Unraveling the genetic landscape of autosomal recessive Charcot-Marie-Tooth neuropathies using a homozygosity mapping approach.

Autosomal recessive forms of Charcot-Marie-Tooth disease (ARCMT) are rare but severe disorders of the peripheral nervous system. Their molecular basis is poorly understood due to the extensive genetic and clinical heterogeneity, posing considerable challenges for patients, physicians, and researchers. We report on the genetic findings from a systematic study of a large collection of 174 independent ARCMT families. Initial sequencing of the three most common ARCMT genes (ganglioside-induced differentiation protein 1­GDAP1, SH3 domain and tetratricopeptide repeats-containing protein 2­SH3TC2, histidine-triad nucleotide binding protein 1­HINT1) identified pathogenic mutations in 41 patients. Subsequently, 87 selected nuclear families underwent single nucleotide polymorphism (SNP) genotyping and homozygosity mapping, followed by targeted screening of known ARCMT genes. This strategy provided molecular diagnosis to 22% of the families. Altogether, our unbiased genetic approach identified pathogenic mutations in ten ARCMT genes in a total of 41.3% patients. Apart from a newly described founder mutation in GDAP1, the majority of variants constitute private molecular defects. Since the gene testing was independent of the clinical phenotype of the patients, we identified mutations in patients with unusual or additional clinical features, extending the phenotypic spectrum of the SH3TC2 gene. Our study provides an overview of the ARCMT genetic landscape and proposes guidelines for tackling the genetic heterogeneity of this group of hereditary neuropathies.

Clinical phenotype of hereditary spastic paraplegia due to KIF1C gene mutations across life span.

Hereditary spastic paraplegias (HSPs) are a group of genetic disorders resulting in pyramidal tract impairment, predominantly in lower limbs. KIF1C gene has recently been identified as one of the genetic causes of HSP and associated with pure or complicated HSP. We present three patients with complicated HSP from two unrelated families, who had early onset progressive cerebellar signs and developed pyramidal tract signs during follow-up. Whole exome sequencing in these patients followed by segregation analysis identified novel truncating KIF1C mutations (c.463C> T; p.R155∗ and c.2478delA; p.Ala828Argfs∗13). Neuroimaging findings showed cerebral and upper cervical spinal atrophy, bilateral symmetrical pyramidal tract involvement, and focal cerebral white matter lesions. Patients with KIF1C mutations may present with cerebellar signs and pyramidal findings may emerge later, therefore complicated HSP should be considered in the differential diagnosis of unidentified cases with cerebellar dysfunction.

Encephalopathy due to carnitine deficiency in an adult patient with gluten enteropathy.

A 48-year-old male patient had two episodes of fever, headache, confusion and seizures following an upper respiratory tract infection. Electroencephalography (EEG) revealed diffuse slowing of background activity. Plasma free carnitine and serum lipid levels were low; fecal fat content and serum antigliadin antibodies were elevated. Duodenal biopsy was compatible with gluten enteropathy. Symptoms improved after the patient was started on a gluten-free diet and carnitine replacement therapy. No recurrence was observed within a four-year follow-up. Carnitine deficiency in adulthood is unusual, and encephalopathy due to carnitine deficiency as a result of celiac disease has not been described previously.

Four novel thymidine phosphorylase gene mutations in mitochondrial neurogastrointestinal encephalomyopathy syndrome (MNGIE) patients.

Mitochondrial neurogastrointestinal encephalomyopathy syndrome (MNGIE) is a rare autosomal recessive neurologic disorder characterised by multiple mitochondrial DNA deletions. In this study, five Turkish MNGIE patients are investigated for mtDNA deletions and TP gene mutations. The probands presented all the clinical criteria of the typical MNGIE phenotype; the muscle biopsy specimens also confirmed the diagnosis with ragged red fibres and cytochrome C oxidase (COX) negative fibres. The mitochondrial DNA analysis revealed no deletions in the probands' skeletal muscle samples. We have identified four novel mutations in the TP gene while one of the patients also harboured a nucleotide change, which was previously reported as a mutation.

A novel homozygous missense mutation in the myotubularin-related protein 2 gene associated with recessive Charcot-Marie-Tooth disease with irregularly folded myelin sheaths.

Mutations in the myotubularin-related protein 2 gene on chromosome 11q22 are known to cause autosomal recessive Charcot-Marie-Tooth disease with irregularly folded myelin sheaths. We screened the coding region of the myotubularin-related protein 2 gene in a Turkish consanguineous Charcot-Marie-Tooth disease family compatible with linkage to chromosome 11q22. A homozygous cytosine to thymine missense mutation at nucleotide position 847, resulting in an amino acid substitution of arginine to tryptophan at codon 283, was detected in exon 9 of the MTMR2 gene. This is the second homozygous missense mutation associated with recessive Charcot-Marie-Tooth disease with focally folded myelin sheaths.

Role of apoptosis in Duchenne's muscular dystrophy.

We investigated the presence of apoptosis in muscle tissues from 24 patients (average age 5.44 +/- 1.81 years) with Duchenne's muscular dystrophy by in situ tailing of nuclear fragmentation. Muscle tissue from 4 children without histologic evidence of myopathy served as normal controls. Muscle fibers positive for nuclear DNA fragmentation were determined quantitatively by counting an area of at least 400 muscle fibers. Eleven of 24 specimens showed no nuclei with DNA fragmentation. On the other hand, 0.37 +/- 0.48% of fibers in patients with Duchenne's muscular dystrophy and none in controls had DNA fragmentation (P > .05). In this study, the percentage of apoptotic nuclei was higher in Duchenne's muscular dystrophy muscle than in normal controls. However, the difference did not reach a statistically significant level, and further studies with larger control groups are warranted.

Andermann syndrome in a Turkish patient.

Agenesis of the corpus callosum with peripheral neuropathy or Andermann syndrome is an autosomal recessive disorder rarely found outside certain regions of the province of Quebec, Canada. We report a 5-year-old Turkish patient with Andermann syndrome born to consanguineous parents. She presented with diffuse hypotonic weakness, predominantly in the distal extremities, and mild mental retardation. Electromyography showed axonal-demyelinating sensorimotor neuropathy. Sural nerve biopsy was compatible with demyelinating neuropathy. Cranial magnetic resonance imaging revealed total agenesis of the corpus callosum, dilatation of the interhemispheric fissure, and enlargement of the cisterna magna. The molecular genetic analysis using microsatellite DNA markers covering the agenesis of the corpus callosum with peripheral neuropathy locus on chromosome 15q13-q15 showed that the patient is homozygous for the whole region. Our findings confirm that Andermann syndrome is a genetically homogeneous disorder.

A new epineural nerve repair technique with external metallic circle.

BACKGROUND: Despite the existence of various nerve coaptation techniques, functional results of nerve repair are still inadequate. Potential benefits of developing modified coaptation techniques cannot be disregarded. METHODS: The authors report a new coaptation technique in which the epineural sutures were performed with an external metallic circle to increase the coaptation surface. The sciatic nerves of 30 male Wistar albino rats were used in the study. RESULTS: The mean Sciatic Function Index values in external metallic circle repair (n:11) and conventional epineural repair (n:10) groups were -42.35 +/- 22.95 and -69.34 +/- 17.96, respectively (p = 0.020). Electrophysiological studies revealed that the duration of compound muscle action potentials (CMAP) was (p = 0,012) shorter in conventional nerve repair group than it was in external metallic nerve repair. When external metallic circle repair and conventional epineural repair groups were examined for distal nerve segments, there were significant findings for the diameter of axons (p = 0.005), diameter of nerves (p = 0.000), and for G ratios (p = 0.000). The mean intraepineural cross sectional areas of external metallic circle repair and conventional epineural repair groups were 3.57 +/- 0.21 and 2.92 +/- 0.23 mm(2), respectively (p = 0.000). CONCLUSION: The external metallic circle repair technique enhances nerve regeneration by enabling a larger sprouting and contact area for nerve fibers.

Identification of a novel Twinkle mutation in a family with infantile onset spinocerebellar ataxia by whole exome sequencing.

Whole exome sequencing combined with homozygosity mapping comprises a genetic diagnostic tool to identify genetic defects in families with multiple affected members, compatible with presumed autosomal recessively inherited neurometabolic/neurogenetic disease. These tools were applied to a family with two individuals manifesting ataxia, associated with peripheral sensory neuropathy, athetosis, seizures, deafness, and ophthalmoplegia. A novel homozygous missense mutation c.1366C>G (L456V) in C10orf2 (the Twinkle gene) was identified, confirming infantile onset spinocerebellar ataxia in the probands. Signs in infantile onset spinocerebellar ataxia follow a fairly distinct pattern, affecting early development, followed by ataxia and loss of skills. However, this very rare disease was previously reported only in Finland. We suggest that infantile onset spinocerebellar ataxia should be more frequently considered in the differential diagnosis of neurometabolic diseases in childhood. Next-generation sequencing and its use along with homozygosity mapping offer highly promising techniques for molecular diagnosis, especially in small families affected with very rare neurometabolic disorders such as infantile onset spinocerebellar ataxia.

A comparison of various vascularization-perfusion venous nerve grafts with conventional nerve grafts in rats.

Peripheral nerves with defective segments can only be repaired using nerve grafts. Among the various nerve graft options, the outcome of vascularized grafts has been shown to be better, especially when used in the hypovascular and scarred recipient bed. The purpose of this study was to compare the regeneration capacities of various types of venous nerve grafts in a rat model. Forty adult male Wistar albino rats were divided into four groups. A 2-cm-long segment of femoral sheath was isolated from the surrounding tissue without disturbing the unity of the femoral sheath contents. Four different nerve graft models were applied: flow-through venous, arterialized venous, prefabricated venous, and conventional nerve graft (control). All nerve grafts were closed with silicone sheets. These neurovascular segments were reopened in postoperative week 10 to determine the viability of the grafted nerves and to assess the degree of nerve healing. Histopathologic examinations, morphometric analysis, and electrophysiological measurements were performed. The degree of nerve healing in the flow-through venous nerve grafts was similar to that observed in the arterialized nerve grafts. Prefabricated flow-through venous grafts were not as successful as flow-through venous grafts or arterialized nerve grafts. All of the vascularized nerve grafts showed better results than the conventional nerve grafts.

Calpain-3 mutations in Turkey.

Autosomal recessive limb-girdle muscular dystrophies (LGMD2s) are a clinically and genetically heterogeneous group of disorders, characterized by progressive involvement of the proximal limb girdle muscles; the group includes at least 10 different genetic entities. The calpainopathies (LGMD2A), a subgroup of LGMD2s, are estimated to be the most common forms of LGMD2 in all populations so far investigated. LGMD2A is usually characterized by symmetrical and selective atrophy of pelvic, scapular and trunk muscles and a moderate to gross elevation of serum CK. However, the course is highly variable. It is caused by mutations in the CAPN3 gene, which encodes for the calpain-3 protein. Until now, 161 pathogenic mutations have been found in the CAPN3 gene. In the present study, through screening of 93 unrelated LGMD2 families, we identified 29 families with LGMD2A, 21 (22.6%) of which were identified as having CAPN3 gene mutations. We detected six novel (p.K211N, p.D230G, p.Y322H, p.R698S, p.Q738X, c.2257delGinsAA) and nine previously reported mutations (c.550delA, c.19_23del, c.1746-20C>G, p.R49H, p.R490Q, p.Y336N, p.A702V, p.Y537X, p.R541Q) in the CAPN3 gene. There may be a wide variety of mutations, but clustering of specific mutations (c.550delA: 40%, p.R490Q: 10%) could be used in the diagnostic scheme in Turkey.

Smooth muscle within incomplete obliterations of processus vaginalis lacks apoptotic nuclei.

INTRODUCTION: Incomplete obliteration of processus vaginalis (PV) has been suggested to result from the persistence of smooth muscle, which should normally disappear after taking part in the descent of testis. Since apoptosis is the mechanism of disappearance, the presence or absence of apoptotic nuclei was evaluated within sacs that result from failed obliteration of PV. MATERIALS AND METHODS: Twenty sacs associated with female inguinal hernia (n = 5), male inguinal hernia (n = 6), hydrocele (n = 5), hydrocele of the cord (n = 2), and undescended testis (n = 2) were evaluated. 10-microm sections were cut from the snap-frozen samples and stained for nuclear DNA fragmentation. RESULTS: Apoptotic nuclei were detected within the vascular structures and mesothelium. However, none of the samples from different diagnostic sources have revealed any apoptotic nucleus within the smooth muscle component. CONCLUSIONS: While the vascular and mesothelial structures within the sacs reveal evidence of apoptosis, the smooth muscle component lacks apoptotic process. The failed apoptosis of smooth muscle may have a role in the persistence of PV.

Reinnervation of denervated muscle in a split-nerve transfer model.

This study was performed to quantify the reinnervation of denervated muscle in a split-nerve transfer model and to determine any possible downgrading effects on the donor nerve and its end organ. Fifty-four adult Wistar rats weighing 200 to 250 g were used. The experimental design consisted of two groups. The motor nerve branch to the anterior tibial muscle and gastrocnemius muscle of the right hind limb were dissected in all rats. In the experimental group (N = 36), the motor nerve branch of the tibial nerve to the gastrocnemius muscle was exposed, cut, and ligated. The motor nerve branch to the anterior tibial muscle was split and transected longitudinally, and the medial half was routed posteriorly. End-to-end neural anastomosis was performed between this medial half of the split nerve and the distal stump of the gastrocnemius nerve. In the control group (N = 18), while the same surgical preparation was performed, the motor nerve branch to the anterior tibial muscle and gastrocnemius nerve were exposed and transected, and the nerve endings were ligated, but neural anastomosis was not performed between these nerves. The left hind limb of all rats served as a normal comparison side without any surgical intervention. Both of the groups were divided into three subgroups (12 rats each for the experimental groups and 6 rats each for the control group) to evaluate the results after periods of 1, 3, and 6 months. Electromyography, light microscopic and morphometric examination, and muscle weight measurements were used to document the results. Although stimulation of the peroneal and tibial nerves did not produce any compound muscle action potential (CMAP) recordings from either the anterior tibial or the gastrocnemius muscle in the control group, the normalized CMAP areas of the tibial nerve were (mean +/- standard deviation) 16.2 +/- 30.8% in the 1-month group, 63.4 +/- 34.7% in the 3-month group, and 72.4 +/- 16.3% in the 6-month group. For the peroneal nerve, the normalized CMAP areas were 17.0 +/- 32.2%, 53.4 +/- 29.4%, and 54.4 +/- 14.5% for the 1-, 3-, and 6-month groups in the experimental groups respectively. A high number of regenerating myelinated nerve fibers was identified in the distal part of the coapted motor nerve branch to the gastrocnemius muscle. The average number of myelinated fibers in the lateral half of the split nerve in the experimental group was 15,108 fibers per square millimeter, 14,167 fibers per square millimeter, and 19,830 fibers per square millimeter at months 1, 3, and 6 respectively. The average number of fibers proximal to the nerve anastomotic site was 15,423 fibers per square millimeter, 19,200 fibers per square millimeter, and 20,774 fibers per square millimeter. Distal to the nerve anastomotic site, the number of myelinated fibers was 17,941 fibers per square millimeter, 18,885 fibers per square millimeter, and 18,895 fibers per square millimeter at 1, 3, and 6 months respectively. There were no myelinated fibers in the control group sections. There were significant differences in muscle weight between the experimental and control groups at the end of month 6. The difference between the experimental side and the untouched normal healthy side was not significant in the weight measurements of both muscles. The results show acceptable reinnervation by split-nerve transfer with minimal functional impairment of the donor muscle. This study confirms that split-nerve transfer is a reliable method of reconstruction for paralyzed muscle with minimal donor area morbidity.

Expression of matrix metalloproteinases in vasculitic neuropathy.

The aim of this study was to investigate the expression pattern and cellular source of matrix metalloproteinases (MMP) in vasculitic neuropathy. Matrix metalloproteinases are endopeptidases degrading components of extracellular matrix proteins, and they have been implicated in the pathogenesis of inflammatory demyelination. They are induced by cytokines, secreted by inflammatory cells, and enhance T cell migration. Vasculitic neuropathy occurs as a component of systemic vasculitis or as an isolated angiitis of the peripheral nervous system, and T cell-mediated inflammation is detected in its pathogenesis. Nerve biopsy sections of eight patients with nonsystemic vasculitic neuropathy (NSVN) and four with systemic vasculitic neuropathy were examined for the presence of CD4+, CD8+, and CD68+ cells and immunohistochemically for MMP-2 and MMP-9 expression. Nerve biopsies of eight patients with noninflammatory neuropathy were used as a control group. Semiquantitative polymerase chain reaction analysis was performed to detect MMP-2 and MMP-9 mRNA. The predominant cells were CD8+ and CD68+ T cells. Expression of MMP-9, but not MMP-2, was increased in perivascular inflammatory infiltrate in nerve tissues of vasculitic neuropathy patients. This MMP-9 expression correlated positively with immunostaining of CD8+ T cells. No difference was detected between immunostaining patterns of nonsystemic and systemic vasculitic neuropathies with the antibodies used, except in MMP-9 immunostaining, which was found to be enhanced in NSVN group. Polymerase chain reaction analysis revealed elevated mRNA levels of MMP-9 and MMP-2 compared with controls, but this did not reach statistical significance. Our results imply a pathogenic role for MMP-9 secreted from CD8+ cells in vasculitic neuropathy.

Effects of ionizing radiation on brain tissue surrounding arteriovenous malformations: an experimental study in a rat caroticojugular fistula model.

Arteriovenous malformation (AVM) may change the cerebral hemodynamics. The purpose of this study was to detect the effects of ionizing radiation (IR) on tissues surrounding AVM in a rat caroticojugular fistula model. Forty rats were divided into four groups. Eight weeks after caroticojugular fistulas and chronic hypoperfusion were created in groups 1 and 2, IR was administered to groups 1 and 3. Group 4 was the control. Brain tissue samples were taken 72 h after irradiation. Comet assay to detect DNA strand breaks (DSB), terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay for apoptosis, and free radical measurement were performed. Although the difference between fistula plus irradiation (group 1) and fistula (group 2) was statistically insignificant in terms of DSB and free radical measurement, apoptotic cell count was significantly higher in group 1. Nonetheless, apoptotic cell count corresponded well with both free radicals and DSB in the irradiated group (group 3). Ionizing radiation resulted in significant apoptosis in both groups with or without fistulas. Chronic hypoperfusion might not prevent cerebral damage after IR. Optimal care should be taken with brain tissue around AVM during radiotherapy, regardless of presence or absence of the "steal" phenomenon.

Mitochondrial neurogastrointestinal encephalomyopathy: diagnostic features of two patients.

Mitochondrial neurogastrointestinal encephalomyopathy is a rare, multisystem disorder characterized by gastrointestinal dysmotility, ptosis, neurologic findings (e.g., peripheral neuropathy), leukoencephalopathy, and thin body habitus. Gastrointestinal motility studies and skeletal muscle biopsy are recommended diagnostic tools. We report two patients that highlight the diagnostic characteristics of this rare entity.

Exercise-induced apoptosis of rat skeletal muscle and the effect of meloxicam.

The aim of this study was to evaluate the effect of exercise on apoptosis in rat gastrocnemius and soleus muscle tissue and to determine the effect of meloxicam, a novel non-steroidal anti-inflammatory drug (NSAID), on the ratio of exercise-induced apoptosis. Forty male Wistar rats were used in the experiments. Spontaneous wheel-running was used as an exercise protocol. Rats were divided randomly into four groups. Group A (n = 10) was the control group, in which rats did not perform any exercise. In group B (n = 10), gastrocnemius and soleus muscles were biopsied immediately after exercise. The rats in group C (n = 10) were placed back in their cages after exercise and allowed to rest for 48 h, after which the gastrocnemius and soleus muscles were biopsied. In group D (n = 10), rats were given 11 mg meloxicam (Mobic, Boehringer Ingelheim) per kilogram body weight per day p.o. for 2 days, after which gastrocnemius and soleus muscles were biopsied 48 h after exercise. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labelling (TUNEL) technique was used to detect DNA fragmentation in situ. TUNEL-positive nuclei were identified and counted. The apoptosis ratio in gastrocnemius muscle was 0.50x10(-3)+/-0.96x10(-3) in group A, 5.42x10(-3)+/-3.58x10(-3) in group B, 3.55x10(-3)+/-3.23x10(-3) in Group C and 3.52x10(-3)+/-1.00 in Group D; the ratios in soleus muscle were 0.98x10(-3)+/-1.83x10(-3), 3.03x10(-3)+/-2.78x10(-3), 4.48x10(-3)+/-3.32x10(-3) and 2.91x10(-3) 1.98x10(-3), respectively. The differences between the apoptosis ratios in group A and B, Group A and C, and Group A and D were statistically significant (P < 0.05). There was no statistically significant difference between group C and D. In conclusion, exercise increased apoptosis in gastrocnemius and soleus muscle tissue, and the apoptosis ratios were not affected by meloxicam.

Mutations in a gene encoding a novel SH3/TPR domain protein cause autosomal recessive Charcot-Marie-Tooth type 4C neuropathy.

Charcot-Marie-Tooth disease type 4C (CMT4C) is a childhood-onset demyelinating form of hereditary motor and sensory neuropathy associated with an early-onset scoliosis and a distinct Schwann cell pathology. CMT4C is inherited as an autosomal recessive trait and has been mapped to a 13-cM linkage interval on chromosome 5q23-q33. By homozygosity mapping and allele-sharing analysis, we refined the CMT4C locus to a suggestive critical region of 1.7 Mb. We subsequently identified mutations in an uncharacterized transcript, KIAA1985, in 12 families with autosomal recessive neuropathy. We observed eight distinct protein-truncating mutations and three nonconservative missense mutations affecting amino acids conserved through evolution. In all families, we identified a mutation on each disease allele, either in the homozygous or in the compound heterozygous state. The CMT4C gene is strongly expressed in neural tissues, including peripheral nerve tissue. The translated protein defines a new protein family of unknown function with putative orthologues in vertebrates. Comparative sequence alignments indicate that members of this protein family contain multiple SH3 and TPR domains that are likely involved in the formation of protein complexes.