Amelie Pandraud

Charcot–Marie–Tooth disease: frequency of genetic subtypes and guidelines for genetic testing

Background Charcot–Marie–Tooth disease (CMT) is a clinically and genetically heterogeneous group of diseases with approximately 45 different causative genes described. The aims of this study were to determine the frequency of different genes in a large cohort of patients with CMT and devise guidelines for genetic testing in practice. Methods The genes known to cause CMT were sequenced in 1607 patients with CMT (425 patients attending an inherited neuropathy clinic and 1182 patients whose DNA was sent to the authors for genetic testing) to determine the proportion of different subtypes in a UK population. Results A molecular diagnosis was achieved in 62.6% of patients with CMT attending the inherited neuropathy clinic; in 80.4% of patients with CMT1 (demyelinating CMT) and in 25.2% of those with CMT2 (axonal CMT). Mutations or rearrangements in PMP22, GJB1, MPZ and MFN2 accounted for over 90% of the molecular diagnoses while mutations in all other genes tested were rare. Conclusion Four commonly available genes account for over 90% of all CMT molecular diagnoses; a diagnostic algorithm is proposed based on these results for use in clinical practice. Any patient with CMT without a mutation in these four genes or with an unusual phenotype should be considered for referral for an expert opinion to maximise the chance of reaching a molecular diagnosis.

Treatable childhood neuronopathy caused by mutations in riboflavin transporter RFVT2

Childhood onset motor neuron diseases or neuronopathies are a clinically heterogeneous group of disorders. A particularly severe subgroup first described in 1894, and subsequently called Brown-Vialetto-Van Laere syndrome, is characterized by progressive pontobulbar palsy, sensorineural hearing loss and respiratory insufficiency. There has been no treatment for this progressive neurodegenerative disorder, which leads to respiratory failure and usually death during childhood. We recently reported the identification of SLC52A2, encoding riboflavin transporter RFVT2, as a new causative gene for Brown-Vialetto-Van Laere syndrome. We used both exome and Sanger sequencing to identify SLC52A2 mutations in patients presenting with cranial neuropathies and sensorimotor neuropathy with or without respiratory insufficiency. We undertook clinical, neurophysiological and biochemical characterization of patients with mutations in SLC52A2, functionally analysed the most prevalent mutations and initiated a regimen of high-dose oral riboflavin. We identified 18 patients from 13 families with compound heterozygous or homozygous mutations in SLC52A2. Affected individuals share a core phenotype of rapidly progressive axonal sensorimotor neuropathy (manifesting with sensory ataxia, severe weakness of the upper limbs and axial muscles with distinctly preserved strength of the lower limbs), hearing loss, optic atrophy and respiratory insufficiency. We demonstrate that SLC52A2 mutations cause reduced riboflavin uptake and reduced riboflavin transporter protein expression, and we report the response to high-dose oral riboflavin therapy in patients with SLC52A2 mutations, including significant and sustained clinical and biochemical improvements in two patients and preliminary clinical response data in 13 patients with associated biochemical improvements in 10 patients. The clinical and biochemical responses of this SLC52A2-specific cohort suggest that riboflavin supplementation can ameliorate the progression of this neurodegenerative condition, particularly when initiated soon after the onset of symptoms.

Exome sequencing reveals riboflavin transporter mutations as a cause of motor neuron disease

Brown-Vialetto-Van Laere syndrome was first described in 1894 as a rare neurodegenerative disorder characterized by progressive sensorineural deafness in combination with childhood amyotrophic lateral sclerosis. Mutations in the gene, SLC52A3 (formerly C20orf54), one of three known riboflavin transporter genes, have recently been shown to underlie a number of severe cases of Brown-Vialetto-Van Laere syndrome; however, cases and families with this disease exist that do not appear to be caused by SLC52A3 mutations. We used a combination of linkage and exome sequencing to identify the disease causing mutation in an extended Lebanese Brown-Vialetto-Van Laere kindred, whose affected members were negative for SLC52A3 mutations. We identified a novel mutation in a second member of the riboflavin transporter gene family (gene symbol: SLC52A2) as the cause of disease in this family. The same mutation was identified in one additional subject, from 44 screened. Within this group of 44 patients, we also identified two additional cases with SLC52A3 mutations, but none with mutations in the remaining member of this gene family, SLC52A1. We believe this strongly supports the notion that defective riboflavin transport plays an important role in Brown-Vialetto-Van Laere syndrome. Initial work has indicated that patients with SLC52A3 defects respond to riboflavin treatment clinically and biochemically. Clearly, this makes an excellent candidate therapy for the SLC52A2 mutation-positive patients identified here. Initial riboflavin treatment of one of these patients shows promising results.

A 6.4 Mb Duplication of the α-Synuclein Locus Causing Frontotemporal Dementia and Parkinsonism: Phenotype-Genotype Correlations

IMPORTANCE α-Synuclein (SNCA) locus duplications are associated with variable clinical features and reduced penetrance but the reasons underlying this variability are unknown. OBJECTIVES To report a novel family carrying a heterozygous 6.4 Mb duplication of the SNCA locus with an atypical clinical presentation strongly reminiscent of frontotemporal dementia and late-onset pallidopyramidal syndromes and study phenotype-genotype correlations in SNCA locus duplications. DESIGN, SETTING, AND PARTICIPANTS We report the clinical and neuropathologic features of a family carrying a 6.4 Mb duplication of the SNCA locus. To identify candidate disease modifiers, we completed a genetic analysis of the family and conducted statistical analysis on previously published cases carrying SNCA locus duplications using regression modeling with robust standard errors to account for clustering at the family level. MAIN OUTCOMES AND MEASURES We assessed whether length of the SNCA locus duplication influences disease penetrance and severity and whether extraduplication factors have a disease-modifying role. RESULTS We identified a large 6.4 Mb duplication of the SNCA locus in this family. Neuropathological analysis showed extensive α-synuclein pathology with minimal phospho-tau pathology. Genetic analysis showed an increased burden of Parkinson disease-related risk factors and the disease-predisposing H1/H1 microtubule-associated protein tau haplotype. Statistical analysis of previously published cases suggested there is a trend toward increasing disease severity and disease penetrance with increasing duplication size. The corresponding odds ratios from the univariable analyses were 1.17 (95% CI, 0.81-1.68) and 1.34 (95% CI, 0.78-2.31), respectively. Sex was significantly associated with both disease risk and severity; men compared with women had increased disease risk and severity and the corresponding odds ratios from the univariable analyses were 8.36 (95% CI, 1.97-35.42) and 5.55 (95% CI, 1.39-22.22), respectively. CONCLUSIONS AND RELEVANCE These findings further expand the phenotypic spectrum of SNCA locus duplications. Increased dosage of genes located within the duplicated region probably cannot increase disease risk and disease severity without the contribution of additional risk factors. Identification of disease modifiers accounting for the substantial phenotypic heterogeneity of patients with SNCA locus duplications could provide insight into molecular events involved in α-synuclein aggregation.

Amyloid precursor protein (APP) contributes to pathology in the SOD1 G93A mouse model of amyotrophic lateral sclerosis

In amyotrophic lateral sclerosis (ALS), the progressive loss of motor neurons is accompanied by extensive muscle denervation, resulting in paralysis and ultimately death. Upregulation of amyloid beta (A4) precursor protein (APP) in muscle fibres coincides with symptom onset in both sporadic ALS patients and the SOD1(G93A) mouse model of familial ALS. We have further characterized this response in SOD1(G93A) mice and also revealed elevated levels of β-amyloid (Aβ) peptides in the SOD1(G93A) spinal cord, which were predominantly localized within motor neurons and their surrounding glial cells. We therefore examined the effect of genetic ablation of APP on disease progression in SOD1(G93A) mice, which significantly improved multiple disease parameters, including innervation, motor function, muscle contractile characteristics, motor unit and motor neuron survival. These results therefore strongly suggest that APP actively contributes to SOD1(G93A)-mediated pathology. Together with observations from ALS cases, this study indicates that APP may contribute to human ALS pathology.

Genetic screening of Greek patients with Huntington’s disease phenocopies identifies an SCA8 expansion.

Huntington’s disease (HD) is an autosomal dominant disorder characterized by a triad of chorea, psychiatric disturbance and cognitive decline. Around 1% of patients with HD-like symptoms lack the causative HD expansion and are considered HD phenocopies. Genetic diseases that can present as HD phenocopies include HD-like syndromes such as HDL1, HDL2 and HDL4 (SCA17), some spinocerebellar ataxias (SCAs) and dentatorubral-pallidoluysian atrophy (DRPLA). In this study we screened a cohort of 21 Greek patients with HD phenocopy syndromes for mutations causing HDL2, SCA17, SCA1, SCA2, SCA3, SCA8, SCA12 and DRPLA. Fifteen patients (71%) had a positive family history. We identified one patient (4.8% of the total cohort) with an expansion of 81 combined CTA/CTG repeats at the SCA8 locus. This falls within what is believed to be the high-penetrance allele range. In addition to the classic HD triad, the patient had features of dystonia and oculomotor apraxia. There were no cases of HDL2, SCA17, SCA1, SCA2, SCA3, SCA12 or DRPLA. Given the controversy surrounding the SCA8 expansion, the present finding may be incidental. However, if pathogenic, it broadens the phenotype that may be associated with SCA8 expansions. The absence of any other mutations in our cohort is not surprising, given the low probability of reaching a genetic diagnosis in HD phenocopy patients.

Madras motor neuron disease (MMND) is distinct from the riboflavin transporter genetic defects that cause Brown–Vialetto–Van Laere syndrome

Introduction Madras motor neuron disease (MMND), MMND variant (MMNDV) and Familial MMND (FMMND) have a unique geographic distribution predominantly reported from Southern India. The characteristic features are onset in young, weakness and wasting of limbs, multiple lower cranial nerve palsies and sensorineural hearing loss. There is a considerable overlap in the phenotype of MMND with Brown–Vialetto–Van Laere syndrome (BVVL) Boltshauser syndrome, Nathalie syndrome and Fazio–Londe syndrome. Recently a number of BVVL cases and families have been described with mutations in two riboflavin transporter genes SLC52A2 and SLC52A3 (solute carrier family 52, riboflavin transporter, member 2 and 3 respectively). Methods and results We describe six families and four sporadic MMND cases that have been clinically characterized in detail with history, examination, imaging and electrophysiological investigations. We sequenced the SLC52A1, SLC52A2 and SLC52A3 in affected probands and sporadic individuals from the MMND series as well as the C9ORF72 expansion. No genetic defects were identified and the C9ORF72 repeats were all less than 10. Conclusions These data suggest that MMND is a distinct clinical subgroup of childhood onset MND patients where the known genetic defects are so far negative. The clinico-genetic features of MMND in comparison with the BVVL group of childhood motor neuron diseases suggest that these diseases are likely to share a common defective biological pathway that may be a combination of genetic and environmental factors.

Novel peripheral myelin protein 22 (PMP22) micromutations associated with variable phenotypes in Greek patients with Charcot-Marie-Tooth disease

ARTICLE Sir, We read with interest the paper by Taioli et al . (2011) reporting eight different micromutations of PMP22 in patients with inherited demyelinating neuropathies. Small frameshift insertions/deletions (indels), nonsense nucleotide substitutions and splice-site mutations led to phenotypically and pathologically variable neuropathies. Patients with mutations causing a premature or delayed stop codon had a phenotype within the hereditary neuropathy with liability to pressure palsies (HNPP) spectrum. One patient with a splice-site mutation causing an in-frame skipping of exon 4 had a CMT1 phenotype. As the authors state, micromutations of PMP22 are considered very rare causes of Charcot–Marie–Tooth (CMT) in most populations and this certainly includes our experience of PMP22 analysis in cases with CMT from the UK. We presently report three novel PMP22 micromutations identified in a Greek cohort of patients with CMT that were associated with variable clinical phenotypes, ranging from severe CMT1 of the Dejerine–Sottas disease spectrum to HNPP. These novel cases, taken together with the findings of Taioli et al . (2011) illustrate that, despite some exceptions, in-frame indels in PMP22 usually lead to CMT1 of varying severity, whereas frameshift changes more often cause HNPP. DNA was extracted from peripheral blood following standard procedures. Patients were previously found negative for the common duplication/deletion in chromosome 17 and for mutations in GJB1 and MPZ . The four coding exons (exons 2–5) of PMP22 with adjacent intronic sequences were amplified by polymerase chain reaction and sequenced on an Applied Biosystems 3730XL genetic analyser. Fragment analysis was performed with GeneMapper® software after running fragments on the 3730XL with a LIZ500 standard. RNA was obtained from peripheral blood using the Qiagen/PreAnalytix™ blood RNA system. Complementary DNA was synthesized using the Applied Biosystems high capacity complementary DNA reverse transcription kit. A 391 bp fragment of complementary DNA spanning the regions of interest …

Mitochondrial deficits and abnormal mitochondrial retrograde axonal transport play a role in the pathogenesis of mutant Hsp27-induced Charcot Marie Tooth Disease

Abstract Mutations in the small heat shock protein Hsp27, encoded by the HSPB1 gene, have been shown to cause Charcot Marie Tooth Disease type 2 (CMT-2) or distal hereditary motor neuropathy (dHMN). Protein aggregation and axonal transport deficits have been implicated in the disease. In this study, we conducted analysis of bidirectional movements of mitochondria in primary motor neuron axons expressing wild type and mutant Hsp27. We found significantly slower retrograde transport of mitochondria in Ser135Phe, Pro39Leu and Arg140Gly mutant Hsp27 expressing motor neurons than in wild type Hsp27 neurons, although anterograde movement velocities remained normal. Retrograde transport of other important cargoes, such as the p75 neurotrophic factor receptor was minimally altered in mutant Hsp27 neurons, implicating that axonal transport deficits primarily affect mitochondria and the axonal transport machinery itself is less affected. Investigation of mitochondrial function revealed a decrease in mitochondrial membrane potential in mutant Hsp27 expressing motor axons, as well as a reduction in mitochondrial complex 1 activity, increased vulnerability of mitochondria to mitochondrial stressors, leading to elevated superoxide release and reduced mitochondrial glutathione (GSH) levels, although cytosolic GSH remained normal. This mitochondrial redox imbalance in mutant Hsp27 motor neurons is likely to cause low level of oxidative stress, which in turn will contribute to, and indeed may be the underlying cause of the deficits in mitochondrial axonal transport. Together, these findings suggest that the mitochondrial abnormalities in mutant Hsp27-induced neuropathies may be a primary cause of pathology, leading to further deficits in the mitochondrial axonal transport and onset of disease.

Mutational analysis of PMP22, EGR2, LITAF and NEFL in Greek Charcot-Marie-Tooth type 1 patients

To the Editor : Charcot–Marie–Tooth (CMT) disease is classified into CMT1 (demyelinating) and CMT2 (axonal) based on motor conduction velocities. Dominant CMT1 is the commonest in most populations, excepting communities with high consanguinity rates, where recessive CMT1 may be common (1). We recently reported on a cohort of Greek CMT1 patients screened for the PMP22 duplication and point mutations in GJB1 and MPZ . This screen elucidated the cause in ∼30% of cases, placing Greece among the countries with a low duplication frequency (2). To further elucidate the cause of CMT1 in the Greek population, we screened 86 undiagnosed patients from the original cohort for point mutations in PMP22 , EGR2 , LITAF and NEFL. Sixty-four cases were familial (50 suggestive of dominant inheritance) and 22 non-familial. Informed consent was obtained and DNA extracted from peripheral blood. Coding exons and adjacent intronic sequences of PMP22 , EGR2 , LITAF and NEFL were sequenced according to established protocols. Three pathogenic mutations were found (3.5%); two recently reported micromutations in PMP22 (3), and one known point mutation in EGR2 (4, 5). No pathogenic mutations were detected in LITAF or NEFL. All variants detected are shown in Table 1. Clinical and genetic details of patients with pathogenic mutations are shown in Table 2. The first PMP22 mutation was a de novo dominant 6 bp deletion in exon 4. The patient had severe CMT1 (Dejerine–Sottas phenotype). The second mutation was a dominantly inherited 21 bp duplication in exon 5. The patient had CMT1 and moderately restricted mobility throughout childhood. The mother carried the same mutation with a milder phenotype. The above mutations cause in-frame changes leading to stable transcripts and result in CMT1 of varying severity (3). The EGR2 mutation (Arg381His) was identified in an adolescent with severe CMT1 and no cranial neuropathies. It has been previously reported in patients with severe CMT1 and occasional cranial nerve involvement (4, 5). In one family, the proband developed cranial neuropathies in his 30s, but his affected daughter had none (4). In the other, the patient had congenital hypomyelination and Duane syndrome (5). It is too early to exclude a late emergence of cranial neuropathies in our patient. These cases show the phenotypic variability associated with the Arg381His mutation, most patients occupying the severe end of the CMT1 spectrum. The finding of two PMP22 (2.3%) and one EGR2 mutation (1.2%) in our cohort is in agreement with previously reported frequencies (1, 6). Given the rarity of mutations in LITAF and NEFL, it is no surprise that none were detected. Our data suggest that a molecular diagnosis can be reached in just over 35% of Greek patients with CMT1 (∼45% in cases suggestive of dominant inheritance) (2). This is considerably lower than most populations, such as the UK, where ∼60–80% of CMT1 patients are accounted for by the genes screened (6). Interestingly, studies from Turkey, Norway and Japan, all three relatively isolated populations, have yielded similar results to ours (2, 7). There is also some evidence that in the general population the PMP22 duplication frequency may be significantly lower than in clinic-based cohorts (7). Limitations in the methodology used to detect the CMT1A duplication and a somewhat permissive electrophysiological cut-off may have contributed to the low frequency observed in our cohort (2). Now that all genes known to cause dominant CMT1 have been screened and after excluding the CMT1A duplication with the most sensitive techniques, it would be important to screen familial cases consistent with recessive inheritance and isolated cases for known genes causing recessive CMT1. In general, recessive forms of CMT are rare in Europe, but in populations with increased consanguinity and in isolated populations this can be significantly higher (1). In addition, the presence of as yet unidentified genes contributing significantly to the pool of Greek CMT patients is a distinct possibility.