Alejandro Horga

Extended phenotypic spectrum of KIF5A mutations From spastic paraplegia to axonal neuropathy

Objective: To establish the phenotypic spectrum of KIF5A mutations and to investigate whether KIF5A mutations cause axonal neuropathy associated with hereditary spastic paraplegia (HSP) or typical Charcot-Marie-Tooth disease type 2 (CMT2). Methods: KIF5A sequencing of the motor-domain coding exons was performed in 186 patients with the clinical diagnosis of HSP and in 215 patients with typical CMT2. Another 66 patients with HSP or CMT2 with pyramidal signs were sequenced for all exons of KIF5A by targeted resequencing. One additional patient was genetically diagnosed by whole-exome sequencing. Results: Five KIF5A mutations were identified in 6 unrelated patients: R204W and D232N were novel mutations; R204Q, R280C, and R280H have been previously reported. Three patients had CMT2 as the predominant and presenting phenotype; 2 of them also had pyramidal signs. The other 3 patients presented with HSP but also had significant axonal neuropathy or other additional features. Conclusion: This is currently the largest study investigating KIF5A mutations. By combining next-generation sequencing and conventional sequencing, we confirm that KIF5A mutations can cause variable phenotypes ranging from HSP to CMT2. The identification of mutations in CMT2 broadens the phenotypic spectrum and underlines the importance of KIF5A mutations, which involve degeneration of both the central and peripheral nervous systems and should be tested in HSP and CMT2.

Prevalence study of genetically defined skeletal muscle channelopathies in England.

Objectives: To obtain minimum point prevalence rates for the skeletal muscle channelopathies and to evaluate the frequency distribution of mutations associated with these disorders. Methods: Analysis of demographic, clinical, electrophysiologic, and genetic data of all patients assessed at our national specialist channelopathy service. Only patients living in the United Kingdom with a genetically defined diagnosis of nondystrophic myotonia or periodic paralysis were eligible for the study. Prevalence rates were estimated for England, December 2011. Results: A total of 665 patients fulfilled the inclusion criteria, of which 593 were living in England, giving a minimum point prevalence of 1.12/100,000 (95% confidence interval [CI] 1.03–1.21). Disease-specific prevalence figures were as follows: myotonia congenita 0.52/100,000 (95% CI 0.46–0.59), paramyotonia congenita 0.17/100,000 (95% CI 0.13–0.20), sodium channel myotonias 0.06/100,000 (95% CI 0.04–0.08), hyperkalemic periodic paralysis 0.17/100,000 (95% CI 0.13–0.20), hypokalemic periodic paralysis 0.13/100,000 (95% CI 0.10–0.17), and Andersen-Tawil syndrome (ATS) 0.08/100,000 (95% CI 0.05–0.10). In the whole sample (665 patients), 15 out of 104 different CLCN1 mutations accounted for 60% of all patients with myotonia congenita, 11 out of 22 SCN4A mutations for 86% of paramyotonia congenita/sodium channel myotonia pedigrees, and 3 out of 17 KCNJ2 mutations for 42% of ATS pedigrees. Conclusion: We describe for the first time the overall prevalence of genetically defined skeletal muscle channelopathies in England. Despite the large variety of mutations observed in patients with nondystrophic myotonia and ATS, a limited number accounted for a large proportion of cases.

Peripheral neuropathy predicts nuclear gene defect in patients with mitochondrial ophthalmoplegia

Mitochondrial ophthalmoplegia is a genetically heterogeneous disorder. Horga et al. investigate whether peripheral neuropathy can predict the underlying genetic defect in patients with progressive external ophthalmoplegia. Results indicate that neuropathy is highly predictive of a nuclear DNA defect and that it is rarely associated with single mitochondrial DNA deletions.

Mutations in noncoding regions of GJB1 are a major cause of X-linked CMT

Objective: To determine the prevalence and clinical and genetic characteristics of patients with X-linked Charcot-Marie-Tooth disease (CMT) due to mutations in noncoding regions of the gap junction β-1 gene (GJB1). Methods: Mutations were identified by bidirectional Sanger sequence analysis of the 595 bases of the upstream promoter region, and 25 bases of the 3′ untranslated region (UTR) sequence in patients in whom mutations in the coding region had been excluded. Clinical and neurophysiologic data were retrospectively collected. Results: Five mutations were detected in 25 individuals from 10 kindreds representing 11.4% of all cases of CMTX1 diagnosed in our neurogenetics laboratory between 1996 and 2016. Four pathogenic mutations, c.-17G>A, c.-17+1G>T, c.-103C>T, and c.-146-90_146-89insT were detected in the 5′UTR. A novel mutation, c.*15C>T, was detected in the 3′ UTR of GJB1 in 2 unrelated families with CMTX1 and is the first pathogenic mutation in the 3′UTR of any myelin-associated CMT gene. Mutations segregated with the phenotype, were at sites predicted to be pathogenic, and were not present in the normal population. Conclusions: Mutations in noncoding DNA are a major cause of CMTX1 and highlight the importance of mutations in noncoding DNA in human disease. Next-generation sequencing platforms for use in inherited neuropathy should therefore include coverage of these regions.

Genetic and clinical characteristics of NEFL-related Charcot-Marie-Tooth disease

Objectives To analyse and describe the clinical and genetic spectrum of Charcot-Marie-Tooth disease (CMT) caused by mutations in the neurofilament light polypeptide gene (NEFL). Methods Combined analysis of newly identified patients with NEFL-related CMT and all previously reported cases from the literature. Results Five new unrelated patients with CMT carrying the NEFL mutations P8R and N98S and the novel variant L311P were identified. Combined data from these cases and 62 kindreds from the literature revealed four common mutations (P8R, P22S, N98S and E396K) and three mutational hotspots accounting for 37 (55%) and 50 (75%) kindreds, respectively. Eight patients had de novo mutations. Loss of large-myelinated fibres was a uniform feature in a total of 21 sural nerve biopsies and ‘onion bulb’ formations and/or thin myelin sheaths were observed in 14 (67%) of them. The neurophysiological phenotype was broad but most patients with E90K and N98S had upper limb motor conduction velocities <38 m/s. Age of onset was ≤3 years in 25 cases. Pyramidal tract signs were described in 13 patients and 7 patients were initially diagnosed with or tested for inherited ataxia. Patients with E90K and N98S frequently presented before age 3 years and developed hearing loss or other neurological features including ataxia and/or cerebellar atrophy on brain MRI. Conclusions NEFL-related CMT is clinically and genetically heterogeneous. Based on this study, however, we propose mutational hotspots and relevant clinical–genetic associations that may be helpful in the evaluation of NEFL sequence variants and the differential diagnosis with other forms of CMT.

Clinicopathologic and molecular spectrum of RNASEH1-related mitochondrial disease.

Objective: Pathologic ribonuclease H1 (RNase H1) causes aberrant mitochondrial DNA (mtDNA) segregation and is associated with multiple mtDNA deletions. We aimed to determine the prevalence of RNase H1 gene (RNASEH1) mutations among patients with mitochondrial disease and establish clinically meaningful genotype-phenotype correlations. Methods: RNASEH1 was analyzed in patients with (1) multiple deletions/depletion of muscle mtDNA and (2) mendelian progressive external ophthalmoplegia (PEO) with neuropathologic evidence of mitochondrial dysfunction, but no detectable multiple deletions/depletion of muscle mtDNA. Clinicopathologic and molecular evaluation of the newly identified and previously reported patients harboring RNASEH1 mutations was subsequently undertaken. Results: Pathogenic c.424G>A p.Val142Ile RNASEH1 mutations were detected in 3 pedigrees among the 74 probands screened. Given that all 3 families had Indian ancestry, RNASEH1 genetic analysis was undertaken in 50 additional Indian probands with variable clinical presentations associated with multiple mtDNA deletions, but no further RNASEH1 mutations were confirmed. RNASEH1-related mitochondrial disease was characterized by PEO (100%), cerebellar ataxia (57%), and dysphagia (50%). The ataxia neuropathy spectrum phenotype was observed in 1 patient. Although the c.424G>A p.Val142Ile mutation underpins all reported RNASEH1-related mitochondrial disease, haplotype analysis suggested an independent origin, rather than a founder event, for the variant in our families. Conclusions: In our cohort, RNASEH1 mutations represent the fourth most common cause of adult mendelian PEO associated with multiple mtDNA deletions, following mutations in POLG, RRM2B, and TWNK. RNASEH1 genetic analysis should also be considered in all patients with POLG-negative ataxia neuropathy spectrum. The pathophysiologic mechanisms by which the c.424G>A p.Val142Ile mutation impairs human RNase H1 warrant further investigation.

SBF1 mutations associated with autosomal recessive axonal neuropathy with cranial nerve involvement

Biallelic mutations in the SBF1 gene have been identified in one family with demyelinating Charcot-Marie-Tooth disease (CMT4B3) and two families with axonal neuropathy and additional neurological and skeletal features. Here we describe novel sequence variants in SBF1 (c.1168C>G and c.2209_2210del) as the potential causative mutations in two siblings with severe axonal neuropathy, hearing loss, facial weakness and bulbar features. Pathogenicity of these variants is supported by co-segregation and in silico analyses and evolutionary conservation. Our findings suggest that SBF1 mutations may cause a syndromic form of autosomal recessive axonal neuropathy (AR-CMT2) in addition to CMT4B3.

IGHMBP2 mutation associated with organ-specific autonomic dysfunction

Highlights • Novel IGHMBP2 variant found in a patient with early onset severe peripheral neuropathy.• IGHMBP2 mutations may cause enteral autonomic dysfunction.• Autonomic dysfunction in IGHMBP2-related disorders may be severe requiring parenteral nutrition.

Mitochondrial impairment and rescue in riboflavin responsive neuropathy

Brown-Vialetto-Van Laere syndrome (BVVLS) represents a phenotypic spectrum of motor, sensory, and cranial nerve neuropathy, often with ataxia, optic atrophy and respiratory problems leading to ventilator-dependence. Loss-of-function mutations in two riboflavin transporter (RFVT) genes, SLC52A2 and SLC52A3, have recently been linked to BVVLS. However, the genetic frequency, neuropathology and downstream consequences of RFVT mutations have previously been undefined. By screening a large cohort of 132 patients with early-onset severe sensory, motor and cranial nerve neuropathy we confirmed the strong genetic link between RFVT mutations and BVVLS, identifying twenty-two pathogenic mutations in SLC52A2 and SLC52A3, fourteen of which were novel. Brain and spinal cord neuropathological examination of two cases with SLC52A3 mutations showed classical symmetrical brainstem lesions resembling pathology seen in mitochondrial disease, including severe neuronal loss in the lower cranial nerve nuclei, anterior horns and corresponding nerves, atrophy of the spinothalamic and spinocerebellar tracts and posterior column-medial lemniscus pathways. Mitochondrial dysfunction has previously been implicated in an array of neurodegenerative disorders. Since riboflavin metabolites are critical components of the mitochondrial electron transport chain (ETC), we hypothesized that reduced riboflavin transport would result in impaired mitochondrial activity, and confirmed this using in vitro and in vivo models. ETC complex I and complex II activity were decreased in SLC52A2 patient fibroblasts, while global knockdown of the single Drosophila RFVT homologue revealed reduced levels of riboflavin, downstream metabolites, and ETC complex I activity. RFVT knockdown in Drosophila also resulted in severely impaired locomotor activity and reduced lifespan, mirroring patient pathology, and these phenotypes could be partially rescued using a novel esterified derivative of riboflavin. Our findings indicate mitochondrial dysfunction as a downstream consequence of RFVT gene defects in BVVLS and validate riboflavin esters as a potential therapeutic strategy.

“Back to the Basics”-Never Forget to Look at the Back

A 46-year-old teacher presented with slowly progressive weakness of facial, proximal upper limb and distal lower limbs muscles since her late 20s. Her brother and father had similar symptoms. Examination revealed scapular winging, asymmetrical weakness of shoulder abduction, weakness of hip flexion and bilateral foot drop. Sensory testing was normal. CK levels were raised at 400–500 IU/L. ECG and EMG at presentation were normal. Molecular genetic testing was consistent with a contraction mutation of the D4Z4 repeat sequence at 4q35, consistent with the diagnosis of facioscapulohumeral muscular dystrophy. The same genetic abnormality was found in the patient’s brother and father.