Distinct diagnostic trajectories in NBAS-associated acute liver failure highlights the need for timely functional studies

Abstract

Variants of uncertain significance (VUS) are commonly found following genomic sequencing, particularly in ethnically diverse populations that are underrepresented in large population databases. Functional characterization of VUS may assist in variant reclassification, however these studies are not readily available and often rely on research funding and good will. We present four individuals from three families at different stages of their diagnostic trajectory with recurrent acute liver failure (RALF) and biallelic NBAS variants, confirmed by either trio analysis or cDNA studies. Functional characterization was undertaken, measuring NBAS and p31 levels by Western blotting, demonstrating reduced NBAS levels in two of three families, and reduced p31 levels in all three families. These results provided functional characterization of the molecular impact of a missense VUS, allowing reclassification of the variant and molecular confirmation of NBAS-associated RALF. Importantly, p31 was decreased in all individuals, including an individual with two missense variants where NBAS protein levels were preserved. These results highlight the importance of access to timely functional studies after identification of putative variants, and the importance of considering a range of assays to validate variants whose pathogenicity is uncertain. We suggest that funding models for genomic sequencing should consider incorporating capabilities for adjunct RNA, protein, biochemical, and other specialized tests to increase the diagnostic yield which will lead to improved medical care, increased equity, and access to molecular diagnoses for all patients.

Keywords: functional genomics; genome sequencing; pediatrics; rapid genomic sequencing; recurrent acute liver failure; variant classification.

FIGURE 1

cDNA studies in Individual B1. The NM_015909.3(NBAS):c.2951T>G, p.(Ile984Ser) variant appeared heterozygous when amplifying the cDNA generated from cells with cycloheximide treatment and as homozygous when amplifying the cDNA generated from cells grown without cycloheximide, suggesting that the allele with the p.(Arg405*) is in trans and largely degraded by nonsense‐mediated decay (NMD)

FIGURE 2

NBAS and p31 protein expression in fibroblasts. Representative Western blot and densitometry analysis suggest NBAS protein levels of 40%–50% of control mean in individual C and B1, and p31 levels of 5% in individual A, 35% in individual B1 and 10% in individual C relative to control mean, GAPDH was used as a protein loading control