RESUMO
The diagnosis of Mendelian disorders has notably advanced with integration of whole exome and genome sequencing (WES and WGS) in clinical practice. However, challenges in variant interpretation and uncovered variants by WES still leave a substantial percentage of patients undiagnosed. In this context, integrating RNA sequencing (RNA-seq) improves diagnostic workflows, particularly for WES inconclusive cases. Additionally, functional studies are often necessary to elucidate the impact of prioritized variants on gene expression and protein function. Our study focused on three unrelated male patients (P1-P3) with ATP6AP1-CDG (congenital disorder of glycosylation), presenting with intellectual disability and varying degrees of hepatopathy, glycosylation defects, and an initially inconclusive diagnosis through WES. Subsequent RNA-seq was pivotal in identifying the underlying genetic causes in P1 and P2, detecting ATP6AP1 underexpression and aberrant splicing. Molecular studies in fibroblasts confirmed these findings and identified the rare intronic variants c.289-233C > T and c.289-289G > A in P1 and P2, respectively. Trio-WGS also revealed the variant c.289-289G > A in P3, which was a de novo change in both patients. Functional assays expressing the mutant alleles in HAP1 cells demonstrated the pathogenic impact of these variants by reproducing the splicing alterations observed in patients. Our study underscores the role of RNA-seq and WGS in enhancing diagnostic rates for genetic diseases such as CDG, providing new insights into ATP6AP1-CDG molecular bases by identifying the first two deep intronic variants in this X-linked gene. Additionally, our study highlights the need to integrate RNA-seq and WGS, followed by functional validation, in routine diagnostics for a comprehensive evaluation of patients with an unidentified molecular etiology.
Assuntos
Íntrons , RNA Mensageiro , Humanos , Masculino , Íntrons/genética , RNA Mensageiro/genética , ATPases Vacuolares Próton-Translocadoras/genética , Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/diagnóstico , Defeitos Congênitos da Glicosilação/patologia , Mutação , Sequenciamento Completo do Genoma , Sequenciamento do Exoma , Análise de Sequência de RNA , Deficiência Intelectual/genética , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/patologia , Criança , Splicing de RNA/genética , Pré-EscolarRESUMO
Glutaric aciduria type 1 (GA1) is a rare inherited metabolic disorder caused by a deficiency of glutaryl-coenzyme A dehydrogenase (GCDH), with accumulation of neurotoxic metabolites, resulting in a complex movement disorder, irreversible brain damage, and premature death in untreated individuals. While early diagnosis and a lysine restricted diet can extend survival, they do not prevent neurological damage in approximately one-third of treated patients, and more effective therapies are required. Here we report the efficacy of adeno-associated virus 9 (AAV9)-mediated systemic delivery of human GCDH at preventing a high lysine diet (HLD)-induced phenotype in Gcdh -/- mice. Neonatal treatment with AAV-GCDH restores GCDH expression and enzyme activity in liver and striatum. This treatment protects the mice from HLD-aggressive phenotype with all mice surviving this exposure; in stark contrast, a lack of treatment on an HLD triggers very high accumulation of glutaric acid, 3-hydroxyglutaric acid, and glutarylcarnitine in tissues, with about 60% death due to brain accumulation of toxic lysine metabolites. AAV-GCDH significantly ameliorates the striatal neuropathology, minimizing neuronal dysfunction, gliosis, and alterations in myelination. Magnetic resonance imaging findings show protection against striatal injury. Altogether, these results provide preclinical evidence to support AAV-GCDH gene therapy for GA1.