Reanalysis of Exome Data Identifies Novel SLC25A46 Variants Associated with Leigh Syndrome.

SLC25A46 (solute carrier family 25 member 46) mutations have been linked to various neurological diseases with recessive inheritance, including Leigh syndrome, optic atrophy, and lethal congenital pontocerebellar hypoplasia. SLC25A46 is expressed in the outer membrane of mitochondria, where it plays a critical role in mitochondrial dynamics. A deceased 7-month-old female infant was suspected to have Leigh syndrome. Clinical exome sequencing was non-diagnostic, but research reanalysis of the sequencing data identified two novel variants in SLC25A46: a missense (c.1039C>T, p.Arg347Cys; NM_138773, hg19) and a donor splice region variant (c.283+5G>A) in intron 1. Both variants were predicted to be damaging. Sanger sequencing of cDNA detected a single missense allele in the patient compared to control, and the SLC25A46 transcript levels were also reduced due to the splice region variant. Additionally, Western blot analysis of whole-cell lysate showed a decrease of SLC25A46 expression in proband fibroblasts, relative to control cells. Further, analysis of mitochondrial morphology revealed evidence of increased fragmentation of the mitochondrial network in proband fibroblasts, compared to control cells. Collectively, our findings suggest that these novel variants in SLC24A46, the donor splice one and the missense variant, are the cause of the neurological phenotype in this proband.

A homozygous stop-gain variant in ARHGAP42 is associated with childhood interstitial lung disease, systemic hypertension, and immunological findings.

ARHGAP42 encodes Rho GTPase activating protein 42 that belongs to a member of the GTPase Regulator Associated with Focal Adhesion Kinase (GRAF) family. ARHGAP42 is involved in blood pressure control by regulating vascular tone. Despite these findings, disorders of human variants in the coding part of ARHGAP42 have not been reported. Here, we describe an 8-year-old girl with childhood interstitial lung disease (chILD), systemic hypertension, and immunological findings who carries a homozygous stop-gain variant (c.469G>T, p.(Glu157Ter)) in the ARHGAP42 gene. The family history is notable for both parents with hypertension. Histopathological examination of the proband lung biopsy showed increased mural smooth muscle in small airways and alveolar septa, and concentric medial hypertrophy in pulmonary arteries. ARHGAP42 stop-gain variant in the proband leads to exon 5 skipping, and reduced ARHGAP42 levels, which was associated with enhanced RhoA and Cdc42 expression. This is the first report linking a homozygous stop-gain variant in ARHGAP42 with a chILD disorder, systemic hypertension, and immunological findings in human patient. Evidence of smooth muscle hypertrophy on lung biopsy and an increase in RhoA/ROCK signaling in patient cells suggests the potential mechanistic link between ARHGAP42 deficiency and the development of chILD disorder.

Striated Preferentially Expressed Protein Kinase (SPEG) in Muscle Development, Function, and Disease.

Mutations in striated preferentially expressed protein kinase (SPEG), a member of the myosin light chain kinase protein family, are associated with centronuclear myopathy (CNM), cardiomyopathy, or a combination of both. Burgeoning evidence suggests that SPEG plays critical roles in the development, maintenance, and function of skeletal and cardiac muscles. Here we review the genotype-phenotype relationships and the molecular mechanisms of SPEG-related diseases. This review will focus on the progress made toward characterizing SPEG and its interacting partners, and its multifaceted functions in muscle regeneration, triad development and maintenance, and excitation-contraction coupling. We will also discuss future directions that are yet to be investigated including understanding of its tissue-specific roles, finding additional interacting proteins and their relationships. Understanding the basic mechanisms by which SPEG regulates muscle development and function will provide critical insights into these essential processes and help identify therapeutic targets in SPEG-related disorders.

Striated Preferentially Expressed Protein Kinase (SPEG)-Deficient Skeletal Muscles Display Fewer Satellite Cells with Reduced Proliferation and Delayed Differentiation.

Centronuclear myopathies (CNMs) are a subtype of congenital myopathies characterized by skeletal muscle weakness and an increase in the number of central myonuclei. SPEG (striated preferentially expressed protein kinase) has been identified as the sixth gene associated with CNM, and it has been shown that striated muscle-specific Speg-knockout (KO) mice have defective triad formation, abnormal excitation-contraction coupling, and calcium mishandling. The impact of SPEG deficiency on the survival and function of myogenic cells remains to be deciphered. In this study, the authors examined the overall population, proliferation, and differentiation of myogenic cells obtained from striated muscle-specific Speg-KO mice and compared them with wild-type (WT) controls. SPEG-deficient skeletal muscles contained fewer myogenic cells, which on further study demonstrated reduced proliferation and delayed differentiation compared with those from WT muscles. Regenerative response to skeletal muscle injury in Speg-KO mice was compared with that of WT mice, leading to the identification of similar abnormalities including fewer satellite cells, fewer dividing cells, and an increase in apoptotic cells in KO mice. Overall, these results reveal specific abnormalities in myogenic cell number and behavior associated with SPEG deficiency. Similar satellite cell defects have been reported in mouse models of MTM1- and DNM2-associated CNM, suggestive of shared underlying pathways.

Knockin mouse model of the human CFL2 p.A35T mutation results in a unique splicing defect and severe myopathy phenotype.

Cofilin-2 is an actin-binding protein that is predominantly expressed in skeletal and cardiac muscles and belongs to the AC group of proteins, which includes cofilin-1 and destrin. In humans, cofilin-2 (CFL2) mutations have been associated with congenital myopathies that include nemaline and myofibrillar myopathy. To understand the pathogenicity of the human CFL2 mutation, p.A35T, that first linked cofilin-2 with the human disease, we created a knock-in mouse model. The Cfl2A35T/A35T (KI) mice were indistinguishable from their wild-type littermates at birth, but they rapidly worsened and died by postnatal day 9. The phenotypic, histopathologic and molecular findings mimicked the constitutive Cfl2-knockout (KO) mice described previously, including sarcomeric disruption and actin accumulations in skeletal muscles and negligible amounts of cofilin-2 protein. In addition, KI mice demonstrated a marked reduction in Cfl2 mRNA levels in various tissues including skeletal muscles. Further investigation revealed evidence of alternative splicing with the presence of two alternate transcripts of smaller size. These alternate transcripts were expressed at very low levels in the wild-type mice and were significantly upregulated in the mutant mice, indicating that pre-translational splicing defects may be a critical component of the disease mechanism associated with the mutation. Evidence of reduced expression of the full-length CFL2 transcript was also observed in the muscle biopsy sample of the patient with p.A35T mutation.

Unique bioinformatic approach and comprehensive reanalysis improve diagnostic yield of clinical exomes.

Clinical exome sequencing (CES) is increasingly being utilized; however, a large proportion of patients remain undiagnosed, creating a need for a systematic approach to increase the diagnostic yield. We have reanalyzed CES data for a clinically heterogeneous cohort of 102 probands with likely Mendelian conditions, including 74 negative cases and 28 cases with candidate variants, but reanalysis requested by clinicians. Reanalysis was performed by an interdisciplinary team using a validated custom-built pipeline, "Variant Explorer Pipeline" (VExP). This reanalysis approach and results were compared with existing literature. Reanalysis of candidate variants from CES in 28 cases revealed 1 interpretation that needed to be reclassified. A confirmed or potential genetic diagnosis was identified in 24 of 75 CES-negative/reclassified cases (32.0%), including variants in known disease-causing genes (n = 6) or candidate genes (n = 18). This yield was higher compared with similar studies demonstrating the utility of this approach. In summary, reanalysis of negative CES in a research setting enhances diagnostic yield by about a third. This study suggests the need for comprehensive, continued reanalysis of exome data when molecular diagnosis is elusive.

Mammalian Hbs1L deficiency causes congenital anomalies and developmental delay associated with Pelota depletion and 80S monosome accumulation.

Hbs1 has been established as a central component of the cell's translational quality control pathways in both yeast and prokaryotic models; however, the functional characteristics of its human ortholog (Hbs1L) have not been well-defined. We recently reported a novel human phenotype resulting from a mutation in the critical coding region of the HBS1L gene characterized by facial dysmorphism, severe growth restriction, axial hypotonia, global developmental delay and retinal pigmentary deposits. Here we further characterize downstream effects of the human HBS1L mutation. HBS1L has three transcripts in humans, and RT-PCR demonstrated reduced mRNA levels corresponding with transcripts V1 and V2 whereas V3 expression was unchanged. Western blot analyses revealed Hbs1L protein was absent in the patient cells. Additionally, polysome profiling revealed an abnormal aggregation of 80S monosomes in patient cells under baseline conditions. RNA and ribosomal sequencing demonstrated an increased translation efficiency of ribosomal RNA in Hbs1L-deficient fibroblasts, suggesting that there may be a compensatory increase in ribosome translation to accommodate the increased 80S monosome levels. This enhanced translation was accompanied by upregulation of mTOR and 4-EBP protein expression, suggesting an mTOR-dependent phenomenon. Furthermore, lack of Hbs1L caused depletion of Pelota protein in both patient cells and mouse tissues, while PELO mRNA levels were unaffected. Inhibition of proteasomal function partially restored Pelota expression in human Hbs1L-deficient cells. We also describe a mouse model harboring a knockdown mutation in the murine Hbs1l gene that shared several of the phenotypic elements observed in the Hbs1L-deficient human including facial dysmorphism, growth restriction and retinal deposits. The Hbs1lKO mice similarly demonstrate diminished Pelota levels that were rescued by proteasome inhibition.

SPEG-deficient skeletal muscles exhibit abnormal triad and defective calcium handling.

Centronuclear myopathies (CNM) are a subtype of congenital myopathies (CM) characterized by skeletal muscle weakness and an increase in the number of central myonuclei. We have previously identified three CNM probands, two with associated dilated cardiomyopathy, carrying striated preferentially expressed gene (SPEG) mutations. Currently, the role of SPEG in skeletal muscle function is unclear as constitutive SPEG-deficient mice developed severe dilated cardiomyopathy and died in utero. We have generated a conditional Speg-KO mouse model and excised Speg by crosses with striated muscle-specific cre-expressing mice (MCK-Cre). The resulting litters had a delay in Speg excision consistent with cre expression starting in early postnatal life and, therefore, an extended lifespan up to a few months. KO mice were significantly smaller and weaker than their littermate-matched controls. Histopathological skeletal muscle analysis revealed smaller myofibers, marked fiber-size variability, and poor integrity and low number of triads. Further, SPEG-deficient muscle fibers were weaker by physiological and in vitro studies and exhibited abnormal Ca2+ handling and excitation-contraction (E-C) coupling. Overall, SPEG deficiency in skeletal muscle is associated with fewer and abnormal triads, and defective calcium handling and excitation-contraction coupling, suggesting that therapies targeting calcium signaling may be beneficial in such patients.