ECHS1 disease in two unrelated families of Samoan descent: Common variant - rare disorder

Abstract

Mutations in the short-chain enoyl-CoA hydratase (SCEH) gene, ECHS1, cause a rare autosomal recessive disorder of valine catabolism. Patients usually present with developmental delay, regression, dystonia, feeding difficulties, and abnormal MRI with bilateral basal ganglia involvement. We present clinical, biochemical, molecular, and functional data for four affected patients from two unrelated families of Samoan descent with identical novel compound heterozygous mutations. Family 1 has three affected boys while Family 2 has an affected daughter, all with clinical and MRI findings of Leigh syndrome and intermittent episodes of acidosis and ketosis. WES identified a single heterozygous variant in ECHS1 at position c.832G > A (p.Ala278Thr). However, western blot revealed significantly reduced ECHS1 protein for all affected family members. Decreased SCEH activity in fibroblasts and a mild increase in marker metabolites in urine further supported ECHS1 as the underlying gene defect. Additional investigations at the DNA (aCGH, WGS) and RNA (qPCR, RT-PCR, RNA-Seq, RNA-Array) level identified a silent, common variant at position c.489G > A (p.Pro163=) as the second mutation. This substitution, present at high frequency in the Samoan population, is associated with decreased levels of normally spliced mRNA. To our understanding, this is the first report of a novel, hypomorphic allele c.489G > A (p.Pro163=), associated with SCEH deficiency.

Keywords: ECHS1; Leigh syndrome; Samoan population; mitochondrial disease; silent variant.

FIGURE 1

Pedigree of Family 1 (F1) and Family 2 (F2) with mutations in ECHS1. Haplotype is indicated below each tested individual. The silent exon 4 mutation is highlighted by red font. Black shading indicates affected status. P indicates patient, M indicates mother and F indicates father, U indicates unaffected, n.d. indicates studies were not done, (a) and (b) indicates phase of the maternal ECHS1 allele in Family 1

FIGURE 2

(a) SCEH enzyme studies on patient fibroblasts. SCEH activity is expressed as percentage of the mean activity in six control cell lines whereas results represent the mean of two independent experiments (b) SDS‐PAGE and ImageJ quantification (bottom) of ECHS1 steady state protein levels of patient and control fibroblast lysates. Values are displayed as percentage of age‐matched control and STD (n of Family 1 = 3 and n of Family 2 = 2). (c) Quantitative PCR of RNA extracted from patient and control fibroblast using TaqMan assay in triplicate. Values were calculated as ΔΔCt and SEM

FIGURE 3

(a) Schematic of proposed impact of c.489G > A (p.Pro163=) on splicing. (b) Cells from adult and child control as well as the mother and Patient 2 of Family 1 were cultured with (+) and without (−) Emetine to halt nonsense mediated decay at 0.1 mg/ml for 5 hr prior to RNA extraction. RT‐PCR was performed using a forward primer overlapping the exon 2/3 junction and a reverse primer in exon 6 (primers are denoted by arrows). PCR products were run on a 2% agarose gel. (c) RT‐PCR to study the impact of the intron 6 variant on splicing using a forward primer in exon 6 and a reverse primer overlapping the exon 7–8 junction, a hypothetical band of 1,094 bp, representing abnormal splicing, was not observed