Genetic Imbalances in Argentinean Patients with Congenital Conotruncal Heart Defects.

Congenital conotruncal heart defects (CCHD) are a subset of serious congenital heart defects (CHD) of the cardiac outflow tracts or great arteries. Its frequency is estimated in 1/1000 live births, accounting for approximately 10⁻30% of all CHD cases. Chromosomal abnormalities and copy number variants (CNVs) contribute to the disease risk in patients with syndromic and/or non-syndromic forms. Although largely studied in several populations, their frequencies are barely reported for Latin American countries. The aim of this study was to analyze chromosomal abnormalities, 22q11 deletions, and other genomic imbalances in a group of Argentinean patients with CCHD of unknown etiology. A cohort of 219 patients with isolated CCHD or associated with other major anomalies were referred from different provinces of Argentina. Cytogenetic studies, Multiplex-Ligation-Probe-Amplification (MLPA) and fluorescent in situ hybridization (FISH) analysis were performed. No cytogenetic abnormalities were found. 22q11 deletion was found in 23.5% of the patients from our cohort, 66% only had CHD with no other major anomalies. None of the patients with transposition of the great vessels (TGV) carried the 22q11 deletion. Other 4 clinically relevant CNVs were also observed: a distal low copy repeat (LCR)D-E 22q11 duplication, and 17p13.3, 4q35 and TBX1 deletions. In summary, 25.8% of CCHD patients presented imbalances associated with the disease.

CYP21A2 mutation update: Comprehensive analysis of databases and published genetic variants.

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders of adrenal steroidogenesis. Disorders in steroid 21-hydroxylation account for over 95% of patients with CAH. Clinically, the 21-hydroxylase deficiency has been classified in a broad spectrum of clinical forms, ranging from severe or classical, to mild late onset or non-classical. Known allelic variants in the disease causing CYP21A2 gene are spread among different sources. Until recently, most variants reported have been identified in the clinical setting, which presumably bias described variants to pathogenic ones, as those found in the CYPAlleles database. Nevertheless, a large number of variants are being described in massive genome projects, many of which are found in dbSNP, but lack functional implications and/or their phenotypic effect. In this work, we gathered a total of 1,340 GVs in the CYP21A2 gene, from which 899 variants were unique and 230 have an effect on human health, and compiled all this information in an integrated database. We also connected CYP21A2 sequence information to phenotypic effects for all available mutations, including double mutants in cis. Data compiled in the present work could help physicians in the genetic counseling of families affected with 21-hydroxylase deficiency.

Misregulation effect of a novel allelic variant in the Z promoter region found in cis with the CYP21A2 p.P482S mutation: implications for 21-hydroxylase deficiency.

The aim of the current study was to search for the presence of genetic variants in the CYP21A2 Z promoter regulatory region in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Screening of the 10 most frequent pseudogene-derived mutations was followed by direct sequencing of the entire coding sequence, the proximal promoter, and a distal regulatory region in DNA samples from patients with at least one non-determined allele. We report three non-classical patients that presented a novel genetic variant-g.15626A>G-within the Z promoter regulatory region. In all the patients, the novel variant was found in cis with the mild, less frequent, p.P482S mutation located in the exon 10 of the CYP21A2 gene. The putative pathogenic implication of the novel variant was assessed by in silico analyses and in vitro assays. Topological analyses showed differences in the curvature and bendability of the DNA region bearing the novel variant. By performing functional studies, a significantly decreased activity of a reporter gene placed downstream from the regulatory region was found by the G transition. Our results may suggest that the activity of an allele bearing the p.P482S mutation may be influenced by the misregulated CYP21A2 transcriptional activity exerted by the Z promoter A>G variation.

Classical and nonclassical 21-hydroxylase deficiency: a molecular study of Argentine patients.

OBJECTIVE: To characterize the molecular basis of the 21-hydroxylase deficiency in a group of Argentine patients presenting the classical and nonclassical forms of the disease. DESIGN: To analyse the frequency of point mutations in the CYP21 gene by DNA amplification and mutation detection. PATIENTS: Forty-one patients from 36 nonrelated families: 25 nonclassical (NC), 11 salt-wasting (SW) and five simple virilizing (SV). A total of 27 parents and 13 nonaffected siblings were also analysed. MEASUREMENTS: Basal steroid hormones and 17-hydroxyprogesterone levels following adrenal stimulation with adrenocorticotrophic hormone were measured, together with an analysis of 10 point mutations in the CYP21 gene. RESULTS: A total of 83% and 74.4% classical and nonclassical chromosomes, respectively, were characterized. The intron 2 mutation was the most prevalent among classical alleles. In addition, a high frequency for R356W was observed in both groups (13.3 and 6.9%, respectively), while V281L was the most frequent mutation among the nonclassical patients with a frequency of 39.5%. No alleles containing P30L were observed, and one de novo mutation (R356W) was found. A total of 68.3% patients were fully genotyped, and all but one showed no genotype/phenotype discrepancy. Though the cut-off value for post-ACTH 17-hydroxyprogesterone stimulation was 30.25 nmol/l (10.00 microg/l), the lowest value observed in the fully genotyped nonclassical group was 42.35 nmol/l (14.00 microg/l). CONCLUSIONS: The high number of unidentified alleles in the nonclassical group suggests that less frequent mutations, or the presence of new ones, might be the cause of the disease in the Argentine population. Alternatively, the cut-off value in the ACTH-stimulated 17-hydroxyprogesterone test might overestimate the diagnosis of the nonclassical form by including some patients with heterozygous status.

In vitro capacitation of spermatozoa in different strains of mice: the acrosome reaction

La reacción acrosómica normal se midió como parámetro indirecto para determinar la capacitación in vitro de espermatozoides en diferentes cepas de ratones. Se utilizaron tres cepas: FI (BALB/c x C57BL/6J-bg). C57BL/6J-bg y C57BL/6J-small. Se hallaron diferencias significativas entre las tres cepas (p<0,05). Se realizó la fertilización in vitro utilizando una concentración de espermatozoides relativamente baja (10 espermatozoide/ml), a fin de averiguar si las diferencias en el porcentaje de espermatozoides que han sufrido reacción acrosómica a los 120min de incubación se correlaciona con las diferencias en las tasas de fertilidad. Se encontraron diferencias en la fertilización (p<0,05), pero sólo en algunas cepas ésto podría ser debido a diferencias en el porcentaje de espermatozoides sin acrosomas (capacitación). Cuando la concentración de espermatozoides fue aumentada a 10 espermatozoide/ml, las diferencias desaparecieron (p>0,05). No se detectaron diferencias entre las cepas utilizadas con respecto a la motilidad o hiperactividad después de la capacitación. La misma cinética de reacción acrosómica se halló en las cepas C57BL/6J-bg y C57BL/6J-small, pero fue diferente en el híbrido. También se observó un alto número (4%) (p<0,05) de espermatozoides morfológicamente anormales en las cepas endocriadas, con respecto a la híbrida (1%)

In vitro capacitation of spermatozoa in different strains of mice: the acrosome reaction

La reacción acrosómica normal se midió como parámetro indirecto para determinar la capacitación in vitro de espermatozoides en diferentes cepas de ratones. Se utilizaron tres cepas: FI (BALB/c x C57BL/6J-bg). C57BL/6J-bg y C57BL/6J-small. Se hallaron diferencias significativas entre las tres cepas (p<0,05). Se realizó la fertilización in vitro utilizando una concentración de espermatozoides relativamente baja (10 espermatozoide/ml), a fin de averiguar si las diferencias en el porcentaje de espermatozoides que han sufrido reacción acrosómica a los 120min de incubación se correlaciona con las diferencias en las tasas de fertilidad. Se encontraron diferencias en la fertilización (p<0,05), pero sólo en algunas cepas ésto podría ser debido a diferencias en el porcentaje de espermatozoides sin acrosomas (capacitación). Cuando la concentración de espermatozoides fue aumentada a 10 espermatozoide/ml, las diferencias desaparecieron (p>0,05). No se detectaron diferencias entre las cepas utilizadas con respecto a la motilidad o hiperactividad después de la capacitación. La misma cinética de reacción acrosómica se halló en las cepas C57BL/6J-bg y C57BL/6J-small, pero fue diferente en el híbrido. También se observó un alto número (4%) (p<0,05) de espermatozoides morfológicamente anormales en las cepas endocriadas, con respecto a la híbrida (1%) (AU)