Presynaptic congenital myasthenic syndrome with altered synaptic vesicle homeostasis linked to compound heterozygous sequence variants in RPH3A

Abstract Background Monogenic defects of synaptic vesicle (SV) homeostasis have been implicated in many neurologic diseases, including autism, epilepsy, and movement disorders. In addition, abnormal vesicle exocytosis has been associated with several endocrine dysfunctions. Methods We report an 11 year old girl with learning disabilities, tremors, ataxia, transient hyperglycemia, and muscle fatigability responsive to albuterol sulfate. Failure of neuromuscular transmission was confirmed by single fiber electromyography. Electron microscopy of motor nerve terminals revealed marked reduction in SV density, double‐membrane‐bound sacs containing SVs, abundant endosomes, and degenerative lamellar bodies. The patient underwent whole exome sequencing (WES) and relevant sequence variants were expressed and studied in a mammalian cell line. Results Chromosomal microarray studies and next generation sequencing (NGS) of mitochondrial DNA were unrevealing; however, NGS of genomic DNA showed two rare sequence variants in the gene encoding rabphilin 3a (RPH3A). The paternally inherited variant c.806 G>A (p.Arg269Gln) involves a substitution of a conserved residue in the linker region, while the maternally inherited variant c.1390 G>T (p.Val464Leu) involves a conserved amino acid substitution in the highly conserved C2A region. Expression studies revealed that p.Arg269Gln strongly impairs the binding of rabphilin 3a to 14‐3‐3, which is a proposed regulator of synaptic transmission and plasticity. In contrast, the binding of rabphilin 3a to 14‐3‐3 is only marginally impaired by p.Val464Leu; thus, the pathogenic role of p.Val464Leu remains unclear. Conclusion In summary, we report a patient with a multisystem neurologic disorder and altered SV regulation attributed to defects in RPH3A, which grants further studies of this gene in human disorders of synaptic transmission.


Whole Exome Sequencing (WES):
The whole exome sequencing was performed in the DNA of the affected patient and her both parents by the commercial laboratory Gene Dx. The Agilent SureSelect XT2 All Exon V4 kit was used to target the exon regions of the genomes. These targeted regions were sequenced using the Illumina HiSeq 2000 sequencing system with 100bp paired-end reads. The DNA sequence was mapped to an analyzed in comparison with the published human genome build UCSC hg19 reference sequence. The targeted coding exons and splice junctions of the known protein-coding RefSeq gene were assessed for the average depth of coverage and data quality threshold values (Mean Depth of Coverage: 132x, Quality threshold: 98.3%). The XomeAnalyzer was used to evaluate sequence changes in the individual compared to other sequenced family members. All reported sequence variants in the proband and parental samples were confirmed by conventional di-deoxyDNA sequence analysis or other appropriate method. The mean depth of coverage refers to the sequence mean read depth across the XomeDx targeted region, defined as coding exons and splice junctions of Agilent SureSelect XT2. All Exon V4 kit targeted protein coding RefSeq genes. The quality threshold refers to the percentage of the XomeDx defined target region where read depth was at least 10x.coverage to permit high quality exome variant base calling and annotation. Analysis: WES data for the patient and her parents was analyzed with the GeneDx's XomeAnalyzer (a variant annotation, filtering, and viewing interface for WES data), which includes nucleotide and amino acid annotations, population frequencies (from the NHLBI Exome Sequencing Project Exome Variant Server and 1000 Genomes), in silico prediction tools, amino acid conservation scores, and mutation references. Variants were filtered on the basis of inheritance patterns, lists of genes of interest, and phenotype and population frequencies, as appropriate. Synonymous variants and common variants (<0.01 in 1000Genomes< 0.01 in the GeneDx database) were ignored. Variants of low quality are also discarded for the analysis. The protein encoded by this gene resides in the nucleus and may play a role in the assembly of the nuclear lamina, and thus help maintain the structural organization of the nuclear envelope. It is highly expressed in lymph nodes.

Dok7
The protein encoded by this gene is essential for neuromuscular synaptogenesis and plays a major role in the aggregation of acetylcholine receptors through the phosphorylation of the muscle specific kinase (MuSK).

Rabphilin 3A
The protein encoded by this gene is thought to be an effector for RAB3A, which is a small G protein that acts in the late stages of neurotransmitter exocytosis. The encoded protein may be involved in Herein reported tremor, ataxia and muscle fatigability.
neurotransmitter release and synaptic vesicle traffic. This gene is only highly expressed in the brain.

DOK7-specific high density deletion/duplication analysis via gene-centric array CGH:
This study was performed by the commercial laboratory Prevention Genetics using genomic DNA from the patient and a sex-matched reference sample labeled and hybridized on a microarray containing probes across the entire DOK7 gene. The study was negative for deletion/duplication. The results are summarized in Figure S2.