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

Abstract

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.

Keywords: ETFDH; MADD; RNA sequencing; deep intronic variant; genome sequencing; pseudo-exon; splicing; transcript processing.

Figure 1

Sanger sequencing results of confirmatory and segregation analysis.

Figure 2

In silico prediction of the potential impact of the reported deep intronic variant on splicing. (a) Outline of ESE changes determined by the three deep ETFDH intron 1 mutations. Box height is proportional to ESE score as computed by the Alamut splicing module. (b) MaxEntScan analysis of 5′ and 3′ splicing site scores for both wild-type exon 1 and exon 2 junctions and the pseudo-exon. In green is the score of the normal sequence; in red, the score of the mutated sequence for the pseudo-exon. (c) SpliceAI analysis. The scores for the wild-type and mutated sequences are indicated for all three mutations in ETFDH intron 1. Boxed in red is the difference (i.e., ∆ score) between normal and mutated sequence scores for the donor site. The light red color indicates that the increase in SpliceAI scores for donor sites is highly significant (for all three mutations > 0.80) as reported by Sagakuchi et al. [24].

Figure 3

Confirmatory targeted RNA analysis on maternal PB-derived cDNA. (a) The blue rectangles represent the first three exons of the ETFDH gene present in both the normal and the aberrant transcript. In yellow, the pseudo-exon included in the abnormal mRNA. For each exon, and for the pseudo-exon, the sequence of first and last nucleotides is indicated. The splicing donor and acceptor sites are also depicted. The additional SAS and SDS created by the intronic mutation (in red) are indicated by the arrows. The aberrant isoform has a start codon within exon 3, with the novel coding sequence in orange, underlined. (b) RT-PCR experiment on ETFDH cDNA. On the left, the gel electrophoresis image is shown. In the first lane, the ladder (L) is present with 200, 300, 400, and 500 bp long DNA fragments. The second (F) and third (M) lane are the PCR products from the father’s and mother’s cDNA, respectively. The fourth and fifth lane (C1, C2) indicate the negative control PCR products. WT isoforms are boxed in light blue, while the novel aberrant transcript is present only in the rectangle. In the M lane, an additional isoform is disclosed (the M lane is boxed in yellow). On the right are the schematic representations of both the normal and aberrant isoforms, with exon 1 and 2 in blue and the pseudo-exon (Ψ) in orange. (c) Sanger sequencing electropherograms of reference (right panel) and abnormal PCR products (left panel) showing the splicing junction between exon 1 and exon 2, and between exon 1 and the pseudo-exon, respectively.