast.

Effects of post-irradiation modulation in presence of 2-deoxy-D-glucose and yeast extract, on chromosomal DNA profile, cell survival, reverse mutation (ILV+) and gene conversion (TRP+), were studied in X-irradiated stationary phase yeast cells (diploid strain D7 of Saccharomyces cerevisiae). The damage and repair in chromosomal DNA bands, resolved by using contour clamped homogeneous electric pulsed-field gel electrophoresis (PFGE) technique, was estimated by calculating intensity ratio, rho n (rho n I(n)/I(t); where I(n) is the intensity of nth band in a lane and I(t) is the sum of intensities of all bands and the well in the lane). The data indicate linear correlation between relative compactness (tau) of a chromosome [chromosome size (Kb)/length of synaptonemal complex (microns)] and DNA damage and repair. The chromosome repair kinetics were biphasic, showing initial decrease followed by an increase in rho n. Variations were observed among different chromosomes with respect to DNA damage, repair and post-irradiation repair modulation. 2-DG inhibited both components of chromosomal DNA repair and also repair of potentially lethal damage but enhanced frequencies of mutants. Relatively the effects on revertants were greater in cells irradiated with lower doses (50 Gy) of X-rays and post-irradiation incubation in presence of phosphate buffer having 2-DG (50 mM) and glucose (10 mM). Yeast extract increased frequencies of revertants and convertants thus promoting error-prone DNA repair. Yeast extract in combination with 2-DG showed complex effects on chromosomal DNA repair and enhanced mutagenesis further.