An Organismal CNV Mutator Phenotype Restricted to Early Human Development.

De novo copy number variants (dnCNVs) arising at multiple loci in a personal genome have usually been considered to reflect cancer somatic genomic instabilities. We describe a multiple dnCNV (MdnCNV) phenomenon in which individuals with genomic disorders carry five to ten constitutional dnCNVs. These CNVs originate from independent formation incidences, are predominantly tandem duplications or complex gains, exhibit breakpoint junction features reminiscent of replicative repair, and show increased de novo point mutations flanking the rearrangement junctions. The active CNV mutation shower appears to be restricted to a transient perizygotic period. We propose that a defect in the CNV formation process is responsible for the "CNV-mutator state," and this state is dampened after early embryogenesis. The constitutional MdnCNV phenomenon resembles chromosomal instability in various cancers. Investigations of this phenomenon may provide unique access to understanding genomic disorders, structural variant mutagenesis, human evolution, and cancer biology.

The role of combined SNV and CNV burden in patients with distal symmetric polyneuropathy.

PURPOSE: Charcot-Marie-Tooth (CMT) disease is a heterogeneous group of genetic disorders of the peripheral nervous system. Copy-number variants (CNVs) contribute significantly to CMT, as duplication of PMP22 underlies the majority of CMT1 cases. We hypothesized that CNVs and/or single-nucleotide variants (SNVs) might exist in patients with CMT with an unknown molecular genetic etiology. METHODS: Two hundred patients with CMT, negative for both SNV mutations in several CMT genes and for CNVs involving PMP22, were screened for CNVs by high-resolution oligonucleotide array comparative genomic hybridization. Whole-exome sequencing was conducted on individuals with rare, potentially pathogenic CNVs. RESULTS: Putatively causative CNVs were identified in five subjects (~2.5%); four of the five map to known neuropathy genes. Breakpoint sequencing revealed Alu-Alu-mediated junctions as a predominant contributor. Exome sequencing identified MFN2 SNVs in two of the individuals. CONCLUSION: Neuropathy-associated CNV outside of the PMP22 locus is rare in CMT. Nevertheless, there is potential clinical utility in testing for CNVs and exome sequencing in CMT cases negative for the CMT1A duplication. These findings suggest that complex phenotypes including neuropathy can potentially be caused by a combination of SNVs and CNVs affecting more than one disease-associated locus and contributing to a mutational burden.Genet Med 18 5, 443-451.

The Variant p.(Arg183Trp) in SPTLC2 Causes Late-Onset Hereditary Sensory Neuropathy.

Hereditary sensory and autonomic neuropathy 1 (HSAN1) is an autosomal dominant disorder that can be caused by variants in SPTLC1 or SPTLC2, encoding subunits of serine palmitoyl-CoA transferase. Disease variants alter the enzyme's substrate specificity and lead to accumulation of neurotoxic 1-deoxysphingolipids. We describe two families with autosomal dominant HSAN1C caused by a new variant in SPTLC2, c.547C>T, p.(Arg183Trp). The variant changed a conserved amino acid and was not found in public variant databases. All patients had a relatively mild progressive distal sensory impairment, with onset after age 50. Small fibers were affected early, leading to abnormalities on quantitative sensory testing. Sural biopsy revealed a severe chronic axonal neuropathy with subtotal loss of myelinated axons, relatively preserved number of non-myelinated fibers and no signs for regeneration. Skin biopsy with PGP9.5 labeling showed lack of intraepidermal nerve endings early in the disease. Motor manifestations developed later in the disease course, but there was no evidence of autonomic involvement. Patients had elevated serum 1-deoxysphingolipids, and the variant protein produced elevated amounts of 1-deoxysphingolipids in vitro, which proved the pathogenicity of the variant. Our results expand the genetic spectrum of HSAN1C and provide further detail about the clinical characteristics. Sequencing of SPTLC2 should be considered in all patients presenting with mild late-onset sensory-predominant small or large fiber neuropathy.

Loss of function mutations in HARS cause a spectrum of inherited peripheral neuropathies.

Inherited peripheral neuropathies are a genetically heterogeneous group of disorders characterized by distal muscle weakness and sensory loss. Mutations in genes encoding aminoacyl-tRNA synthetases have been implicated in peripheral neuropathies, suggesting that these tRNA charging enzymes are uniquely important for the peripheral nerve. Recently, a mutation in histidyl-tRNA synthetase (HARS) was identified in a single patient with a late-onset, sensory-predominant peripheral neuropathy; however, the genetic evidence was lacking, making the significance of the finding unclear. Here, we present clinical, genetic, and functional data that implicate HARS mutations in inherited peripheral neuropathies. The associated phenotypic spectrum is broad and encompasses axonal and demyelinating motor and sensory neuropathies, including four young patients presenting with pure motor axonal neuropathy. Genome-wide linkage studies in combination with whole-exome and conventional sequencing revealed four distinct and previously unreported heterozygous HARS mutations segregating with autosomal dominant peripheral neuropathy in four unrelated families (p.Thr132Ile, p.Pro134His, p.Asp175Glu and p.Asp364Tyr). All mutations cause a loss of function in yeast complementation assays, and p.Asp364Tyr is dominantly neurotoxic in a Caenorhabditis elegans model. This study demonstrates the role of HARS mutations in peripheral neuropathy and expands the genetic and clinical spectrum of aminoacyl-tRNA synthetase-related human disease.

Transcriptional regulator PRDM12 is essential for human pain perception.

Pain perception has evolved as a warning mechanism to alert organisms to tissue damage and dangerous environments. In humans, however, undesirable, excessive or chronic pain is a common and major societal burden for which available medical treatments are currently suboptimal. New therapeutic options have recently been derived from studies of individuals with congenital insensitivity to pain (CIP). Here we identified 10 different homozygous mutations in PRDM12 (encoding PRDI-BF1 and RIZ homology domain-containing protein 12) in subjects with CIP from 11 families. Prdm proteins are a family of epigenetic regulators that control neural specification and neurogenesis. We determined that Prdm12 is expressed in nociceptors and their progenitors and participates in the development of sensory neurons in Xenopus embryos. Moreover, CIP-associated mutants abrogate the histone-modifying potential associated with wild-type Prdm12. Prdm12 emerges as a key factor in the orchestration of sensory neurogenesis and may hold promise as a target for new pain therapeutics.

A Costa Rican family affected with Charcot-Marie-Tooth disease due to the myelin protein zero (MPZ) p.Thr124Met mutation shares the Belgian haplotype / Una familia costarricense afectada por la enfermedad de Charcot-Marie-Tooth, debido a la mutación p.thr124Met en la proteína mielina cero (MPZ) compartida con el haplotipo belga

The p.Thr124Met mutation in the myelin protein zero (MPZ) causes the Charcot-Marie-Tooth disease type 2J, a peripheral neuropathy with additional symptoms as pupillary alterations and deafness. It was observed in several families around the world originating e. g. from Germany, Belgium, Japan, Italy and North America. Here we report Central American patients originating from a family in Costa Rica carrying this mutation. Clinical, electrophysiological and molecular analysis of patients and controls were performed, including gene and linked markers´ sequencing. Carriers share almost the entire haplotype with two non related Belgian CMT patients. As a result of the haplotype analysis, based on ten markers (seven SNPs, two microsatellites and an intronic polyA stretch), the founder effect hypothesis for this allele migration is suggestive. Rev. Biol. Trop. 62 (4): 1285-1293. Epub 2014 December 01.

La mutación p.thr124Met en la proteína mielina cero (MPZ) causa la enfermedad de Charcot-Marie-Tooth tipo 2J, una neuropatía periférica con síntomas adicionales como alteraciones pupilares y sordera. Se ha observado en varias familias alrededor del mundo, originarias de Alemania, Bélgica, Japón, Italia y Norteamérica, entre otras. Aquí reportamos a pacientes centroamericanos provenientes de Costa Rica que acarrean esta mutación. Se realizaron análisis clínico, electrofisiológico y molecular de pacientes y controles, incluyendo secuenciación del gen y de marcadores ligados a éste. Estos pacientes comparten casi por completo el haplotipo con dos pacientes belgas no emparentados. Como resultado del análisis de los haplotipos, basado en diez marcadores (siete SNPs, dos microsatélites y un elemento poli-A intrónico), se sugiere que se ha dado un efecto fundador en la migración de este alelo.

Selected items from the Charcot-Marie-Tooth (CMT) Neuropathy Score and secondary clinical outcome measures serve as sensitive clinical markers of disease severity in CMT1A patients.

This study evaluates primary and secondary clinical outcome measures in Charcot-Marie-Tooth disease type 1A (CMT1A) with regard to their contribution towards discrimination of disease severity. The nine components of the composite Charcot-Marie-Tooth disease Neuropathy Score and six additional secondary clinical outcome measures were assessed in 479 adult patients with genetically proven CMT1A and 126 healthy controls. Using hierarchical clustering, we identified four significant clusters of patients according to clinical severity. We then tested the impact of each of the CMTNS components and of the secondary clinical parameters with regard to their power to differentiate these four clusters. The CMTNS components ulnar sensory nerve action potential (SNAP), pin sensibility, vibration and strength of arms did not increase the discriminant value of the remaining five CMTNS components (Ulnar compound motor action potential [CMAP], leg motor symptoms, arm motor symptoms, leg strength and sensory symptoms). However, three of the six additional clinical outcome measures - the 10m-timed walking test (T10MW), 9 hole-peg test (9HPT), and foot dorsal flexion dynamometry - further improved discrimination between severely and mildly affected patients. From these findings, we identified three different composite measures as score hypotheses and compared their discriminant power with that of the CMTNS. A composite of eight components CMAP, Motor symptoms legs, Motor symptoms arms, Strength of Legs, Sensory symptoms), displayed the strongest power to discriminate between the clusters. As a conclusion, five items from the CMTNS and three secondary clinical outcome measures improve the clinical assessment of patients with CMT1A significantly and are beneficial for upcoming clinical and therapeutic trials.

The instability of the BTB-KELCH protein Gigaxonin causes Giant Axonal Neuropathy and constitutes a new penetrant and specific diagnostic test.

BACKGROUND: The BTB-KELCH protein Gigaxonin plays key roles in sustaining neuron survival and cytoskeleton architecture. Indeed, recessive mutations in the Gigaxonin-encoding gene cause Giant Axonal Neuropathy (GAN), a severe neurodegenerative disorder characterized by a wide disorganization of the Intermediate Filament network. Growing evidences suggest that GAN is a continuum with the peripheral neuropathy Charcot-Marie-Tooth diseases type 2 (CMT2). Sharing similar sensory-motor alterations and aggregation of Neurofilaments, few reports have revealed that GAN and some CMT2 forms can be misdiagnosed on clinical and histopathological examination. The goal of this study is to propose a new differential diagnostic test for GAN/CMT2. Moreover, we aim at identifying the mechanisms causing the loss-of-function of Gigaxonin, which has been proposed to bind CUL3 and substrates as part of an E3 ligase complex. RESULTS: We establish that determining Gigaxonin level constitutes a very valuable diagnostic test in discriminating new GAN cases from clinically related inherited neuropathies. Indeed, in a set of seven new families presenting a neuropathy resembling GAN/CMT2, only five exhibiting a reduced Gigaxonin abundance have been subsequently genetically linked to GAN. Generating the homology modeling of Gigaxonin, we suggest that disease mutations would lead to a range of defects in Gigaxonin stability, impairing its homodimerization, BTB or KELCH domain folding, or CUL3 and substrate binding. We further demonstrate that regardless of the mutations or the severity of the disease, Gigaxonin abundance is severely reduced in all GAN patients due to both mRNA and protein instability mechanisms. CONCLUSIONS: In this study, we developed a new penetrant and specific test to diagnose GAN among a set of individuals exhibiting CMT2 of unknown etiology to suggest that the prevalence of GAN is probably under-evaluated among peripheral neuropathies. We propose to use this new test in concert with the clinical examination and prior to the systematic screening of GAN mutations that has shown strong limitations for large deletions. Combining the generation of the structural modeling of Gigaxonin to an analysis of Gigaxonin transcripts and proteins in patients, we provide the first evidences of the instability of this E3 ligase adaptor in disease.

Clinical spectrum in three families with familial hemiplegic migraine type 2 including a novel mutation in the ATP1A2 gene.

INTRODUCTION: Familial hemiplegic migraine (FHM) is a rare subtype of migraine with transient hemiplegic aura. PATIENTS AND METHODS: We describe three unrelated families with familial hemiplegic migraine type II (FHM2). Retrospectively, information on 47 family members could be obtained, 15 by personal examination and 32 by indirect anamnesis from relatives. Genetic analyses were performed in 13 patients. RESULTS: One family had a novel missense mutation in the ATP1A2 gene (c.659C>T, p.Ser220Leu) that segregated with the phenotype in three generations. Two further unrelated families with different ethnic backgrounds (one from Germany and one from Russia) had a missense mutation that has not been described as yet in FHM, but occurred in only a single patient with sporadic hemiplegic migraine (c.2723G>A, p.Arg908Gln). Clinically the patients had severe attacks lasting up to several weeks as well as epileptic seizures. Three patients with a proven mutation in the ATP1A2 gene clinically presented without hemiparesis. Furthermore, there was a possible relation of FHM2 to mental retardation in another two patients. CONCLUSION: Clinical symptoms may last for several weeks in some patients. Patients with FHM2 may also present without hemiplegia. Therefore, the full family history has to be taken into account to establish the diagnosis of FHM.

A Costa Rican family affected with Charcot-Marie-Tooth disease due to the myelin protein zero (MPZ) p.Thr124Met mutation shares the Belgian haplotype.

The p.Thr124Met mutation in the myelin protein zero (MPZ) causes the Charcot-Marie-Tooth disease type 2J, a peripheral neuropathy with additional symptoms as pupillary alterations and deafness. It was observed in several families around the world originating e. g. from Germany, Belgium, Japan, Italy and North America. Here we report Central American patients originating from a family in Costa Rica carrying this mutation. Clinical, electrophysiological and molecular analysis of patients and controls were performed, including gene and linked markers' sequencing. Carriers share almost the entire haplotype with two non related Belgian CMT patients. As a result of the haplotype analysis, based on ten markers (seven SNPs, two microsatellites and an intronic polyA stretch), the founder effect hypothesis for this allele migration is suggestive.

Facioscapulohumeral muscular dystrophy and Charcot-Marie-Tooth neuropathy 1A - evidence for "double trouble" overlapping syndromes.

BACKGROUND: We report on a patient with genetically confirmed overlapping diagnoses of CMT1A and FSHD. This case adds to the increasing number of unique patients presenting with atypical phenotypes, particularly in FSHD. Even if a mutation in one disease gene has been found, further genetic testing might be warranted in cases with unusual clinical presentation. CASE PRESENTATION: The reported 53 years old male patient suffered from walking difficulties and foot deformities first noticed at age 20. Later on, he developed scapuloperoneal and truncal muscle weakness, along with atrophy of the intrinsic hand and foot muscles, pes cavus, claw toes and a distal symmetric hypoesthesia. Motor nerve conduction velocities were reduced to 20 m/s in the upper extremities, and not educible in the lower extremities, sensory nerve conduction velocities were not attainable. Electromyography showed both, myopathic and neurogenic changes. A muscle biopsy taken from the tibialis anterior muscle showed a mild myopathy with some neurogenic findings and hypertrophic type 1 fibers. Whole-body muscle MRI revealed severe changes in the lower leg muscles, tibialis anterior and gastrocnemius muscles were highly replaced by fatty tissue. Additionally, fatty degeneration of shoulder girdle and straight back muscles, and atrophy of dorsal upper leg muscles were seen. Taken together, the presenting features suggested both, a neuropathy and a myopathy. Patient's family history suggested an autosomal dominant inheritance.Molecular testing revealed both, a hereditary motor and sensory neuropathy type 1A (HMSN1A, also called Charcot-Marie-Tooth neuropathy 1A, CMT1A) due to a PMP22 gene duplication and facioscapulohumeral muscular dystrophy (FSHD) due to a partial deletion of the D4Z4 locus (19 kb). CONCLUSION: Molecular testing in hereditary neuromuscular disorders has led to the identification of an increasing number of atypical phenotypes. Nevertheless, finding the right diagnosis is crucial for the patient in order to obtain adequate medical care and appropriate genetic counseling, especially in the background of arising curative therapies.

Genetic and phenotypic variability of optic neuropathies.

Hereditary optic neuropathies comprise a group of clinically and genetically heterogeneous disorders. Two subgroups can be formed: isolated hereditary optic atrophies and optic neuropathy as part of complex disorders. In group 1 of hereditary optic neuropathies, optic nerve dysfunction is typically the only manifestation of the disease. This group comprises autosomal dominant, autosomal recessive and X-linked recessive optic atrophy and the maternally inherited Leber's hereditary optic neuropathy. Among the autosomal-dominant forms of optic atrophy, Kjer's disease is most frequently observed. In the second group of complex disorders, various neurologic and other systemic abnormalities are regularly observed. Most frequent in this group are mtDNA mutations, inherited peripheral neuropathies, Charcot-Marie-Tooth disorders (CMT2A2, CMTX5), hereditary sensory neuropathy type 3 (HSAN3), Friedreich's ataxia, leukodystrophies, sphingolipidoses, ceroid-lipofuscinoses and neurodegeneration with brain iron accumulation. We review current knowledge about the underlying genetic predispositions, the most urgent open questions and how this may affect our management of this heterogeneous group of disorders in the future.

Charcot-Marie-Tooth neuropathy due to a novel EGR2 gene mutation with mild phenotype--usefulness of human mapping chip linkage analysis in a Czech family.

Charcot-Marie-Tooth neuropathies (CMT) are a group of clinically and genetically heterogeneous disorders of the peripheral nervous system. Selection of candidate disease genes for mutation analysis is sometimes difficult since more than 40 genes and loci are known to be associated with CMT neuropathies. Hence a Czech family Cz-CMT with demyelinating type of autosomal dominant CMT disease was investigated by genome-wide linkage analysis by means of single-nucleotide polymorphism (SNP) arrays. Among 35 regions with linkage, five carried known CMT genes. In the final result a novel early growth response 2 - missense mutation c.1235 A>G, p.Glu412Gly was found. Surprisingly, the more severely affected proband carried an additional heterozygous myelin protein zero variant p.Asp246Asn detected previously, which may modify the phenotype. However, this MPZ variant is benign in heterozygous state alone, because it is also carried by the patient's healthy father.

A new phenotype of brain iron accumulation with dystonia, optic atrophy, and peripheral neuropathy.

BACKGROUND: Neurodegeneration with brain iron accumulation is clinically and genetically heterogeneous because of mutations in at least 7 nuclear genes. METHODS: We performed homozygosity mapping and whole-exome sequencing in 2 brothers with brain iron accumulation from a consanguineous family. RESULTS: We identified a homozygous missense mutation in both brothers in the very recently identified chromosome 19 open-reading frame 12 gene. The disease presented before age 10 with slowly progressive tremor, dystonia, and spasticity. Additional features were optic atrophy, peripheral neuropathy, and learning difficulties. A raised serum creatine kinase indicated neuromuscular involvement, and compensatory mitochondrial proliferation implicated mitochondrial dysfunction as a pathological mechanism. CONCLUSIONS: Further studies are needed to explore the function of the chromosome 19 open-reading frame 12 gene, and extended genetic analysis on larger patient cohorts will provide more information about the presentation and frequency of this disease.

A novel mutation of myelin protein zero associated with late-onset predominantly axonal Charcot-Marie-Tooth disease.

We report a case of late-onset predominantly axonal Charcot-Marie-Tooth disease resulting from a novel mutation in the MPZ gene encoding myelin protein zero (P0). Neurological examination, electrophysiological examination and genetic testing were performed on three members of a Finnish family (family A) and one member of a German family (family B). Three other members of the Finnish family were interviewed and genetically tested. Genetic testing was also performed on 95 healthy Finnish controls. Three members in two generations of family A and the member of family B were affected with late-onset axonal more than demyelinating, motor and sensory polyneuropathy. Heterozygous c.316C>T mutation in MPZ leading to p.Arg106Cys in P0 was found in all the affected subjects, but not in the three unaffected members of the Finnish family. None of 95 healthy Finnish controls harbored the mutation. The findings of this study indicate that p.Arg106Cys allele in MPZ causes late-onset predominantly axonal sensory and motor neuropathy.

A rat model of Charcot-Marie-Tooth disease 1A recapitulates disease variability and supplies biomarkers of axonal loss in patients.

Charcot-Marie-Tooth disease is the most common inherited neuropathy and a duplication of the peripheral myelin protein 22 gene causes the most frequent subform Charcot-Marie-Tooth 1A. Patients develop a slowly progressive dysmyelinating and demyelinating peripheral neuropathy and distally pronounced muscle atrophy. The amount of axonal loss determines disease severity. Although patients share an identical monogenetic defect, the disease progression is strikingly variable and the impending disease course can not be predicted in individual patients. Despite promising experimental data, recent therapy trials have failed. Established clinical outcome measures are thought to be too insensitive to detect amelioration within trials. Surrogate biomarkers of disease severity in Charcot-Marie-Tooth 1A are thus urgently needed. Peripheral myelin protein 22 transgenic rats harbouring additional copies of the peripheral myelin protein 22 gene ('Charcot-Marie-Tooth rats'), which were kept on an outbred background mimic disease hallmarks and phenocopy the variable disease severity of patients with Charcot-Marie-Tooth 1A. Hence, we used the Charcot-Marie-Tooth rat to dissect prospective and surrogate markers of disease severity derived from sciatic nerve and skin tissue messenger RNA extracts. Gene set enrichment analysis of sciatic nerve transcriptomes revealed that dysregulation of lipid metabolism associated genes such as peroxisome proliferator-activated receptor gamma constitutes a modifier of present and future disease severity. Importantly, we directly validated disease severity markers from the Charcot-Marie-Tooth rats in 46 patients with Charcot-Marie-Tooth 1A. Our data suggest that the combination of age and cutaneous messenger RNA levels of glutathione S-transferase theta 2 and cathepsin A composes a strong indicator of disease severity in patients with Charcot-Marie-Tooth 1A, as quantified by the Charcot-Marie-Tooth Neuropathy Score. This translational approach, utilizing a transgenic animal model, demonstrates that transcriptional analysis of skin biopsy is suitable to identify biomarkers of Charcot-Marie-Tooth 1A.

High frequency of mutations in the PIK3CA gene helical and kinase coding regions in a group of Iranian patients with high-grade glioblastomas: five novel mutations.

Glioblastoma multiform (GBM; World Health Organization (WHO) grade IV) and anaplastic astrocytomas (AA; WHO grade III) are highly aggressive and lethal astrocytic brain tumors. To detect cancer-specific somatic mutations in two hot-spot regions of PIK3CA gene, the helical and kinase domains (encoded by exons 9 and 20, respectively) in GBM and AA, the authors examined the respective sequences 31 paraffin-embedded samples (23 GBM and 8 AA). The samples were obtained from a group of Iranian patients affected with high-grade glioblastoma (HGG). The overall prevalence of PIK3CA mutations was 23% (7/31) for both tumor types (22% in GBM, and 25% in AA). Five mutations were detected in exon 20, p.Arg992Gln (c.2976G→A), p.Met1005Val (c.3014A→G), p.Ile1019→Val (c.3056A→G), p.Ser1008Cys (c.3024C→G), and p.Asn1044Asp (c.3130A→G), and one mutation in exon 9, p.Glu545Ala (c.1634A→C). Additionally exons 4-8 of P53 gene in four unrelated young patients, who showed no mutations in PIK3CA exons 9 and 20, were analyzed. Three mutations were identified: p.Pro72Ala (c.214C→G), g.11608G→T (homozygote splice mutation), and p.Thr170Thr (c.510G→A silent mutation). In conclusion, mutation detection in PIK3CA in patients with a high degree of malignancy and early age at diagnosis should be included in molecular diagnostic protocols, also with regard to possible upcoming therapies.