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Homozygous splice-variants in human ARV1 cause GPI-anchor synthesis deficiency

https://doi.org/10.1016/j.ymgme.2020.02.005Get rights and content

Abstract

Background

Mutations in the ARV1 Homolog, Fatty Acid Homeostasis Modulator (ARV1), have recently been described in association with early infantile epileptic encephalopathy 38. Affected individuals presented with epilepsy, ataxia, profound intellectual disability, visual impairment, and central hypotonia. In S. cerevisiae, Arv1 is thought to be involved in sphingolipid metabolism and glycophosphatidylinositol (GPI)-anchor synthesis. The function of ARV1 in human cells, however, has not been elucidated.

Methods

Mutations were discovered through whole exome sequencing and alternate splicing was validated on the cDNA level. Expression of the variants was determined by qPCR and Western blot. Expression of GPI-anchored proteins on neutrophils and fibroblasts was analyzed by FACS and immunofluorescence microscopy, respectively.

Results

Here we describe seven patients from two unrelated families with biallelic splice mutations in ARV1. The patients presented with early onset epilepsy, global developmental delays, profound hypotonia, delayed speech development, cortical visual impairment, and severe generalized cerebral and cerebellar atrophy. The splice variants resulted in decreased ARV1 expression and significant decreases in GPI-anchored protein on the membranes of neutrophils and fibroblasts, indicating that the loss of ARV1 results in impaired GPI-anchor synthesis.

Conclusion

Loss of GPI-anchored proteins on our patients' cells confirms that the yeast Arv1 function of GPI-anchor synthesis is conserved in humans. Overlap between the phenotypes in our patients and those reported for other GPI-anchor disorders suggests that ARV1-deficiency is a GPI-anchor synthesis disorder.

Introduction

Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors proteins involved in signal transduction and immune response to the outer membrane of cells [1,2]. The GPI-anchor consists of a phospholipid tail, a glycan core and a phosphoethanolamine linker, and is added to proteins as a post-translational modification [1,2]. Biosynthesis of GPI-anchors, a process that involves at least 26 genes, occurs in the endoplasmic reticulum (ER) and Golgi [3,4]. The process starts with the formation of a phospholipid tail on the cytoplasmic side of the ER, during which the first glycosylation steps occur; the GPI-precursor is then flipped into the ER lumen to access the mannosyltransferases [[3], [4], [5]]. After the GPI-anchor is formed and attached to the target protein, it travels through the Golgi, where additional modifications are made [3].

Thus far 19 genes involved in GPI-anchor biosynthesis have been associated with human disease [4,[6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27]]; all present with a multitude of symptoms, including seizures, hematologic and metabolic abnormalities, developmental delays, intellectual disabilities and occasionally dysmorphic features and congenital anomalies [4,28].

Studies in S. cerevisiae suggest that ACAT related enzyme 2 required for viability 1 (Arv1) may be involved in the process of flipping the GPI-precursors into the ER lumen [5], a role that is conserved in the human homolog ARV1 (Arv1 homolog, or Fatty Acid Homeostasis Modulator: MIM 611647; NM_022786.1) [29]. A homozygous missense mutation in ARV1 (c.565 G>A, p.Gly189Arg) was first described in affected members of a consanguineous family who presented with phenotypes resembling GPI-anchor disorders, including severe intellectual disability, early onset seizures, poor head control and ataxia [6]. An additional homozygous splicing mutation (c.294+1 G>A, p.59-98del) was described in a second report in an individual with a similar phenotype, including visual impairment, central hypotonia, and dystonia [27]; that condition was classified as early infantile epileptic encephalopathy 38 (EIEE38, MIM 617020). These cases did not include a description of GPI anchor abnormalities due to ARV1 mutations.

Here we describe seven additional patients from two unrelated families with homozygous splice mutations in ARV1 and EIEE38. In addition to what has been described in literature, we show that, similar to Arv1 in S. cerevisiae, GPI-anchor synthesis in humans is impaired by the loss of ARV1 protein, causing a significant decrease in membrane bound GPI-anchored proteins measured in neutrophils and fibroblasts.

Section snippets

Subjects and samples

Patient 1 and 2 (Family I) were enrolled at the National Institutes of Health Clinical Center (NIH-CC) under the protocol 14-HG-0071: “Clinical and Basic Investigations Into Known and Suspected Congenital Disorders of Glycosylation” (NCT02089789) and protocol 15-HG-0130: “Clinical and Genetic Evaluation of Individuals With Undiagnosed Disorders Through the Undiagnosed Diseases Network” (NCT02450851) [[30], [31], [32], [33]]. Both clinical protocols were approved by the National Human Genome

Family I

Patient 1, is a 17 years old female, the first child to American-Mexican parents (Fig. 1A, II.1) who was born after a 41-week gestation. At 6 months of age, she had her first seizure, followed by frequent episodes of both febrile and non-febrile seizures, some requiring hospitalization. Patient 1 never developed a social smile or met any motor milestones. She has been dependent on a gastric tube feeding since age 2 years and is wheelchair dependent. She has cortical blindness and bilateral

Discussion

Here we describe seven patients from two unrelated families with early infantile epileptic encephalopathy 38 due to homozygous splice mutations in ARV1. Similar to two previous reports on patients with variants in ARV1, our patients present with early onset seizures, central hypotonia, severe developmental delay, visual impairment and feeding difficulties, supporting the clinical diagnosis of IEE38. The probands in this study had severe speech delay, spasticity, scoliosis and deformity of the

Disclosures

None.

Acknowledgemnts

The authors would like to thank all patients and their families for their participation in this research. We also thank Yan Huang for establishing primary fibroblast cultures and for sample preparations. This research was in part supported by the Intramural Research Program of the National Human Genome Research Institute and the NIH Office of the Director's Common Fund. The research conducted at the Murdoch Children's Research Institute was supported by the Victorian Government‘s Operational

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