Whole-exome sequencing (WES) has transformed our ability to detect mutations causing rare diseases. FORGE (Finding Of Rare disease GEnes) and Care4Rare Canada are nation-wide projects focused on identifying disease genes using WES and translating this technology to patient care. Rare forms of epilepsy are well-suited for WES and we retrospectively selected FORGE and Care4Rare families with clinical descriptions that included childhood-onset epilepsy or seizures not part of a recognizable syndrome or an early-onset encephalopathy where standard-of-care investigations were unrevealing. Nine families met these criteria and a diagnosis was made in seven, and potentially eight, of the families. In the eight families we identified mutations in genes associated with known neurological and epilepsy disorders: ASAH1, FOLR1, GRIN2A (two families), SCN8A, SYNGAP1 and SYNJ1. A novel and rare mutation was identified in KCNQ2 and was likely responsible for the benign seizures segregating in the family though additional evidence would be required to be definitive. In retrospect, the clinical presentation of four of the patients was considered atypical, thereby broadening the phenotypic spectrum of these conditions. Given the extensive clinical and genetic heterogeneity associated with epilepsy, our findings suggest that WES may be considered when a specific gene is not immediately suspected as causal.
Background: Mutations in the slow skeletal muscle troponin T (TNNT1) gene cause a congenital nemaline myopathy resulting in death from respiratory insufficiency in early infancy. We report on four French Canadians with a novel congenital TNNT1 myopathy. Methods: Patients underwent lower extremity and paraspinal MRI, quadriceps biopsy and genetic testing. TNNT1 expression in muscle was assessed by quantitative PCR and immunoblotting. Wild type or mutated TNNT1 mRNAs were co-injected with morpholinos in a zebrafish knockdown model to assess for rescue of the morphant phenotype. Results: Four patients shared a novel missense homozygous mutation in TNNT1. They developed from childhood slowly progressive limb-girdle weakness with spinal rigidity and contractures. They suffered from restrictive lung disease and recurrent episodes of rhabdomyolysis. Older patients remained ambulatory into their sixties. Lower extremity MRI showed symmetrical myopathic changes. Paraspinal MRI showed diffuse fibro-fatty involution. Biopsies showed multi-minicores. Nemaline rods were seen in half the patients. TNNT1 mRNA expression was similar in controls and patients, while levels of TNNT1 protein were reduced in patients. Wild type TNNT1 mRNA rescued the zebrafish morphants but mutant transcripts failed to do so. Conclusions: This study expands the spectrum of TNNT1-related myopathy to include a milder clinical phenotype caused by a functionally-confirmed novel mutation.
Background: Spinal muscular atrophy (SMA) is a children’s neuromuscular disorder. Although motor neuron loss is a major feature of the disease, we have identified fatty acid abnormalities in SMA patients and in preclinical animal models, suggesting metabolic perturbation is also an important component of SMA. Methods: Biochemical, histological, proteomic, and high resolution respirometry were used. Results: SMA patients are more susceptible to dyslipidemia than the average population as determined by a standard lipid profile in a cohort of 72 pediatric patients. As well, we observed a non-alcoholic liver disease phenotype in apreclinical mouse model. Denervation alone was not sufficient to induce liver steatosis, as a mouse model of ALS, did not develop fatty liver. Hyperglucagonemia in Smn2B/-mice could explain the hepatic steatosis by increasing plasma substrate availability via glycogen depletion and peripheral lipolysis. Proteomic analysis identified mitochondrion and lipid metabolism as major clusters. Alterations in mitochondrial function were revealed by high-resolution respirometry. Finally, low-fat diets led to increased survival in Smn2B/-mice. Conclusions: These results provide strong evidence for lipid metabolism defects in SMA. Further investigation will be required to establish the primary mechanism of these alterations and understand how they lead to additional co-morbidities in SMA patients.
Polyneuropathies are amongst the most common neurological conditions worldwide affecting over 20 million people. However, 40% of patients with primary polyneuropathies have no disease-causing mutation identified.We investigated patients with gene-negative primary polyneuropathies using a combination of whole genome sequencing, homozygosity mapping and segregation analysis. Pathogenicity was confirmed via enzymatic assays and mass spectroscopy on recombinant protein and patient-derived fibroblasts, plasma and erythrocytes. We used circular dichroism to show secondary structure changes and isothermal titration calorimetry to investigate the ATP binding.We report that biallelic mutations in human PDXK are associated with primary axonal polyneuropathy and optic atrophy. Pyridoxal kinase (PDXK) is involved in converting vitamin B6 to its active form, pyridoxal 5’-phosphate (PLP). We show that PDXK mutations lead to disease via decreased plasma PLP concentrations. Our functional studies revealed conformational rearrangement in the mutant enzyme around the kinase ATP-binding pocket with impaired PDXK ability to bind ATP and leading to reduced erythrocyte PDXK activity. We show that both the human clinical picture and biochemical profile in PDXK mutations are rescued by PLP supplementation. Patients regained their ability to walk independently. Furthermore, treatment-led normalisation of plasma PLP levels, correlated with reduction of neurofilament light chain concentrations, a biomarker of axonal breakdown.In conclusion, biallelic mutations in human PDXK are associated with a novel disorder leading to treatable primary axonal polyneuropathy and optic atrophy and identifies PLP as therapeutic target.
Background: Mutations in the slow skeletal muscle troponin T (TNNT1) gene cause a congenital nemaline myopathy resulting in death from respiratory insufficiency in early infancy. We report on four French Canadians with a novel congenital TNNT1 myopathy. Methods: Patients underwent lower extremity and paraspinal MRI, quadriceps biopsy and genetic testing. TNNT1 expression in muscle was assessed by quantitative PCR and immunoblotting. Wild type or mutated TNNT1 mRNAs were co-injected with morpholinos in a zebrafish knockdown model to assess for rescue of the morphant phenotype. Results: Four patients shared a novel missense homozygous mutation in TNNT1. They developed from childhood slowly progressive limb-girdle weakness with spinal rigidity and contractures. They suffered from restrictive lung disease and recurrent episodes of rhabdomyolysis. Older patients remained ambulatory into their sixties. Lower extremity MRI showed symmetrical myopathic changes. Paraspinal MRI showed diffuse fibro-fatty involution. Biopsies showed multi-minicores. Nemaline rods were seen in half the patients. TNNT1 mRNA expression was similar in controls and patients, while levels of TNNT1 protein were reduced in patients. Wild type TNNT1 mRNA rescued the zebrafish morphants but mutant transcripts failed to do so. Conclusions: This study expands the spectrum of TNNT1-related myopathy to include a milder clinical phenotype caused by a functionally-confirmed novel mutation.
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