RESUMO
PURPOSE: Patients with Fanconi anaemia (FA), a rare DNA repair genetic disease, exhibit chromosome fragility, bone marrow failure, malformations and cancer susceptibility. FA molecular diagnosis is challenging since FA is caused by point mutations and large deletions in 22 genes following three heritability patterns. To optimise FA patients' characterisation, we developed a simplified but effective methodology based on whole exome sequencing (WES) and functional studies. METHODS: 68 patients with FA were analysed by commercial WES services. Copy number variations were evaluated by sequencing data analysis with RStudio. To test FANCA missense variants, wt FANCA cDNA was cloned and variants were introduced by site-directed mutagenesis. Vectors were then tested for their ability to complement DNA repair defects of a FANCA-KO human cell line generated by TALEN technologies. RESULTS: We identified 93.3% of mutated alleles including large deletions. We determined the pathogenicity of three FANCA missense variants and demonstrated that two FANCA variants reported in mutations databases as 'affecting functions' are SNPs. Deep analysis of sequencing data revealed patients' true mutations, highlighting the importance of functional analysis. In one patient, no pathogenic variant could be identified in any of the 22 known FA genes, and in seven patients, only one deleterious variant could be identified (three patients each with FANCA and FANCD2 and one patient with FANCE mutations) CONCLUSION: WES and proper bioinformatics analysis are sufficient to effectively characterise patients with FA regardless of the rarity of their complementation group, type of mutations, mosaic condition and DNA source.
Assuntos
Sequenciamento do Exoma , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Anemia de Fanconi/genética , Predisposição Genética para Doença , Linhagem Celular , Variações do Número de Cópias de DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Anemia de Fanconi/patologia , Feminino , Técnicas de Inativação de Genes , Humanos , Masculino , Mutação de Sentido Incorreto/genética , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
OBJECTIVES: (1) To evaluate the effectiveness of adenotonsillectomy for the treatment of Obstructive Sleep Apnea Hypopnea Syndrome (OSAHS) in children. (2) To evaluate the usefulness of respiratory polygraphy (RP) for controlling post-adenotonsillectomy effects. METHODS: The children studied were referred to the Burgos Sleep Unit (SU) with clinical suspicion of OSAHS before undergoing adenotonsillectomy. For all patients, a clinical history was taken and a general physical examination, as well as a specific ear, nose, and throat examination was done. RP before adenotonsillectomy, and seven months afterwards, was also done. OSAHS was diagnosed if the Apnea Hypopnea Index (AHI) was ≥ 4.6. RESULTS: Of the 100 children studied, 68 were male and 32 female, with an age of 4.17 ± 2.05 years. Using RP, 86 of them were diagnosed with OSAHS before undergoing adenotonsillectomy. There was a significant improvement in all clinical and polygraphic variables after adenotonsillectomy. The pre and post surgery AHI index was 11.9 ± 11.0 and 2.6 ± 1.5, respectively, with a significant mean difference (9.4 ± 10.9, p<0.01). The residual OSAHS was 11.6% (CI 95%: 4.3-19%). CONCLUSIONS: Respiratory polygraphy is a useful tool for monitoring the effectiveness of surgical treatment and the detection of residual OSAHS in children with adenotonsillar hypertrophy.
