A Compound Heterozygous Mutation in Calpain 1 Identifies a New Genetic Cause for Spinal Muscular Atrophy Type 4 (SMA4).

Spinal Muscular Atrophy (SMA) is a heterogeneous group of neuromuscular diseases characterized by degeneration of anterior horn cells of the spinal cord, leading to muscular atrophy and weakness. Although the major cause of SMA is autosomal recessive exon deletions or loss-of-function mutations of survival motor neuron 1 (SMN1) gene, next generation sequencing technologies are increasing the genetic heterogeneity of SMA. SMA type 4 (SMA4) is an adult onset, less severe form of SMA for which genetic and pathogenic causes remain elusive.Whole exome sequencing in a 30-year-old brother and sister with SMA4 identified a compound heterozygous mutation (p. G492R/p. F610C) in calpain-1 (CAPN1). Mutations in CAPN1 have been previously associated with cerebellar ataxia and hereditary spastic paraplegia. Using skin fibroblasts from a patient bearing the p. G492R/p. F610C mutation, we demonstrate reduced levels of CAPN1 protein and protease activity. Functional characterization of the SMA4 fibroblasts revealed no changes in SMN protein levels and subcellular distribution. Additional cellular pathways associated with SMA remain unaffected in the patient fibroblasts, highlighting the tissue specificity of CAPN1 dysfunction in SMA4 pathophysiology. This study provides genetic and functional evidence of CAPN1 as a novel gene for the SMA4 phenotype and expands the phenotype of CAPN1 mutation disorders.

Energy metabolism and mitochondrial defects in X-linked Charcot-Marie-Tooth (CMTX6) iPSC-derived motor neurons with the p.R158H PDK3 mutation.

Charcot-Marie-Tooth (CMT) is a group of inherited diseases clinically and genetically heterogenous, characterised by length dependent degeneration of axons of the peripheral nervous system. A missense mutation (p.R158H) in the pyruvate dehydrogenase kinase 3 gene (PDK3) has been identified as the genetic cause for an X-linked form of CMT (CMTX6) in two unrelated families. PDK3 is one of four PDK isoenzymes that regulate the activity of the pyruvate dehydrogenase complex (PDC). The balance between kinases (PDKs) and phosphatases (PDPs) determines the extend of oxidative decarboxylation of pyruvate to generate acetyl CoA, critically linking glycolysis and the energy producing Krebs cycle. We had shown the p.R158H mutation causes hyperactivity of PDK3 and CMTX6 fibroblasts show hyperphosphorylation of PDC, leading to reduced PDC activity and ATP production. In this manuscript we have generated induced pluripotent stem cells (iPSCs) by re-programming CMTX6 fibroblasts (iPSCCMTX6). We also have engineered an isogenic control (iPSCisogenic) and demonstrated that genetic correction of the p.R158H mutation reverses the CMTX6 phenotype. Patient-derived motor neurons (MNCMTX6) show increased phosphorylation of the PDC, energy metabolism defects and mitochondrial abnormalities, including reduced velocity of trafficking mitochondria in the affected axons. Treatment of the MNCMTX6 with a PDK inhibitor reverses PDC hyperphosphorylation and the associated functional deficits founds in the patient motor neurons, demonstrating that the MNCMTX6 and MNisogenic motor neurons provide an excellent neuronal system for compound screening approaches to identify drugs for the treatment of CMTX6.

Quantitative muscle ultrasound as a biomarker in Charcot-Marie-Tooth neuropathy.

OBJECTIVE: The utility of quantitative muscle ultrasound as a marker of disease severity in Charcot-Marie-Tooth (CMT) disease subtypes was investigated. METHODS: Muscle ultrasound was prospectively performed on 252 individual muscles from 21 CMT patients (9 CMT1A, 8 CMTX1, 4 CMT2A) and compared to 120 muscles from 10 age and gender-matched controls. Muscle ultrasound recorded echogenicity and thickness in representative muscles including first dorsal interosseus (FDI) and tibialis anterior (TA). RESULTS: Muscle volume of FDI and thickness of TA correlated with MRC strength. Muscle echogenicity was significantly increased in FDI (65.05 vs 47.09; p<0.0001) and TA (89.45 vs 66.30; p<0.0001) of CMT patients. In TA, there was significantly higher muscle thickness (23 vs 18 vs 16mm; p<0.0001) and lower muscle echogenicity (80 vs 95 vs 108; p<0.0001) in CMT1A compared to CMTX1 and CMT2A. This corresponded to disease severity based on muscle strength (MRC grading CMT1A vs CMTX1 vs CMT2A: 59 vs 48 vs 44; p=0.002). CONCLUSION: In CMT, quantitative muscle ultrasound of FDI and TA is a useful marker of disease severity. SIGNIFICANCE: The current findings suggest that quantitative muscle ultrasound has potential as a surrogate marker of disease progression in future interventional trials in CMT.

Mutation analysis of genes within the dynactin complex in a cohort of hereditary peripheral neuropathies.

The cytoplasmic dynein-dynactin genes are attractive candidates for neurodegenerative disorders given their functional role in retrograde transport along neurons. The cytoplasmic dynein heavy chain (DYNC1H1) gene has been implicated in various neurodegenerative disorders, and dynactin 1 (DCTN1) genes have been implicated in a wide spectrum of disorders including motor neuron disease, Parkinson's disease, spinobulbar muscular atrophy and hereditary spastic paraplegia. However, the involvement of other dynactin genes with inherited peripheral neuropathies (IPN) namely, hereditary sensory neuropathy, hereditary motor neuropathy and Charcot-Marie-Tooth disease is under reported. We screened eight genes; DCTN1-6 and ACTR1A and ACTR1B in 136 IPN patients using whole-exome sequencing and high-resolution melt (HRM) analysis. Eight non-synonymous variants (including one novel variant) and three synonymous variants were identified. Four variants have been reported previously in other studies, however segregation analysis within family members excluded them from causing IPN in these families. No variants of disease significance were identified in this study suggesting the dynactin genes are unlikely to be a common cause of IPNs. However, with the ease of querying gene variants from exome data, these genes remain worthwhile candidates to assess unsolved IPN families for variants that may affect the function of the proteins.

Proof of genetic heterogeneity in X-linked Charcot-Marie-Tooth disease.

OBJECTIVE: To characterize a large family with X-linked Charcot-Marie-Tooth (CMT) neuropathy without mutations in the gap junction protein B1 (GJB1) gene, which has an unusual phenotype that is different in some aspects from classic CMTX1. METHODS: We tested CMT families consistent with X-linked inheritance for GJB1 mutations. We compared the largest family (CMT623) without GJB1 mutation and with linkage excluding the CMTX1 locus to CMTX1 and normal individuals. RESULTS: Only 51% of probable X-linked CMT families had mutations in GJB1. Family CMT623 shows linkage to Xq26.3-q27.1 (lod score z = 6.58), a region within the previously identified locus for CMTX3, Xq26-q28. Unlike CMTX1, affected males in family CMT623 report pain and paraesthesia before the onset of sensory loss, and women are usually asymptomatic. As in CMTX1, affected males have widely ranging intermediate motor conduction velocities. The coding regions of 14 positional candidate genes within the narrowed CMTX3 locus have been excluded for a pathogenic role in the disease. CONCLUSION: This study is the first to confirm the CMTX3 locus and to refine the genetic interval to a 5.7-Mb region flanked by the markers DXS1041 and DXS8106. GJB1 mutation-negative forms of X-linked CMT, such as CMTX3, may account for a significant proportion of X-linked CMT.

A rapid and definitive test for Charcot-Marie-Tooth 1A and hereditary neuropathy with liability to pressure palsies using multiplexed real-time PCR.

Alterations in gene copy number have been shown to cause disease in humans. Two of the most common inherited peripheral neuropathies, Charcot-Marie-Tooth 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP), are two such diseases resulting from alteration in gene copy number of the dosage sensitive peripheral myelin protein 22 (PMP22) gene. Many complicated and laborious diagnostic tests exist for the diagnosis of these diseases. The aim of our study was to develop the first quantitative multiplex real-time PCR assay for the diagnosis of CMT1A and HNPP. A total of 160 individuals who were known to have CMT1A, HNPP, or were normal from previous testing were assayed by our multiplex real-time PCR method. The results confirmed the previously determined gene copy number of all patient and control individuals tested. The range of ratio values between the disease and control groups were easily defined. The assay is accurate, simple, and cost effective and can detect a 50% change in gene copy number. This represents an ideal assay for any small diagnostic laboratory.

A locus for hereditary sensory neuropathy with cough and gastroesophageal reflux on chromosome 3p22-p24.

Hereditary sensory neuropathy type I (HSN I) is a group of dominantly inherited degenerative disorders of peripheral nerve in which sensory features are more prominent than motor involvement. We have described a new form of HSN I that is associated with cough and gastroesophageal reflux. To map the chromosomal location of the gene causing the disorder, a 10-cM genome screen was undertaken in a large Australian family. Two-point analysis showed linkage to chromosome 3p22-p24 (Zmax=3.51 at recombination fraction (theta) 0.0 for marker D3S2338). A second family with a similar phenotype shares a different disease haplotype but segregates at the same locus. Extended haplotype analysis has refined the region to a 3.42-cM interval, flanked by markers D3S2336 and D3S1266.

A new autosomal dominant pure cerebellar ataxia.

A kindred is described with a dominantly inherited "pure" cerebellar ataxia in which the currently known spinocerebellar ataxias have been excluded. In the eight subjects studied, a notable clinical feature is slow progression, with the three least affected having only a mild degree of gait ataxia after three or more decades of disease duration. Pending an actual chromosomal locus discovery, the name spinocerebellar ataxia (SCA)15 is expectantly applied.

Dominant intermediate Charcot-Marie-Tooth neuropathy maps to chromosome 19p12-p13.2.

The hereditary disorders of peripheral nerve form one of the most common groups of human genetic diseases, collectively called Charcot-Marie-Tooth (CMT) neuropathy. Using linkage analysis we have identified a new locus for a form of CMT that we have called "dominant intermediate CMT" (DI-CMT). A genomewide screen using 383 microsatellite markers showed strong linkage to the short arm of chromosome 19 (maximum LOD score 4.3, with a recombination fraction (straight theta) of 0, at D19S221 and maximum LOD score 5.28, straight theta=0, at D19S226). Haplotype analysis performed with 14 additional markers placed the DI-CMT locus within a 16.8-cM region flanked by the markers D19S586 and D19S546. Multipoint linkage analysis suggested the most likely location at D19S226 (maximum multipoint LOD score 6.77), within a 10-cM confidence interval. This study establishes the presence of a locus for DI-CMT on chromosome 19p12-p13.2.

Genomic structure and physical mapping of C17orf1: a gene associated with the proximal element of the CMT1A-REP binary repeat.

C17orf1, a gene expressed in skeletal muscle and heart, was initially isolated from a fetal brain cDNA library and localized centromeric to and partially within the proximal CMT1A-REP element. A second gene, COX10, spans the distal CMT1A-REP element, and a duplicated exon of this gene is present in the proximal CMT1A-REP element. C17orf1 includes this duplicated COX10 exon within its sequence; however, the DNA strand opposite to that of the COX10 gene is utilized. We have determined the genomic organization of C17orf1 and found it to be oriented in the direction opposite to COX10. Analysis of the genomic structure of C17orf1 has revealed that it contains at least six exons and spans a length of at least 17 kb. All but one of the splice sites conform to the GT/AG rule, and in this case the splice acceptor site within intron 1 is GA instead of the expected AG. Sequencing and mapping analyses have shown that the centromeric boundary of the proximal CMT1A-REP element lies within intron 5. A 7-bp insertion, identified from genomic sequencing of cosmid clones and verified in the original cDNA clone and RT-PCR products, has extended the previously reported open reading frame from 591 to 756 bp. C17orf1 therefore encodes a 252-amino-acid protein.

The Charcot-Marie-Tooth binary repeat contains a gene transcribed from the opposite strand of a partially duplicated region of the COX10 gene.

Misalignment between the two elements of the CMT1A-REP binary repeat on chromosome 17p11.2-p12 causes two inherited peripheral neuropathies, Charcot-Marie-Tooth type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies. This binary repeat contains repetitive DNA elements that include LINES, SINES, medium reiteration frequency repeats, and a transposon-like element. The COX10 gene has been mapped 10 kb centromeric to the distal CMT1A-REP element, and a portion of this gene is present in both the proximal and the distal CMT1A-REP elements. We report the isolation and characterization of a novel cDNA (C170RF1), which maps centromeric to and partially within the proximal CMT1A-REP element. Part of C170RF1 is transcribed from the opposite strand of the COX10 partial gene duplication present in the proximal CMT1A-REP element. This finding shows that C170RF1 and COX10 are being transcribed from opposite strands of identical DNA sequences that are separated by 1.5 Mb in the genome. RT-PCR analysis showed the proximal transcript was expressed in skeletal muscle. Sequence analysis identified an open reading frame encoding a 199-amino-acid protein. Zoo blot analysis showed that the transcript is conserved in nonhuman primates. The presence of a binary repeat contributes to the instability of this region of chromosome 17, yet two CMT1A-REP elements are present in the chimpanzee and all human populations. The presence of expressed sequences in both elements of the CMT1A-REP binary repeat could explain the maintenance of this repeat in humans.

The gene for hereditary sensory neuropathy type I (HSN-I) maps to chromosome 9q22.1-q22.3.

Hereditary sensory neuropathy type I (HSN-I, also known as hereditary sensory and autonomic neuropathy type I (HSAN-I), or hereditary sensory radicular neuropathy) is an autosomal dominant disorder that is the most common of a group of degenerative disorders of sensory neurons. HSN-I was initially recognized as a disease that produced mutilating ulceration leading to amputation of digits (Fig. 1). It was given names such as familial ulcers with mutilating lesions of the extremities and perforating ulcers with osseous atrophy. The disease involves a progressive degeneration of dorsal root ganglion and motor neurons, leading to distal sensory loss and later distal muscle wasting and weakness and variable neural deafness. Sensory deficits include loss of all modalities, particularly loss of sensation to pain and temperature. Skin injuries may lead to chronic skin ulcers, osteomyelitis, and extrusion of bone fragments, especially the metatarsals. Onset of symptoms is in the second or later decades. We undertook a genome screen using linkage analysis in four Australian HSN-I kindreds. We now show that the HSN1 gene maps to an 8-centiMorgan (cM) region flanked by D9S318 and D9S176 on chromosome 9q22.1-q22.3. Multipoint linkage analysis suggests a most likely location at D9S287, within a 4.9-cM confidence interval.

Detection of Charcot-Marie-Tooth type 1A duplication by the polymerase chain reaction.

Charcot-Marie-Tooth disease type 1A (CMT1A) is a hereditary peripheral neuropathy with a genetic locus on chromosome 17p11.2. The majority of patients carry a duplicated DNA segment that encompasses the gene PMP22, which encodes a peripheral myelin protein. PMP22 is the crucial gene involved in the pathogenesis of CMT1A. Molecular diagnosis of CMT1A requires detection of this duplicated segment. Existing methods for detection of the duplication are laborious and time consuming. We have developed a set of polymorphic (AC)n repeat markers (contained within the duplication) for use in the polymerase chain reaction, which give a high probability of detecting three unique alleles in affected individuals. This test detected 85% of a panel of 52 CMT1A patients in which the duplication had previously been demonstrated.

Phenotypic expression of benign familial neonatal convulsions linked to chromosome 20.

OBJECTIVES: To determine whether the syndrome of benign familial neonatal convulsions in a large family was linked to markers on chromosome 20q and to study the seizure patterns in affected individuals. DESIGN: A clinical and molecular biologic study of a single large family in which the probands were identical twins with benign familial neonatal convulsions. PATIENTS: Thirteen living affected family members and 27 living unaffected family members were evaluated. RESULTS: Multipoint linkage analysis with use of the chromosome 20q markers CMM6 and RMR6 gave a maximum lod score of 3.13 at theta = 0.063, indicating linkage in this family. Of the 13 affected members, 10 had known neonatal seizures. Four subjects had febrile seizures, of whom only two had known neonatal seizures. Two members had afebrile seizures later, one of whom had not previously suffered neonatal or febrile seizures. CONCLUSION: The phenotypic heterogeneity in this family, with an epilepsy syndrome determined by a single gene, was striking. This suggests that molecular genetic approaches to the common forms of idiopathic epilepsy, involving patients with clinically similar phenotypes from unrelated families, may be inappropriate.

A frame shift mutation in the PMP22 gene in hereditary neuropathy with liability to pressure palsies.

Hereditary neuropathy with liability to pressure palsies (HNPP) has been a associated with a deletion of 1.5 megabases of chromosome 17p. One of four biopsy proven HNPP families that we have studied did not possess this deletion. As the deleted DNA region includes the coding region for a peripheral myelin gene (PMP22), we used single strand conformation analysis to examine this gene for mutations in the non-deleted HNPP family. An abnormal fragment in exon 1 was identified, and sequencing revealed a two base pair deletion in all affected family members. The deletion results in a frame shift, providing strong evidence that this gene has an important role in the pathogenesis of the disease.

Charcot-Marie-Tooth neuropathy type 1A mutation: apparent crossovers with D17S122 are due to a duplication.

A locus for the slow conducting form of Charcot-Marie-Tooth neuropathy (CMT1A) was localised to the proximal short arm of chromosome 17, in band p11.2, distal to D17S58. Linkage studies of CMT1A in 3 large Australian families with the marker loci D17S58, D17S71, and D17S57 suggested the order, pter-CMT1A-D17S71-D17S58-centromere-D17S57. However, the estimate of the recombination fraction between CMT1A and D17S122, also assigned to p11.2, was incompatible with known map distances. The impasse was resolved when the D17S122 genotypes were revised to take into account a dosage effect due to a duplication. After correction of the genotypes, the maximum lod score between CMT1A and D17S122 increased from 0.53 at a recombination fraction of 0.3 to 34.28 at zero recombination. This result emphasizes that genotypes for markers in the p12-p11.2 region should be examined very carefully as ignoring the duplication changes the linkage results dramatically. The fact that no crossovers were found between CMT1A and D17S122 suggests that the duplication may cause the disease phenotype.