Compound heterozygous splicing variants expand the genotypic spectrum of EMC1‐related disorders

EMC1 encodes subunit 1 of the endoplasmic reticulum (ER) membrane protein complex (EMC), a transmembrane domain insertase involved in membrane protein biosynthesis. Variants in EMC1 are described as a cause of global developmental delay, hypotonia, cortical visual impairment, and commonly, cerebral atrophy on MRI scan. We report an individual with severe global developmental delay and progressive cerebellar atrophy in whom exome sequencing identified a heterozygous essential splice‐site variant in intron‐3 of EMC1 (NM_015047.3:c.287‐1G>A). Whole genome sequencing (WGS) identified a deep intronic variant in intron‐20 of EMC1 (NM_015047.3:c.2588‐771C>G) that was poorly predicted by in silico programs to disrupt pre‐mRNA splicing. Reverse Transcription‐PCR (RT‐PCR) revealed stochastic activation of a pseudo‐exon associated with the c.2588‐771C>G variant and mis‐splicing arising from the c.287‐1G>A variant. This case highlights the utility of WGS and RNA studies to identify and assess likely pathogenicity of deep intronic variants and expands the genotypic and phenotypic spectrum of EMC1‐related disorders.


| INTRODUCTION
EMC1 encodes subunit 1 of the highly conserved, multi-functional endoplasmic reticulum (ER) membrane protein complex (EMC). 1 Comprised of 10 subunits, the EMC plays an important role in the biosynthesis of integral membrane proteins by facilitating insertion of transmembrane domains into the ER membrane. 2 The EMC is suggested to have chaperone activity acting as a quality control checkpoint for transmembrane proteins transiting the secretory pathway, with additional roles related to ER-associated degradation, sterol regulation and autophagy. 3 Variants in EMC1 4-7 are associated with syndromic neurodevelopmental disorders.
We describe a child with biallelic variants in EMC1, including a novel deep intronic single nucleotide variant that activates inclusion of a pseudoexon, to expand the genotypic and phenotypic spectrum of EMC1-related disorders.

See supplementary information.
3 | RESULTS

| Clinical History
The proband was the first child of NZ M aori/Samoan parents ( Figure 1C). She presented at 5 months of age with hypotonia, feeding difficulties and global developmental delay without history of regression. Rolling was achieved at 4 years and sitting at 6 years of age.
Speech was severely delayed with only five spoken words.
Cortical visual impairment was present with structurally normal ophthalmic examination. Bilateral cataracts developed at 6 years of age, possibly related to self-injurious behaviour. Severe bulbar dysfunction and aspiration necessitated nasogastric feeding from 5 months, followed by gastrostomy insertion. Severe obstructive sleep apnoea was documented at 3 years of age. She had recurrent chest infections and died at 7 years of age from a severe respiratory illness.
She had no history of seizures.
Examination findings included mild brachycephaly and midtemporal narrowing. Head circumference was on the 50th centile. She had a high palate and intermittent esotropia but was not otherwise dysmorphic. She had profound hypotonia, decreased muscle bulk and absent deep tendon reflexes. She had mild pectus excavatum, positional kyphosis, but no scoliosis.
Extensive neurometabolic investigation did not yield a clinical diagnosis. MRI at 7 months was normal with repeat MRI at 6 years of age showing mild atrophy of the superior vermis and symmetrical atrophy of the superior aspect of the cerebellar hemispheres ( Figure 1D). Nerve conduction studies were normal.

| Skeletal muscle histopathology
Collectively, muscle biopsy findings were interpreted as non-specific ( Figure 1E, supplementary results, quantified in Table S2).

| Genetic investigations and findings
Exome sequencing identified a maternal acceptor splice-site variant in  Figure S1 and Table S3).

| Western blot of EMC1 protein
Western blot of primary fibroblasts shows a 50% reduction in EMC1 protein expression in the proband compared to two age-and sex-matched controls ( Figure 1F i/ Figure S2), with no rescue of EMC1 protein levels in fibroblasts treated with 5 μM MG132 for 5 hours to inhibit proteosomal degradation ( Figure 1F ii). Validation of three EMC1 antibodies (Table S4)  skeletal muscle using all EMC1 antibodies was uninformative, with numerous non-specific bands complicating confident interpretation of results ( Figure S4).

| EMC1 RT-PCR
RT-PCR was performed on mRNA extracted from proband skeletal muscle (Figures 2 and 3)