Homozygous frame shift variant in ATP7B exon 1 leads to bypass of nonsense-mediated mRNA decay and to a protein capable of copper export

Abstract

Wilson disease (WD) is an autosomal recessive disease of copper excess due to pathogenic variants in the ATP7B gene coding for a copper-transporting ATPase. We present a 5-year-old girl with the homozygous frame shift variant NM_000053.3: c.19_20del in exon 1 of ATP7B (consecutive exon numbering with c.1 as first nucleotide of exon 1), detected by whole-exome sequencing as a secondary finding. The variant leads to a premature termination codon in exon 2. The girl exhibited no WD symptoms and no abnormalities in liver biopsy. ATP7B liver mRNA expression was comparable to healthy controls suggesting that nonsense-mediated mRNA decay (NMD) could be bypassed by the mechanism of translation reinitiation. To verify this hypothesis, a CMV-driven ATP7B minigene (pcDNA3) was equipped with the authentic ATP7B 5′ untranslated region and a truncated intron 2. We introduced c.19_20del by site-directed mutagenesis and overexpressed the constructs in HEK293T cells. We analyzed ATP7B expression by qRT-PCR, northern and western blot, and examined protein function by copper export capacity assays. Northern blot, qRT-PCR, and western blot revealed that c.19_20del ATP7B mRNA and protein is expressed in size and amount comparable to wild-type. Copper export capacity was also comparable to wild-type. Our results indicate that c.19_20del in ATP7B is able to bypass NMD by translation reinitiation, demonstrating that the classification of truncating variants as pathogenic without additional investigations should be done carefully.

Molecular diagnostics of Mendelian disorders via combined DNA and RNA sequencing

“…In cases of a homozygous frameshift mutation in exon 2 of ATP7B, we detected mRNA expression comparable with healthy controls, suggesting that NMD could be bypassed by the mechanism of translation re-initiation. This was confirmed by Western blot and functional tests of copper export capacity [15]. In another case, we identified bi-allelic frameshift variants in FLAD1, which encodes FAD synthase.…”

“…Identification of possible protein-truncating variants in genes for which nontruncating/in-frame pathogenic loss-offunction variants are known can already be challenging. If transcripts affected by such variants escape nonsense-mediated mRNA decay (NMD), they may still produce a functional protein by the mechanism of translation re-initiation [15], by functional alternative transcript isoforms [13], or by preserved/residual function of the truncated protein [7]. A recent systematic study shows that exons present only in tissue specific isoforms may not be essential for protein function [5].…”

SRSF7 maintains its homeostasis through the expression of Split-ORFs and nuclear body assembly

“…Patients with nonsense mutations in genes causing several diseases have been shown to express NMD-resistant PTC-containing transcripts. In all cases, reinitiation at an AUG downstream of the PTC caused the expression of an N-terminally truncated protein, which delayed the symptoms or acted as a dominant-negative variant [55][56][57][58] . Ribosome profiling in human hearts further revealed that translation frequently occurs downstream of PTCs, which might prevent the degradation of transcripts with nonsense mutations 42 .…”

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“…There is a preconceived idea that while synonymous variants are rather innocuous, frameshift mutations create a premature stop codon generating truncated non-functional product. However, at least for a few such (Zhou et al 2021) and exon skipping (Wang et al 2018), effect of the premature stop codon due to a frame shift mutation may be overcome by bypassing nonsense mediated mRNA decay through translation re-initiation (Stalke et al 2019). Since few observations contrary to the expected outcome have been reported, this should act as a caveat while applying a generalized interpretation regarding functional consequence of such variants without appropriate investigation.…”