A Novel Homozygous Founder Variant of RTN4IP1 in Two Consanguineous Saudi Families

Abstract

The genetic architecture of mitochondrial disease continues to expand and currently exceeds more than 350 disease-causing genes. Bi-allelic variants in RTN4IP1, also known as Optic Atrophy-10 (OPA10), lead to early-onset recessive optic neuropathy, atrophy, and encephalopathy in the afflicted patients. The gene is known to encode a mitochondrial ubiquinol oxidoreductase that interacts with reticulon 4 and is thought to be a mitochondrial antioxidant NADPH oxidoreductase. Here, we describe two unrelated consanguineous families from the northern region of Saudi Arabia harboring a missense variant (RTN4IP1:NM_032730.5; c.475G<T, p.Val159Phe) in the gene. Clinically affected individuals presented with intellectual disability, encephalopathy, ataxia, optic atrophy, and seizures. Based on whole exome sequencing and confirmatory Sanger sequencing, the variant was fully segregated with the phenotype in the families, absent among large ethnically matching controls as well as numerous in-house exomes, and predicted to be pathogenic by different in silico classifiers. Structural modeling and immunoblot analyses strongly indicated this variant to be pathogenic. Since the families belong to one of the tribal inhabitants of Saudi Arabia, we postulate that the variant is likely to be a founder. We provide the estimated age of the variant and present data confirming the disease-causality of this founder variant.

Keywords: RTN4IP1; age of variant; encephalopathy; founder variant; in silico pathogenicity prediction; missense; optic atrophy; structural modeling.

Figure 1

Histochemical and immunohistochemical analyses of muscle biopsy. (A) Gomori trichrome staining shows prominent subsarcolemmal accumulation of mitochondria in most fibers with ragged red-like fibers. (B) COX staining shows no COX-negative fibers, but there is marked subsarcolemmal staining. (C) SDH staining shows marked subsarcolemmal staining. (D) Oil red staining indicates moderate increase in lipid stores.

Figure 2

Brain MRI results. Selected MRI sequences and planes showing optic chiasm atrophy (white/red/black arrows) in patient 1 (image (1B) coronal CISS), Patient 2 (image (1C) coronal T2), Patient 4 (image (1E) sagittal T1), and Patient 5 (image (1F) coronal T2). Other selected images also show bilateral optic nerve atrophy (white arrows) in patient 1 (image (1A) coronal T2), Patient 3 (image (1D) axial T2), and Patient 6 (image (1G) axial T2; image (1H) coronal T2).

Figure 3

Brain MRS findings of the patients. Multiple single-voxel MRS studies showed small lactate doublets at 1.3 ppm (white arrows) in Patient 1 (image (2A)), Patient 3 (image (2B)), Patient 5 (image (2D)), and Patient 6 (image (2E)). There is no obvious lactate doublet seen in Patient 4 (image (2C)). An MRS study was not performed for Patient 2.

Figure 4

Genetic analysis results. (A) Pedigree of the Family 1. Affected individuals are labeled with black-colored (filled) symbols. Carriers are represented with half-filled symbols; squares represent males, circles represent females, and double line indicates consanguinity. (B) Pedigree for the family 2. Brief Sanger sequencing results of the c.475 G>T variant under the pedigrees show the results for the Families. (C) Homozygosity results for all the affected individuals in both families. The patients are aligned with the normal individuals in both families. The shared ROH block is shown in the red bracket on chromosome 6 where RTN4IP1 is located. Numbers 1-6 indicate family members from Family 1, individuals 1 to Family 2, and individual 3, (F1-II-1, F1-II-2, F1-II-3, F2-II-1, F2-II-2, and F2-II-3), respectively. (D) Protein sequence alignment of RTN4IP1, the blue arrow shows the affected Valine amino acids at position 159 by the novel variant. The alignment showed that the amino acid in this region is highly conserved among different species.

Figure 5

Structural analysis and immunoblotting experiment results. (A) The location of the variant within the 3-D crystal structure of RTN4IP1 (PDB ID: 2VN8). RTN4IP1 forms a dimer (green and blue chains). Val159 is shown as a red stick model. The cofactor NADPH is shown as a stick model with carbon atoms in white. The zoom view shows the effect of the p.Val159Phe substitution. Clashes of the Phenylalanine side chain (yellow) are shown as red discs. Key side chains in the vicinity are highlighted as stick models. (B) Immunoblotting analysis results. The Western blotting analysis revealed significantly decreased RTN4IP1 protein levels in the patient’s extracts from cultured fibroblasts in comparison to controls. A polyclonal antibody was targeted against the protein, and beta-actin was used as a control-loading marker. (C) Schematic presentation of RTN4IP1. The top panel shows localization of all previously reported variants (black) and the newly identified novel variant (red) distributed among the nine exons (blue). The bottom panel shows both the alcohol dehydrogenase domain (ADH_N) and the zinc-binding motif (ADH_Zinc).