Novel frameshift variant of the CFTR gene: S511Lfs*2 from phenotype to molecular predictions.

Cystic fibrosis (CF) is a genetic disease caused by variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. There are over 2,000 different pathogenic and non-pathogenic variants described in association with a broad clinical heterogeneity. In this work, we identified a novel variant S511Lfs*2 in CFTR gene that has not been reported in patients with CF. The patient was a female genotyped with c.1000C>T (legacy name: R334W) variant (pathogenic, CF-causing) and the novel variant (S511Lfs*2). We verified the amino acid sequence, the protein structure, and predicted the pathogenicity employing computational analysis. Our findings showed that S511Lfs*2 is a frameshift variant and suggest that it is associated with severe CF phenotype, as it leads to a lack of CFTR protein synthesis, and consequently the loss of its functional activity.

Development of CYP21A2 Genotyping Assay for the Diagnosis of Congenital Adrenal Hyperplasia.

BACKGROUND: Steroid 21-hydroxylase deficiency due to CYP21A2 gene mutations represents more than 90% of all congenital adrenal hyperplasia cases. This deficiency is screened by measuring levels of 17-hydroxyprogesterone, which may vary, causing false positive or false negative results. In order to assist the diagnosis, molecular methodologies have been employed. This work aimed to perform genotyping assays to detect mutations in the CYP21A2 gene and compare the findings with other population studies. METHODS: The SNaPshot assay was developed to simultaneously detect 12 frequent point mutations in the CYP21A2 gene (p.Arg409Cys, p.Gln319Ter, p.Arg357Trp, p.Leu308PhefsTer6, p.Val237Glu, IVS2-13A/C > G, p.Ile173Asn, p.Pro31Leu, p.Pro454Ser, p.Val282Leu, p.Gly111ValfsTer21 and p.His63Leu). The direct sequencing and multiplex ligation-dependent probe amplification assays were used to confirm point mutations present in the developed method. The latter was also used to search large deletions and gene conversion, complementing the investigation. A total of 166 cases were studied. RESULTS: The SNaPshot assay was successfully developed to detect the 12 mutations. The results of mutation analysis indicated 84 pathogenic alleles in 48 cases, with p.Val282Leu (27.1%) and IVS2-13A/C > G (20.8%) being the most frequently found mutations. Between the findings of this study and those of other South American studies, there were significant differences in frequency for p.Pro31Leu and p.Val282Leu (p < 0.001). A new variant T in IVS2-13A/C > G was identified in two patients via the SNaPshot assay. CONCLUSION: The molecular strategy developed for CYP21A2 gene mutation screening allowed us to detect the principle mutations described around the world. Furthermore, the first Southern Brazilian mutation frequencies concerning the CYP21A2 gene were obtained.

Neonatal screening for congenital adrenal hyperplasia in Southern Brazil: a population based study with 108,409 infants.

BACKGROUND: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder associated with inborn errors of steroid metabolism. 21-hydroxylase enzyme deficiency occurs in 90 to 95% of all cases of CAH, with accumulation of 17 hydroxyprogesterone (17-OHP). Early diagnosis of CAH based on newborn screening is possible before the development of symptoms and allows proper treatment, correct sex assignment, and reduced mortality rates. This study describes the results obtained in the first year of a public CAH screening program in the state of Rio Grande do Sul, Brazil. METHODS: We reviewed the screening database in search of babies with suspected CAH, that is, altered birth-weight adjusted 17-OHP values at screening. The following data were analyzed for this population: screening 17-OHP values, retest 17-OHP values, serum 17-OHP values for those with confirmed CAH on retest, maternal and newborn data, and family history of CAH. For the screening program, 17-OHP levels are determined on dried blood spots obtained in filter paper with GSP solid phase time-resolved immunofluorescence. RESULTS: Of 108,409 newborns screened, eight were diagnosed with CAH (four males, four females). The incidence of CAH in the state was 1:13,551. Six cases were identified as classic salt-wasting CAH and two were cases of virilizing CAH. The positive predictive value (PPV) of the initial screening (before diagnostic confirmation) was 1.6%. The overall rate of false positive results was 0.47%. The number of false positive results was higher among newborns with birth weight < 2000 g. CONCLUSION: The present results support the need for CAH screening by the public health care system in the state, and show that the strategy adopted is adequate. PPV and false positive results were similar to those reported for other states of Brazil with similar ethnic backgrounds.