Mitochondrial respiratory chain disorders are a heterogeneous group of disorders in which the underlying genetic defect is often unknown. We have identified a pathogenic mutation (c.156C>G [p.F52L]) in YARS2, located at chromosome 12p11.21, by using genome-wide SNP-based homozygosity analysis of a family with affected members displaying myopathy, lactic acidosis, and sideroblastic anemia (MLASA). We subsequently identified the same mutation in another unrelated MLASA patient. The YARS2 gene product, mitochondrial tyrosyl-tRNA synthetase (YARS2), was present at lower levels in skeletal muscle whereas fibroblasts were relatively normal. Complex I, III, and IV were dysfunctional as indicated by enzyme analysis, immunoblotting, and immunohistochemistry. A mitochondrial protein-synthesis assay showed reduced levels of respiratory chain subunits in myotubes generated from patient cell lines. A tRNA aminoacylation assay revealed that mutant YARS2 was still active; however, enzyme kinetics were abnormal compared to the wild-type protein. We propose that the reduced aminoacylation activity of mutant YARS2 enzyme leads to decreased mitochondrial protein synthesis, resulting in mitochondrial respiratory chain dysfunction. MLASA has previously been associated with PUS1 mutations; hence, the YARS2 mutation reported here is an alternative cause of MLASA.

One Sentence Summary: Transcriptome sequencing improves the diagnostic rate for Mendelian disease in patients for whom genetic analysis has not returned a diagnosis. AbstractExome and whole-genome sequencing are becoming increasingly routine approaches in Mendelian disease diagnosis. Despite their success, the current diagnostic rate for genomic analyses across a variety of rare diseases is approximately 25-50%. Here, we explore the utility of transcriptome sequencing (RNA-seq) as a complementary diagnostic tool in a cohort of 50 patients with genetically undiagnosed rare muscle disorders. We describe an integrated approach to analyze patient muscle RNA-seq, leveraging an analysis framework focused on the detection of transcript-level changes that are unique to the patient compared to over 180 control skeletal muscle samples. We demonstrate the power of RNA-seq to validate candidate splice-disrupting mutations and to identify splice-altering variants in both exonic and deep intronic regions, yielding an overall diagnosis rate of 35%. We also report the discovery of a highly recurrent de novo intronic mutation in COL6A1 that results in a dominantly acting splice-gain event, disrupting the critical glycine repeat motif of the triple helical domain. We identify this pathogenic variant in a total of 27 genetically unsolved patients in an external collagen VI-like dystrophy cohort, thus explaining approximately 25% of patients clinically suggestive of collagen VI dystrophy in whom prior genetic analysis is negative. Overall, this study represents a large systematic application of transcriptome sequencing to rare disease diagnosis and highlights its utility for the detection and interpretation of variants missed by current standard diagnostic approaches. IntroductionThe advent of exome (WES) and whole genome (WGS) sequencing has greatly accelerated our capacity to identify variants that explain many Mendelian diseases in both known and new disease genes. While these technologies are mainstays in Mendelian disease diagnosis, their success rate for detecting causal variants is far from complete, ranging from 25-50% (1-4). The primary challenge of these genome-based diagnostics is that the capacity of WES and WGS to discover genetic variants substantially exceeds our ability to interpret their functional and clinical impact (5-7).

Expression of the cloned neuronal nicotinic acetylcholine receptor (nAChR) α7 subunit in several cultured mammalian cell lines has revealed that the folding, assembly, and subcellular localization of this protein are critically dependent upon the nature of the host cell. In all cell lines that were examined, high levels of α7 protein were detected by metabolic labelling and immunoprecipitation after transfection with the cloned α7 cDNA. In contrast, elevated levels of α‐bungarotoxin binding could be detected in only two of the nine cell lines. Both of these “α7‐permissive” cell lines [rat phaeochromocytoma (PC12) and human neuroblastoma (SH‐SY5Y)] express an endogenous α7 subunit. However, by expression of an epitope‐tagged α7 subunit, it has been possible to show that the elevation in surface α‐bungarotoxin binding in these two cell lines is due to expression of cDNA‐encoded α7. The cell‐specific misfolding of the neuronal nAChR α7 subunit is a phenomenon that is not shared by either the hetero‐oligomeric muscle nAChR or the homo‐oligomeric serotonin receptor 5‐HT3 subunit. Our data also indicate that the cell‐specific misfolding cannot be explained by a requirement for the coassembly with other known nAChR subunits and cannot be alleviated by treatments that have been reported to affect the assembly efficiency of other neurotransmitter‐gated ion channels.

The predominant nicotinic acetylcholine receptor (nAChR) expressed in vertebrate brain is a pentamer containing ␣4 and ␤2 subunits. In this study we have examined how temperature and the expression of subunit chimeras can influence the efficiency of cell-surface expression of the rat ␣4␤2 nAChR. In addition to the relatively well characterized nicotinic acetylcholine receptor (nAChR) 1 expressed at the vertebrate neuromuscular junction, a family of pharmacologically distinct "neuronal" nAChRs is expressed within the central and peripheral nervous system (1, 2). Whereas the muscle-type nAChR is a pentameric complex of known subunit composition (␣ 2 ␤␥␦ in fetal muscle and ␣ 2 ␤⑀␦ in adult), the precise subunit composition of the various neuronal nAChR subtypes is less certain. To date, 11 neuronal nAChR subunits (␣2-␣9 and ␤2-␤4) have been identified and cloned. There is evidence to suggest that the predominant neuronal nAChR subtype expressed in the vertebrate brain contains the ␣4 and ␤2 subunits (3, 4). When co-expressed in Xenopus oocytes, ␣4 and ␤2 co-assemble to form functional nAChRs (5) with a subunit stoichiometry of (␣4) 2 (␤2) 3 (6, 7).Several studies have demonstrated that relatively high levels of functional nAChRs are expressed on the cell surface of mammalian fibroblasts transfected with muscle (␣ 2 ␤␥␦ or ␣ 2 ␤⑀␦) nAChR subunit cDNAs (8, 9). In contrast, it appears that some neuronal nAChR subunit combinations are expressed considerably less efficiently when expressed heterologously in mammalian cell lines. In particular, the neuronal nAChR ␣7 and ␣8 subunits, which readily form functional homo-oligomeric nAChRs when expressed in Xenopus oocytes, appear to fold and assemble very inefficiently in many mammalian cell types (10 -15). In contrast, chimeric subunits containing the extracellular domain of the ␣7 or ␣8 subunits, together with the transmembrane and intracellular regions of the 5HT 3 receptor subunit, produce very high levels of cellsurface expression in all cell types examined (11,12,14,16,17).Functional expression of recombinant ␣4␤2 nAChRs in mammalian cell lines has been demonstrated previously (18 -20), but detailed characterization has been hindered somewhat by relatively low levels of cell-surface expression. Chronic exposure to nicotine has been shown to result in an increase in radioligand binding sites in cell lines expressing recombinant ␣4␤2 nAChRs (21-23), and correlates with an up-regulation (by ϳ2-fold) of the number of cell-surface nAChRs (21). However, despite up-regulation of cell-surface nAChRs, chronic treatment with nicotine has been reported to result in persistent functional inactivation of both recombinant ␣4␤2 and native nAChRs (21,24,25). It has been suggested that this "persistent inactivation" may be a consequence of the receptor adopting a long-lasting desensitized state. A 2-fold up-regulation in the level of cell-surface ␣4␤2 nAChR has also been reported as a consequence of treatments which elevate intracellular cAMP (26). It has also been shown previously ...