EMC10 homozygous variant identified in a family with global developmental delay, mild intellectual disability, and speech delay

Abstract In recent years, several genes have been implicated in the variable disease presentation of global developmental delay (GDD) and intellectual disability (ID). The endoplasmic reticulum membrane protein complex (EMC) family is known to be involved in GDD and ID. Homozygous variants of EMC1 are associated with GDD, scoliosis, and cerebellar atrophy, indicating the relevance of this pathway for neurogenetic disorders. EMC10 is a bone marrow‐derived angiogenic growth factor that plays an important role in infarct vascularization and promoting tissue repair. However, this gene has not been previously associated with human disease. Herein, we describe a Saudi family with two individuals segregating a recessive neurodevelopmental disorder. Both of the affected individuals showed mild ID, speech delay, and GDD. Whole‐exome sequencing (WES) and Sanger sequencing were performed to identify candidate genes. Further, to elucidate the functional effects of the variant, quantitative real‐time PCR (RT‐qPCR)‐based expression analysis was performed. WES revealed a homozygous splice acceptor site variant (c.679‐1G>A) in EMC10 (chromosome 19q13.33) that segregated perfectly within the family. RT‐qPCR showed a substantial decrease in the relative EMC10 gene expression in the patients, indicating the pathogenicity of the identified variant. For the first time in the literature, the EMC10 gene variant was associated with mild ID, speech delay, and GDD. Thus, this gene plays a key role in developmental milestones, with the potential to cause neurodevelopmental disorders in humans.


| Genomic DNA and peripheral blood mononuclear cell extraction
Genomic DNA was extracted from the fresh blood of all available individuals using the QIAamp DNA Micro kit according to the manufacturer's instructions. DNA quantification was performed using a NanoDrop spectrophotometer. Density gradient centrifugation was used to separate peripheral blood mononuclear cells (PBMCs) using Ficoll-prefilled Leucosep tubes using the standard methods. The percentage of PBMC viability was assessed using the Trypan blue exclusion method (85%-90%).

| Whole exome sequencing
Whole exome sequencing (WES) was performed by Centogene Data analysis and interpretation were performed by Centogene using an end-to-end in-house bioinformatics pipeline with applications including base calling, filtering of low-quality reads, and alignment of reads to the GRCh37/hg19 (http://genome.ucsc.edu/) genome assembly.

| Filtration of variants
Primary filtering was performed using the standard methods, including filtering out low-quality reads and potential artifacts. Subsequently, variant annotation was performed using the standard methods. All disease-causing variants reported in the HGMD, ClinVar, CentoMD, PubMed, and variants with minor allele frequency (MAF) of >1% in the gnomAD/ExAC database were given predilection.
Variant filtration steps also focused on the autosomal recessive mode of inheritance, and coding exons along with flanking ±20 intronic base pairs were given priority. All pertinent inheritance patterns were considered. However, a recessive pattern was given preference based on the pedigree analysis. Patient clinical reports were evaluated to perform genotype-phenotype correlations. In silico prediction tools were used to analyze the pathogenicity of the identified variants, including NNSplice

| Sanger sequencing
The identified homozygous variant was Sanger sequenced in all available members of the family. Sanger sequencing was performed as previously described. 9 Primer pairs were designed using the Primer3 online tool (http://bioinfo.ut.ee/primer3-0.4.0/). Primers included annealing temperature, 60.5 C).

| RNA extraction
Total RNA was extracted from PBMCs after the addition of TRIzol reagent (Invitrogen), followed by chloroform (200 μL) for organic and aqueous phase separation. After centrifugation at 4 C for 15 min, RNA in the aqueous phase was transferred into RNase-free tubes.
Washing was performed using isopropanol, followed by precipitation with 75% ethanol. Quantification and purity tests were performed using standard methods. 10

| Quantitative real-time PCR
Total RNA was extracted to quantitatively monitor the EMC10 . PCR cycle conditions were the same as described previously, 10 and GAPDH was used as the endogenous control.

| Statistical analysis
The quantitative real-time PCR results were analyzed using GraphPad Prism (version 8.1). The analysis of variance (one-way ANOVA) statistical test was applied. P < .05 was considered significant.
Patient IV-1 is a 14-year-old male Saudi patient. He was born to consanguineous healthy parents after an unremarkable pregnancy course at full term by cesarean section due to fetal distress. His birth weight was 5 kg (>95th percentile) and the APGAR score was 9 and 9 at 1 and 5 min, respectively. After delivery, the patient was discharged immediately from the hospital.

| DISCUSSION
The ER membrane protein complex (EMC) was first discovered in yeast as a 6-subunit (Emc1-6) transmembrane protein complex, which helps in proper ER protein folding. Subsequently, 10 subunits (EMC1-10) were observed in different animal species. 1,13 EMC is an evolutionarily conserved and multifunctional protein complex with a significant functional importance in ER-mitochondria tethering, ERassociated degradation, and the proper assembly of different transmembrane proteins. 12 EMC6 is reported to regulate cell autophagy in humans, and modulates the ER-associated degradation of particular proteins in Caenorhabditis elegans. 14,15 Pathogenic homozygous variants in the EMC1 gene have been associated with a recessive neurodevelopmental disorder, characterized by pathognomonic GDD, cerebellar atrophy, ID, visual impairment, psychomotor retardation with epilepsy, and scoliosis. 16,17 EMC1 also belongs to the EMC family of proteins and interacts with EMC10 ( Figure 1F). Here, we investigated two Saudi patients with hallmark features of anxiety disorder, speech delay, tics, and mild ID. Using WES following Sanger sequencing, we identified a homozygous splice acceptor site variant (c.679-1G>A) in the EMC10 gene located on chromosome 19q13.33, consisting of a total of seven Since our knowledge of EMC biology is limited, the proper functional role of EMC and its association with human diseases remain to be fully elucidated. However, it has been suggested that EMC10 is a secreted protein and is found in pancreatic beta cells. The EMC10 gene is regulated by glucose, indicating its key role in glucose metabolism. 18 In an in vitro study, EMC10 was suggested as a potential therapeutic target for malignant glioblastoma after being found to exert cell proliferation inhibition, invasion, angiogenesis in endothelial cells, and cell migration in glioma cell lines. 19,20 In schizophrenia mouse models, reduced Mirta22 (human EMC10 ortholog) levels completely rescued the dendritic deficits and spine formation at the hippocampal pyramidal neurons, thus suggesting a key role in neuronal dendrites and spine development. 21 Furthermore, studies revealed that knockout mouse (emc10 −/− ) showed effects on their behavior (hyperactivity, abnormal gait, and abnormal vocalization), decreased bone mineral content, persistence of hyaloid vascular system, cardiovascular issues (decreased heart rate), deceased corpuscular volume in females, thrombocytopenia, metabolic effects, and infertility phenotypes in males (MGI: 5548589). 22 The EMC has been associated with protein folding and ERmitochondria crosstalk, and variants in EMC genes such as EMC1 have been previously associated with syndromic neurodegeneration. 5,8,[23][24][25] Variants in the COPA genes (OMIM 601924) have been associated with impaired intracellular communication, which resulted in impaired inter-organelle transport, increased ER stress, and cytokine generation, causing hereditary autoimmune lung disease and arthritis. 26 Similarly, variants in TANGO2 (OMIM 616878) have been associated with recurrent metabolic encephalomyopathic crises with rhabdomyolysis, cardiac arrhythmias, and neurodegeneration, resulting due to vesicular Golgi-ER transport and an increased ER stress, highlighting the importance of maintaining proper intracellular organelle crosstalk. 27,28 In conclusion, this study is the first to define the phenotypic spectrum of EMC10-associated disorders, including GDD, mild ID, and speech delay. Our work provides evidence that homozygous variants in EMC10 may lead to neurodevelopmental disorders in humans.
However, further study will be needed to identify the exact mechanism, which may involve the dysregulation of ER-mitochondria tethers or proper protein folding.