LAUSR

We read the paper “Multiple acyl‐CoA dehydrogenase defi‐ ciency [MADD] in elderly carriers” by Macchione et al. with great interest, where they describe two elderly individuals, each with only one heterozygous variant in ETFDH gene [1]. After sequencing the exons and exon–intron bounda‐ ries of genes known to cause MADD and performing copy number analyses, they justifiably discuss that they “cannot formally exclude the presence of deep intronic mutations,” but suggest that these patients are symptomatic carriers [1]. However, the authors’ claim may be unsubstantiated, for lack of adequate functional or genetic evidence. Mutations in deep intronic, promoter or other regula‐ tory regions are implicated in several monogenic disorders and deserve a more thorough discussion [2]. For example, it has been estimated that 1–5% of cystic fibrosis alleles in CFTR gene are believed to be located in regulatory regions [3]. These variants are not rare enough to be ignored, espe‐ cially if one proposes an alternative inheritance pattern to a well‐characterized disease. In addition, numerous MADD patients with a single heterozygous variant in ETFDH have been published previously [4], simply assuming that the other variant could not be identified using the meth‐ ods at hand. MADD is such a heterogeneous disease that septuagenarians with biallelic (homozygous or compound heterozygous) mutations becoming symptomatic would not be that big a stretch, since an Italian patient starting to exhibit symptoms at the age of 68 has already been reported (although not confirmed by genetic analysis) [5]. To claim that the new patients are heterozygotes, and not merely cases where the second variant eludes detection, it is insufficient to exclude copy number variations; more evidence is required. One such evidence could come from segregation analysis. In their paper cited by Macchione et al., Fanin et al. describe eight individuals with autosomal recessive carnitine palmi‐ toyltransferase II deficiency, who seem to be symptomatic although they only harbor a single heterozygous variant in CPT2 gene. By analyzing the segregation of the alleles in the families, they show that only two are true heterozygotes, whereas the other six are probably compound heterozygotes whose second mutations remain undetectable [6]. Similar pitfalls might have been overlooked by Macchione et al., since no pedigree data are presented. Although DNA from the parents of the two new patients might not be available, analyzing the segregation of the variant and the phenotype among siblings or children of the index cases could still give an idea about the nature of the other allele. Protein and mRNA studies from skeletal muscle tissue could have provided another line of evidence for or against the authors’ claim. For example, their argument would be convincing if the amount of wild‐type ETFDH protein in skeletal muscle was similar to that in heterozygotes. In contrast, by analyzing the mRNA and protein products of ETFDH gene, multiple affected patients with only a single detectable heterozygous variant have been shown to com‐ pletely lack the ETFDH protein, which clearly indicates the presence of an undetectable, but severe (null) variant on the other allele [7]. In short, we are doubtful that the paper’s title is accurate, since the presented data focus on the inability to find the sec‐ ond mutation rather than establishing carrier status. None‐ theless, we are excited about the prospect of a new inherit‐ ance pattern for this already very interesting disease and we congratulate the authors for their observation. We will be on the lookout for confirmation of this novel observation, This comment refers to the article available online at https ://doi. org/10.1007/s0041 5‐020‐09729 ‐z.