Role of RNA in Molecular Diagnosis of MADD Patients

Abstract

The electron-transfer flavoprotein dehydrogenase gene (ETFDH) encodes the ETF-ubiquinone oxidoreductase (ETF-QO) and has been reported to be the major cause of multiple acyl-CoA dehydrogenase deficiency (MADD). In this study, we present the clinical and molecular diagnostic challenges, at the DNA and RNA levels, involved in establishing the genotype of four MADD patients with novel ETFDH variants: a missense variant, two deep intronic variants and a gross deletion. RNA sequencing allowed the identification of the second causative allele in all studied patients. Simultaneous DNA and RNA investigation can increase the number of MADD patients that can be confirmed following the suggestive data results of an expanded newborn screening program. In clinical practice, accurate identification of pathogenic mutations is fundamental, particularly with regard to diagnostic, prognostic, therapeutic and ethical issues. Our study highlights the importance of RNA studies for a definitive molecular diagnosis of MADD patients, expands the background of ETFDH mutations and will be important in providing an accurate genetic counseling and a prenatal diagnosis for the affected families.

Keywords: ETFDH; MADD; NBS; RNA; glutaric aciduria type II; β-oxidation.

Figure 1

Families’ pedigrees and genotype data from four MADD families. Symbols: square, male; circle, female; filled, affected individual; diagonal line, deceased; NA, not available. Mutations found in ETFDH gene are shown below each symbol: -, wild-type allele; M1, p.Val324Met (c.970A>G); M2, c.35-768A>G; M3, p.Arg41* (c.121C>T); M4, c.35-1008 T>G; M5, p.Leu550Valfs*4 (c.1648_1649delCT); M6, c.34_607del; M7, p.Arg155Gly (c.463A>G).

Figure 2

Electropherograms of ETFDH mutations from (a) family 1, (b) family 2, (c) family 3 and (d) family 4, showing the deletion detected by Copy Number Variation analysis. (e) Sequence alignment among 8 vertebrates around the sites of the four exonic mutations. The four identified mutations are conserved across species.

Figure 3

Schematic representation of ETFDH region including exon 1, intron 1 and exon 2. WT: structure of wild-type ETFDH transcript (exons 1–2). MUT: structure of ETFDH transcript generated due to (a) c.35-768A>G mutation (red, arrow) and (b) c.35-1008T>G mutation (red, arrow). Intron splice site acceptor and donor sequences are shown. In the two cases (a,b) the pseudoexons (blue) result from activation of the 5′ donor site that also results in preferential use of the existing 3′ acceptor site (GATAG) at the 5′ end of the pseudoexon to effect splicing. (c,d) Electropherograms showing the cDNA sequences of patients with the heterozygous mutations c.35-768A>G and c.35-1008T>G, respectively.