Missense variants in the N-terminal domain of the A isoform of FHF2/FGF13 cause an X-linked developmental and epileptic encephalopathy.

Fibroblast growth factor homologous factors (FHFs) are intracellular proteins which regulate voltage-gated sodium (Nav) channels in the brain and other tissues. FHF dysfunction has been linked to neurological disorders including epilepsy. Here, we describe two sibling pairs and three unrelated males who presented in infancy with intractable focal seizures and severe developmental delay. Whole-exome sequencing identified hemi- and heterozygous variants in the N-terminal domain of the A isoform of FHF2 (FHF2A). The X-linked FHF2 gene (also known as FGF13) has alternative first exons which produce multiple protein isoforms that differ in their N-terminal sequence. The variants were located at highly conserved residues in the FHF2A inactivation particle that competes with the intrinsic fast inactivation mechanism of Nav channels. Functional characterization of mutant FHF2A co-expressed with wild-type Nav1.6 (SCN8A) revealed that mutant FHF2A proteins lost the ability to induce rapid-onset, long-term blockade of the channel while retaining pro-excitatory properties. These gain-of-function effects are likely to increase neuronal excitability consistent with the epileptic potential of FHF2 variants. Our findings demonstrate that FHF2 variants are a cause of infantile-onset developmental and epileptic encephalopathy and underline the critical role of the FHF2A isoform in regulating Nav channel function.

Comprehensive genetic sequence and copy number analysis for Charcot-Marie-Tooth disease in a Canadian cohort of 2517 patients.

Charcot-Marie-Tooth disease (CMT) is one of the most common Mendelian disorders characterised by genetic heterogeneity, progressive distal muscle weakness and atrophy, foot deformities and distal sensory loss. In this report, we describe genetic testing data including comprehensive sequencing and copy number analysis of 34 CMT-related genes in a Canadian cohort of patients with suspected CMT. We have demonstrated a notable gender testing bias, with an overall diagnostic yield of 15% in males and 21% in females. We have identified a large number of novel pathogenic variants as well as variants of unknown clinical significance in CMT-related genes. In this largest to date analysis of gene CNVs in CMT, in addition to the common PMP22 deletion/duplication, we have described a significant contribution of pathogenic CNVs in several CMT-related genes. This study significantly expand the mutational spectrum of CMT genes, while demonstrating the clinical utility of a comprehensive sequence and copy number next-generation sequencing-based clinical genetic testing in patients with suspected diagnosis of CMT.

Dual molecular diagnoses in a neurometabolic specialty clinic.

Reports of patients with concomitant diagnoses of two inherited genetic disorders, sometimes referred to as "double trouble," have appeared intermittently in the medical literature. We report eight additional cases with dual diagnoses of two genetic conditions. All cases had a phenotype atypical for their primary diagnosis, leading to the search for a second genetic diagnosis. These cases highlight the importance of the history, physical examination and continued work-up if the phenotype of the patient falls drastically outside what has been reported with their primary diagnosis. Some of the diagnoses of the patients presented here (e.g., Myotonic Dystrophy Type 1, fascioscapulohumeral muscular dystrophy) would not have been identified by genetic testing done on a next generation sequencing backbone (e.g., panel or exome sequencing). When the clinical picture is atypical or more severe than expected the possibility of a dual diagnosis (double trouble) should be considered. Identification of a second genetic condition can impact management and genetic counseling.

The Efficacy of Whole Genome Sequencing and RNA-Seq in the Diagnosis of Whole Exome Sequencing Negative Patients with Complex Neurological Phenotypes.

Whole-genome sequencing (WGS) is being increasingly utilized for the diagnosis of neurological disease by sequencing both the exome and the remaining 98 to 99% of the genetic code. In addition to more complete coverage, WGS can detect structural variants (SVs) and intronic variants (SNVs) that cannot be identified by whole exome sequencing (WES) or chromosome microarray (CMA). Other multi-omics tools, such as RNA sequencing (RNA-Seq), can be used in conjunction with WGS to functionally validate certain variants by detecting changes in gene expression and splicing. The objective of this retrospective study was to measure the diagnostic yield of duo/trio-based WGS and RNA-Seq in a cohort of 22 patients (20 families) with pediatric onset neurological phenotypes and negative or inconclusive WES results in lieu of reanalysis. WGS with RNA-Seq resulted in a definite diagnosis of an additional 25% of cases. Sixty percent of these solved cases arose from the identification of variants that were missed by WES. Variants that could not be unequivocally proven to be causative of the patients' condition were identified in an additional 5% of cases.

Pre- and Postnatal Characterization of Autosomal Recessive KIDINS220-Associated Ventriculomegaly.

Introduction: Heterozygous loss-of-function variants in the last 2 exons of KIDINS220 have been associated with spastic paraplegia, intellectual disability, nystagmus, and obesity (SINO). Syndromic features of this condition include macrocephaly and dilatation of the lateral ventricles. Homozygous variants in the more proximal exons of KIDINS220 have been reported in several fetuses with a similar but more severe phenotype of limb contractures and severe ventriculomegaly identified in the second trimester of pregnancy. Case Presentation: We present here a 2.5-year-old female with profound global developmental delays and spasticity who was found by fetal ultrasound in week 19 of gestation to have bilateral talipes equinovarus and severe bilateral ventriculomegaly. Postnatal genetic testing revealed biallelic variants in KIDINS220. Discussion: To our knowledge, this is the first living individual reported with the autosomal recessive form of a KIDINS220-associated condition. This case provides additional information about the postnatal phenotype and a detailed history of development from prenatal ultrasonography.

De novo pathogenic variant in SETX causes a rapidly progressive neurodegenerative disorder of early childhood-onset with severe axonal polyneuropathy.

Pathogenic variants in SETX cause two distinct neurological diseases, a loss-of-function recessive disorder, ataxia with oculomotor apraxia type 2 (AOA2), and a dominant gain-of-function motor neuron disorder, amyotrophic lateral sclerosis type 4 (ALS4). We identified two unrelated patients with the same de novo c.23C > T (p.Thr8Met) variant in SETX presenting with an early-onset, severe polyneuropathy. As rare private gene variation is often difficult to link to genetic neurological disease by DNA sequence alone, we used transcriptional network analysis to functionally validate these patients with severe de novo SETX-related neurodegenerative disorder. Weighted gene co-expression network analysis (WGCNA) was used to identify disease-associated modules from two different ALS4 mouse models and compared to confirmed ALS4 patient data to derive an ALS4-specific transcriptional signature. WGCNA of whole blood RNA-sequencing data from a patient with the p.Thr8Met SETX variant was compared to ALS4 and control patients to determine if this signature could be used to identify affected patients. WGCNA identified overlapping disease-associated modules in ALS4 mouse model data and ALS4 patient data. Mouse ALS4 disease-associated modules were not associated with AOA2 disease modules, confirming distinct disease-specific signatures. The expression profile of a patient carrying the c.23C > T (p.Thr8Met) variant was significantly associated with the human and mouse ALS4 signature, confirming the relationship between this SETX variant and disease. The similar clinical presentations of the two unrelated patients with the same de novo p.Thr8Met variant and the functional data provide strong evidence that the p.Thr8Met variant is pathogenic. The distinct phenotype expands the clinical spectrum of SETX-related disorders.

POLRMT mutations impair mitochondrial transcription causing neurological disease.

While >300 disease-causing variants have been identified in the mitochondrial DNA (mtDNA) polymerase γ, no mitochondrial phenotypes have been associated with POLRMT, the RNA polymerase responsible for transcription of the mitochondrial genome. Here, we characterise the clinical and molecular nature of POLRMT variants in eight individuals from seven unrelated families. Patients present with global developmental delay, hypotonia, short stature, and speech/intellectual disability in childhood; one subject displayed an indolent progressive external ophthalmoplegia phenotype. Massive parallel sequencing of all subjects identifies recessive and dominant variants in the POLRMT gene. Patient fibroblasts have a defect in mitochondrial mRNA synthesis, but no mtDNA deletions or copy number abnormalities. The in vitro characterisation of the recombinant POLRMT mutants reveals variable, but deleterious effects on mitochondrial transcription. Together, our in vivo and in vitro functional studies of POLRMT variants establish defective mitochondrial transcription as an important disease mechanism.

A mitochondrial disorder with ptosis and exercise intolerance without ophthalmoparesis secondary to m.5865 T > C variant.

We describe a novel mitochondrial variant (m.5865 T > C) in a patient with decreased exercise endurance and juvenile onset slowly progressive bilateral ptosis without ophthamloparesis. The m.5865 T > C variant was seen in 82.9% of mtDNA molecules in skeletal muscle tissue and ~8% of mtDNA molecules in urine epithelium, but was not detected in blood leukocytes. The proband does not demonstrate any additional features often seen in individuals with a mitochondrial disorder (i.e., sensorineural hearing loss, type 2 diabetes, stroke-like episodes, muscle weakness, ophthalmoparesis, cardiomyopathy or cardiac arrhythmias). This case suggests that ptosis and exercise intolerance, without ophthalmoparesis, are the primary clinical features of the m.5865 T > C mtDNA variant.

Clinical and demographic features of chronic progressive external ophthalmoplegia in a large adult-onset cohort.

Chronic progressive external ophthalmoplegia (CPEO) is a common mitochondrial disease. We evaluated the impact of sex and smoking status upon knee extension strength and the phenotypic spectrum of disease in a large cohort of adult-onset CPEO patients (N=116) using retrospective chart analysis. The CPEO patients showed significantly lower knee extension strength as compared to the age- and sex-matched control population (-37%, P<0.05). Smoking also negatively impacted knee extension strength only in women with CPEO (-26%, P<0.05). We conclude that smoking and female sex interact negatively in CPEO patients.

Genotypes of chronic progressive external ophthalmoplegia in a large adult-onset cohort.

Chronic progressive external ophthalmoplegia (CPEO) is a common presentation of mitochondrial disease. We performed a retrospective evaluation of the molecular genetic testing and genotype-phenotype correlations in a large cohort of adult-onset CPEO patients (N = 111). One hundred percent of patients tested had at least one mitochondrial DNA (mtDNA) deletion. Genetic testing of nuclear genes encoding mitochondrial proteins identified pathogenic/likely pathogenic variants likely to be associated with CPEO in 7.6% of patients. As expected, the nuclear gene most associated with DNA variation was POLG. A single likely pathogenic mitochondrial DNA variant (m.12278T>C) was identified in two unrelated patients. No significant differences were noted in the clinical phenotypes of patients with pathogenic or likely pathogenic nuclear variants in comparison to those with negative nuclear gene testing. Analysis of deletion size and heteroplasmy in muscle-derived mtDNA showed significant correlations with age of symptom onset but not disease severity (number of canonical CPEO features). Results suggest that smaller mtDNA deletions (p = 0.0127, r2 = 0.1201) and higher heteroplasmy of single mtDNA deletions (p = 0.0112, r2 = 0.2483) are associated with an earlier age of onset in CPEO patients.

Males With MECP2 C-terminal-Related Atypical Rett Syndromes and Their Carrier Mothers.

BACKGROUND: This communication examines the expanding phenotypes of the MECP2 C-terminal atypical Rett syndromes in males and their affected carrier mothers. DESCRIPTIONS: We describe three males with normal karyotypes who presented with congenital evolving complex neurodevelopmental encephalopathies with multifaceted symptomatology of hypotonia, epilepsy, ataxia, spasticity, movement disorders, behavioral issues, severe intellectual impairment, and communication skills, and a protracted regression phase followed by stabilization. These phenotypes did not prompt us to identify atypical Rett syndrome early in childhood. RESULTS: Genetic analysis identified the two brothers with C-terminal truncation and the third male with C-terminal missense mutations. These mutations were inherited from their mothers, both of whom had incompletely characterized modest intellectual, mental health, social, and gastrointestinal impairments. Neither was independently able to care properly for their son(s). CONCLUSIONS: Mutations of the MECP2 gene should be considered early in males with hypotonia, developmental delay, profound intellectual impairment, and seizures, associated with a mother with psychosocial, cognitive, and gastrointestinal impairments. Counseling and supporting mildly affected mothers requires both medical and social efforts.

A mutation in the TMEM65 gene results in mitochondrial myopathy with severe neurological manifestations.

Recent research has suggested that transmembrane protein 65 (TMEM65) is localized within the inner mitochondrial membrane. Little else is known about its function. In this study we investigated the location and function of TMEM65. Further, we report the functional consequences of a novel homozygous splice variant (c.472+1G>A) in the TMEM65 gene in a patient with mitochondrial encephalomyopathy. Here we investigated the location of TMEM65 by immunofluorescence staining of the protein and by immunoblotting of the isolated mitochondrial fractions in healthy fibroblasts and those from the patient. To study the function of TMEM65 we knocked down mRNA using TMEM65-specific siRNA, and measured mitochondrial function by enzymology, protein abundance and oxygen consumption rate in fibroblasts. Subcellular fractionation confirmed that the TMEM65 protein was present in the inner mitochondrial membrane. Knocking down TMEM65 expression in dermal fibroblasts severely affected mitochondrial content and respiration rate. Further evidence for the essential role of TMEM65 in mitochondrial function came from the demonstration of severe cellular and clinical consequences resulting from the novel TMEM65 gene mutation. In conclusion, these findings suggest that TMEM65, an inner mitochondrial membrane protein, plays a significant role in mitochondrial respiratory chain function. We also provide the first evidence that a mutation in the TMEM65 gene results in mitochondrial dysfunction and a severe mitochondrial encephalomyopathy phenotype.

Impact of habitual exercise on the strength of individuals with myotonic dystrophy type 1.

OBJECTIVE: It remains unclear whether habitual physical activity can attenuate the rate of progressive muscle strength loss in individuals with myotonic dystrophy type 1 (DM1). The aim of this study was to identify whether there were any strength differences between DM1 patients who were habitually active or sedentary. DESIGN: Knee extension, handgrip, and elbow flexion quantitative strength measurements were investigated in the DM1 patients using isokinetic dynamometry. Strength was compared between the patients who followed self-selected formal exercise plans for at least 1 yr, those who were sedentary (controls), and those who initiated or terminated a formal exercise routine. RESULTS: Physically active DM1 patients with midrange CTG repeat size (100-500 CTG repeat sizes) had significantly stronger handgrip and knee extension and elbow flexion torques as compared with their sedentary counterparts with the same CTG repeat range. The DM1 patients who began a formal exercise routine experienced a significant improvement in knee extension torque measurements (+24.3%) in comparison with those who were habitually active or sedentary. CONCLUSIONS: These data suggest that there is an association between physical activity and strength. This may be shown to be a useful tool for the management of this condition. Further investigations into the relationships between physical exercise, muscle weakness, and genetic factors are needed before evidence-based recommendations can be made.