Deep Intronic ETFDH Variants Represent a Recurrent Pathogenic Event in Multiple Acyl-CoA Dehydrogenase Deficiency.

Multiple acyl-CoA dehydrogenase deficiency (MADD) is a rare inborn error of metabolism affecting fatty acid and amino acid oxidation with an incidence of 1 in 200,000 live births. MADD has three clinical phenotypes: severe neonatal-onset with or without congenital anomalies, and a milder late-onset form. Clinical diagnosis is supported by urinary organic acid and blood acylcarnitine analysis using tandem mass spectrometry in newborn screening programs. MADD is an autosomal recessive trait caused by biallelic mutations in the ETFA, ETFB, and ETFDH genes encoding the alpha and beta subunits of the electron transfer flavoprotein (ETF) and ETF-coenzyme Q oxidoreductase enzymes. Despite significant advancements in sequencing techniques, many patients remain undiagnosed, impacting their access to clinical care and genetic counseling. In this report, we achieved a definitive molecular diagnosis in a newborn by combining whole-genome sequencing (WGS) with RNA sequencing (RNA-seq). Whole-exome sequencing and next-generation gene panels fail to detect variants, possibly affecting splicing, in deep intronic regions. Here, we report a unique deep intronic mutation in intron 1 of the ETFDH gene, c.35-959A>G, in a patient with early-onset lethal MADD, resulting in pseudo-exon inclusion. The identified variant is the third mutation reported in this region, highlighting ETFDH intron 1 vulnerability. It cannot be excluded that these intronic sequence features may be more common in other genes than is currently believed. This study highlights the importance of incorporating RNA analysis into genome-wide testing to reveal the functional consequences of intronic mutations.

Impact of High-to-Moderate Penetrance Genes on Genetic Testing: Looking over Breast Cancer.

Breast cancer (BC) is the most common cancer and the leading cause of cancer death in women worldwide. Since the discovery of the highly penetrant susceptibility genes BRCA1 and BRCA2, many other predisposition genes that confer a moderate risk of BC have been identified. Advances in multigene panel testing have allowed the simultaneous sequencing of BRCA1/2 with these genes in a cost-effective way. Germline DNA from 521 cases with BC fulfilling diagnostic criteria for hereditary BC were screened with multigene NGS testing. Pathogenic (PVs) and likely pathogenic (LPVs) variants in moderate penetrance genes were identified in 15 out of 521 patients (2.9%), including 2 missense, 7 non-sense, 1 indel, and 3 splice variants, as well as two different exon deletions, as follows: ATM (n = 4), CHEK2 (n = 5), PALB2 (n = 2), RAD51C (n = 1), and RAD51D (n = 3). Moreover, the segregation analysis of PVs and LPVs into first-degree relatives allowed the detection of CHEK2 variant carriers diagnosed with in situ melanoma and clear cell renal cell carcinoma (ccRCC), respectively. Extended testing beyond BRCA1/2 identified PVs and LPVs in a further 2.9% of BC patients. In conclusion, panel testing yields more accurate genetic information for appropriate counselling, risk management, and preventive options than assessing BRCA1/2 alone.