P.01 A KLHL40 3′UTR splice-altering variant causes milder NEM8

Abstract

Nemaline myopathy 8 (NEM8) is typically a severe autosomal recessive disorder associated with variants in the kelch-like family member 40 gene (KLHL40). To date, only protein-altering pathogenic variants in <i>KLHL40</i> have been implicated in NEM8. Common features include fetal akinesia/hypokinesia, fractures, contractures, dysphagia, respiratory failure, and neonatal death. Here, we describe a 26-year-old man with relatively mild NEM8. He presented with hypotonia and bilateral femur fractures at birth, later developing bilateral Achilles' contractures, scoliosis, and elbow and knee contractures. He had walking difficulties throughout his childhood and became wheelchair bound from age 13 after prolonged immobilization. Muscle MRI at age 13 indicated prominent fat replacement in his pelvic girdle, posterior compartments of thighs, vastus intermedius and legs. Muscle biopsy revealed nemaline bodies and intranuclear rods. RNA sequencing and western blotting of patient skeletal muscle indicated significant reduction in <i>KLHL40</i> mRNA and protein respectively. Using gene panel screening, exome sequencing and RNA sequencing, we identified compound heterozygous variants in <i>KLHL40</i>; a truncating 10.9 kb deletion in trans with a likely pathogenic variant (c.*152G>T) in the 3' untranslated region (UTR). Computational tools SpliceAI and Introme predicted the c.*152G>T variant created a cryptic donor splice site. RNA sequencing and in vitro analyses indicated that the c.*152G>T variant induces multiple de novo splicing events. Nonsense mediated decay of <i>KLHL40</i> mRNA, provoked by introduction of 3' UTR introns, likely explains the loss of both mRNA and protein expression in the patient. Our analysis of 3' UTR variants in ClinVar suggests that SNPs that introduce aberrant 3'UTR splicing may be under recognised in Mendelian disease. We encourage consideration of this mechanism during disease gene screening.