Genetic landscape of congenital insensitivity to pain and hereditary sensory and autonomic neuropathies.

Congenital insensitivity to pain (CIP) and hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders exclusively or predominantly affecting the sensory and autonomic neurons. Due to the rarity of the diseases and findings based mainly on single case reports or small case series, knowledge about these disorders is limited. Here, we describe the molecular workup of a large international cohort of CIP/HSAN patients including patients from normally under-represented countries. We identify 80 previously unreported pathogenic or likely pathogenic variants in a total of 73 families in the >20 known CIP/HSAN-associated genes. The data expand the spectrum of disease-relevant alterations in CIP/HSAN, including novel variants in previously rarely recognized entities such as ATL3-, FLVCR1- and NGF-associated neuropathies and previously under-recognized mutation types such as larger deletions. In silico predictions, heterologous expression studies, segregation analyses and metabolic tests helped to overcome limitations of current variant classification schemes that often fail to categorize a variant as disease-related or benign. The study sheds light on the genetic causes and disease-relevant changes within individual genes in CIP/HSAN. This is becoming increasingly important with emerging clinical trials investigating subtype or gene-specific treatment strategies.

Genetic findings in Czech patients with limb girdle muscular dystrophy.

Limb girdle muscular dystrophies (LGMD) are a genetically heterogeneous group of muscular dystrophies. The study presents an overview of molecular characteristics of a large cohort of LGMD patients who are representative of the Czech LGMD population. We present 226 LGMD probands in which 433 mutant alleles carrying 157 different variants with a supposed pathogenic effect were identified. Fifty-four variants have been described only in the Czech LGMD population so far. LGMD R1 caplain3-related is the most frequent subtype of LGMD involving 53.1% of patients with genetically confirmed LGMD, followed by LGMD R9 FKRP-related (11.1%), and LGMD R12 anoctamin5-related (7.1%). If we consider identified variants, then all but five were small-scale variants. One large gene deletion was identified in the LAMA2 gene and two deletions in each of CAPN3 and SGCG. We performed comparison our result with other published studies. The results obtained in the Czech LGMD population clearly differ from the outcome of other LGMD populations in two aspects-we have a more significant proportion of patients with LGMD R1 calpain3-related and a smaller proportion of LGMD R2 dysferlin-related.

Diagnostic utility of neurofilament markers for MND is limited in restricted disease phenotype and for differentiation from compressive myeloradiculopathies.

Misdiagnosis is frequent in early motor neuron disease (MND), typically compressive radiculopathy, or in patients with restricted MND phenotype. In this retrospective, single tertiary centre study, we measured levels of neurofilament light (NfL) and phosphorylated neurofilament heavy (p-NfH) chain in cerebrospinal fluid (CSF) and of p-NfH in serum with commercially available ELISA kits and assessed their respective diagnostic performance as a marker of MND. The entire study population (n = 164) comprised 71 MND patients, 30 patients with compressive myelo- or radiculopathy, and 63 disease controls (DC). Among MND patients, we specified subgroups with only lower motoneuron involvement (MND-LMN, n = 15) and with confounding nerve roots or spinal cord compression (MND-C, n = 18), representing clinical diagnostic pitfalls. MND-LMN displayed significantly lower CSF NfL (p = 0.003) and p-NFH (p = 0.017), but not serum p-NfH (p = 0.347) levels compared to other MND patients (n = 56). The discriminative ability (area under the curve-AUC) of both CSF Nfs towards all MND patients was comparable to each other but significantly higher than that of p-NfH in serum (ps < 0.001). AUC of both CSF Nfs between MND-LMN and DC and also between MND-C and myelo-/radiculopathies were reduced, as compared to AUC between other MND and DC or myelo-/radiculopathies, respectively. Our results suggest that both Nfs in CSF represent a reliable diagnostic marker in a general MND population, fulfilling Awaji criteria. As for diagnostic pitfalls, and also for p-NfH in serum, their discriminative ability and, therefore, clinical utility appears to be limited.

Thermal quantitative sensory testing as a screening tool for cardiac autonomic neuropathy in patients with diabetes mellitus.

INTRODUCTION: Electrophysiological diagnosis of cardiac autonomic neuropathy (CAN) is based on the evaluation of cardiovascular autonomic reflex tests (CARTs). CARTs are relatively time consuming and must be performed under standardized conditions. This study aimed to determine whether thermal quantitative sensory testing (TQST) can be used as a screening tool to identify patients with diabetes at a higher risk of CAN. METHODS: Eighty-five patients with diabetes and 49 healthy controls were included in the study. Neurological examination, CARTs, TQST, biochemical analyses, and neuropathy symptom questionnaires were performed. RESULTS: CAN was diagnosed in 46 patients with diabetes (54%). CAN-positive patients with diabetes had significantly higher warm detection thresholds (WDT) and significantly lower cold detection thresholds (CDT) in all tested regions (thenar, tibia, and the dorsum of the foot). CDT on the dorsum < 21.8°C in combination with CDT on the tibia < 23.15°C showed the best diagnostic ability in CAN prediction, with 97.4 % specificity, 60.9% sensitivity, 96.6% positive predictive value, and 67.3% negative predictive value. CONCLUSION: TQST can be used as a screening tool for CAN before CART.

Risk factors for depression and anxiety in painful and painless diabetic polyneuropathy: A multicentre observational cross-sectional study.

BACKGROUND: Despite the high prevalence of depression and anxiety in chronic pain conditions, current knowledge concerning emotional distress among painful diabetic polyneuropathy (pDSPN) and other diabetes mellitus (DM) sufferers is limited. METHODS: This observational multicentre cohort study employed the Hospital Anxiety and Depression Scale, the Beck Depression Inventory II and the State-Trait Anxiety Inventory to assess symptoms of depression and anxiety in several groups with diabetes, as well as in a control group. The study cohort included 347 pDSPN patients aged 63.4 years (median), 55.9% males; 311 pain-free diabetic polyneuropathy (nDSPN) patients aged 63.7 years, 57.9% males; 50 diabetes mellitus (DM) patients without polyneuropathy aged 61.5 years, 44.0% males; and 71 healthy controls (HC) aged 63.0 years, 42.3% males. The roles played in emotional distress were explored in terms of the biological, the clinical (diabetes-, neuropathy- and pain-related), the socio-economic and the cognitive factors (catastrophizing). RESULTS: The study disclosed a significantly higher prevalence of the symptoms of depression and anxiety not only in pDSPN (46.7% and 60.7%, respectively), but also in patients with nDSPN (24.4% and 44.4%) and DM without polyneuropathy (22.0% and 30.0%) compared with HCs (7.0% and 14.1%, p < 0.001). Multiple regression analysis demonstrated the severity of pain and neuropathy, catastrophic thinking, type 2 DM, lower age and female sex as independent contributors to depression and anxiety. CONCLUSIONS: In addition to the severity of neuropathic pain and its cognitive processing, the severity of diabetic polyneuropathy and demographic factors are key independent contributors to emotional distress in diabetic individuals. SIGNIFICANCE: In large cohorts of well-defined painless and painful diabetic polyneuropathy patients and diabetic subjects without polyneuropathy, we found a high prevalence of the symptoms of depression and anxiety, mainly in painful individuals. We have confirmed neuropathic pain, its severity and cognitive processing (pain catastrophizing) as dominant risk factors for depression and anxiety. Furthermore, some demographic factors (lower age, female sex), type 2 diabetes mellitus and severity of diabetic polyneuropathy were newly identified as important contributors to emotional distress independent of pain.

Antibodies to the Caspr1/contactin-1 complex in chronic inflammatory demyelinating polyradiculoneuropathy.

Previous studies have described the clinical, serological and pathological features of patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and antibodies directed against the paranodal proteins neurofascin-155, contactin-1 (CNTN1), contactin-associated protein-1 (Caspr1), or nodal forms of neurofascin. Such antibodies are useful for diagnosis and potentially treatment selection. However, antibodies targeting Caspr1 only or the Caspr1/CNTN1 complex have been reported in few patients with CIDP. Moreover, it is unclear if these patients belong to the same pathophysiological subgroup. Using cell-based assays in routine clinical testing, we identified sera from patients with CIDP showing strong membrane reactivity when both CNTN1 and Caspr1 were co-transfected (but not when CNTN1 was transfected alone). Fifteen patients (10 male; aged between 40 and 75) with antibodies targeting Caspr1/CNTN1 co-transfected cells were enrolled for characterization. The prevalence of anti-Caspr1/CNTN1 antibodies was 1.9% (1/52) in the Sant Pau CIDP cohort, and 4.3% (1/23) in a German cohort of acute-onset CIDP. All patients fulfilled European Federation of Neurological Societies/Peripheral Nerve Society (EFNS/PNS) definite diagnostic criteria for CIDP. Seven (47%) were initially diagnosed with Guillain-Barré syndrome due to an acute-subacute onset. Six (40%) patients had cranial nerve involvement, eight (53%) reported neuropathic pain and 12 (80%) ataxia. Axonal involvement and acute denervation were frequent in electrophysiological studies. Complete response to intravenous immunoglobulin was not observed, while most (90%) responded well to rituximab. Enzyme-linked immunosorbent assay (ELISA) and teased nerve fibre immunohistochemistry confirmed reactivity against the paranodal Caspr1/CNTN1 complex. Weaker reactivity against Caspr1 transfected alone was also detected in 10/15 (67%). Sera from 13 of these patients were available for testing by ELISA. All 13 samples reacted against Caspr1 by ELISA and this reactivity was enhanced when CNTN1 was added to the Caspr1 ELISA. IgG subclasses were also investigated by ELISA. IgG4 was the predominant subclass in 10 patients, while IgG3 was predominant in other three patients. In conclusion, patients with antibodies to the Caspr1/CNTN1 complex display similar serological and clinical features and constitute a single subgroup within the CIDP syndrome. These antibodies likely target Caspr1 primarily and are detected with Caspr1-only ELISA, but reactivity is optimal when CNTN1 is added to Caspr1 in cell-based assays and ELISA.

Anti-Müllerian hormone as an ovarian reserve marker in women with the most frequent muscular dystrophies.

Some muscular dystrophies may have a negative impact on fertility. A decreased ovarian reserve is 1 of the factors assumed to be involved in fertility impairment. AMH (anti-Müllerian hormone) is currently considered the best measure of ovarian reserve.A total of 21 females with myotonic dystrophy type 1 (MD1), 25 females with myotonic dystrophy type 2 (MD2), 12 females with facioscapulohumeral muscular dystrophy (FSHD), 12 female carriers of Duchenne muscular dystrophy mutations (cDMD) and 86 age-matched healthy controls of reproductive age (range 18 - 44 years) were included in this case control study. An enzymatically amplified 2-site immunoassay was used to measure serum AMH level.The MD1 group shows a significant decrease of AMH values (median 0.7 ng/mL; range 0 - 4.9 ng/mL) compared with age-matched healthy controls (P < .01). AMH levels were similar between patients and controls in terms of females with MD2 (P = .98), FSHD (P = .55) and cDMD (P = .60).This study suggests decreased ovarian reserve in women with MD1, but not in MD2, FSHD and cDMD.

Correction to: Eight years after an international workshop on myotonic dystrophy patient registries: case study of a global collaboration for a rare disease.

The original version of this article [1] unfortunately included an error to an author's name. Author Jordi Díaz-Manera was erroneously presented as Jorge Alberto Diaz Manera. The correct author name has been included in the author list of this Correction article.

Amyotrophy, cerebellar impairment and psychiatric disease are the main symptoms in a cohort of 14 Czech patients with the late-onset form of Tay-Sachs disease.

BACKGROUND: Tay-Sachs disease (TSD) is an inherited neurodegenerative disorder caused by a lysosomal ß-hexosaminidase A deficiency due to mutations in the HEXA gene. The late-onset form of disease (LOTS) is considered rare, and only a limited number of cases have been reported. The clinical course of LOTS differs substantially from classic infantile TSD. METHODS: Comprehensive data from 14 Czech patients with LOTS were collated, including results of enzyme assays and genetic analyses. RESULTS: 14 patients (9 females, 5 males) with LOTS were diagnosed between 2002 and 2018 in the Czech Republic (a calculated birth prevalence of 1 per 325,175 live births). The median age of first symptoms was 21 years (range 10-33 years), and the median diagnostic delay was 10.5 years (range 0-29 years). The main clinical symptoms at the time of manifestation were stammering or slurred speech, proximal weakness of the lower extremities due to anterior horn cell neuronopathy, signs of neo- and paleocerebellar dysfunction and/or psychiatric disorders. Cerebellar atrophy detected through brain MRI was a common finding. Residual enzyme activity was 1.8-4.1% of controls. All patients carried the typical LOTS-associated c.805G>A (p.Gly269Ser) mutation on at least one allele, while a novel point mutation, c.754C>T (p.Arg252Cys) was found in two siblings. CONCLUSION: LOTS seems to be an underdiagnosed cause of progressive distal motor neuron disease, with variably expressed cerebellar impairment and psychiatric symptomatology in our group of adolescent and adult patients. The enzyme assay of ß-hexosaminidase A in serum/plasma is a rapid and reliable tool to verify clinical suspicions.

Autosomal recessive hereditary spastic paraplegia type SPG35 due to a novel variant in the FA2H gene in a Czech patient.

Biallelic pathogenic variants in FA2H gene have been repeatedly described as a cause of hereditary spastic paraplegia (HSP) type35 (SPG35). Targeted massive parallel sequencing (MPS) of the HSP genes panel revealed a novel homozygous variant c.130C > T (p.P44S) in the FA2H gene in the 30-year-old patient presenting with spastic paraplegia. The patient originated form the Czech minority in Romania. The patient manifests typical clinical signs for SPG35 (youth onset gait impairment, progressive spastic paraparesis on lower limbs, dysarthria, white matter changes in MRI).

Eight years after an international workshop on myotonic dystrophy patient registries: case study of a global collaboration for a rare disease.

BACKGROUND: Myotonic Dystrophy is the most common form of muscular dystrophy in adults, affecting an estimated 10 per 100,000 people. It is a multisystemic disorder affecting multiple generations with increasing severity. There are currently no licenced therapies to reverse, slow down or cure its symptoms. In 2009 TREAT-NMD (a global alliance with the mission of improving trial readiness for neuromuscular diseases) and the Marigold Foundation held a workshop of key opinion leaders to agree a minimal dataset for patient registries in myotonic dystrophy. Eight years after this workshop, we surveyed 22 registries collecting information on myotonic dystrophy patients to assess the proliferation and utility the dataset agreed in 2009. These registries represent over 10,000 myotonic dystrophy patients worldwide (Europe, North America, Asia and Oceania). RESULTS: The registries use a variety of data collection methods (e.g. online patient surveys or clinician led) and have a variety of budgets (from being run by volunteers to annual budgets over €200,000). All registries collect at least some of the originally agreed data items, and a number of additional items have been suggested in particular items on cognitive impact. CONCLUSIONS: The community should consider how to maximise this collective resource in future therapeutic programmes.

Mutations in ATP1A1 Cause Dominant Charcot-Marie-Tooth Type 2.

Although mutations in more than 90 genes are known to cause CMT, the underlying genetic cause of CMT remains unknown in more than 50% of affected individuals. The discovery of additional genes that harbor CMT2-causing mutations increasingly depends on sharing sequence data on a global level. In this way-by combining data from seven countries on four continents-we were able to define mutations in ATP1A1, which encodes the alpha1 subunit of the Na+,K+-ATPase, as a cause of autosomal-dominant CMT2. Seven missense changes were identified that segregated within individual pedigrees: c.143T>G (p.Leu48Arg), c.1775T>C (p.Ile592Thr), c.1789G>A (p.Ala597Thr), c.1801_1802delinsTT (p.Asp601Phe), c.1798C>G (p.Pro600Ala), c.1798C>A (p.Pro600Thr), and c.2432A>C (p.Asp811Ala). Immunostaining peripheral nerve axons localized ATP1A1 to the axolemma of myelinated sensory and motor axons and to Schmidt-Lanterman incisures of myelin sheaths. Two-electrode voltage clamp measurements on Xenopus oocytes demonstrated significant reduction in Na+ current activity in some, but not all, ouabain-insensitive ATP1A1 mutants, suggesting a loss-of-function defect of the Na+,K+ pump. Five mutants fall into a remarkably narrow motif within the helical linker region that couples the nucleotide-binding and phosphorylation domains. These findings identify a CMT pathway and a potential target for therapy development in degenerative diseases of peripheral nerve axons.

Biomarkers predict outcome in Charcot-Marie-Tooth disease 1A.

BACKGROUND: Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited neuropathy, a debilitating disease without known cure. Among patients with CMT1A, disease manifestation, progression and severity are strikingly variable, which poses major challenges for the development of new therapies. Hence, there is a strong need for sensitive outcome measures such as disease and progression biomarkers, which would add powerful tools to monitor therapeutic effects in CMT1A. METHODS: We established a pan-European and American consortium comprising nine clinical centres including 311 patients with CMT1A in total. From all patients, the CMT neuropathy score and secondary outcome measures were obtained and a skin biopsy collected. In order to assess and validate disease severity and progression biomarkers, we performed qPCR on a set of 16 animal model-derived potential biomarkers in skin biopsy mRNA extracts. RESULTS: In 266 patients with CMT1A, a cluster of eight cutaneous transcripts differentiates disease severity with a sensitivity and specificity of 90% and 76.1%, respectively. In an additional cohort of 45 patients with CMT1A, from whom a second skin biopsy was taken after 2-3 years, the cutaneous mRNA expression of GSTT2, CTSA, PPARG, CDA, ENPP1 and NRG1-Iis changing over time and correlates with disease progression. CONCLUSIONS: In summary, we provide evidence that cutaneous transcripts in patients with CMT1A serve as disease severity and progression biomarkers and, if implemented into clinical trials, they could markedly accelerate the development of a therapy for CMT1A.

Confirmation of the GNB4 gene as causal for Charcot-Marie-Tooth disease by a novel de novo mutation in a Czech patient.

The association of GNB4 with Charcot-Marie-Tooth (CMT) has recently been described in a publication by Soong et al. (Soong, et al., 2013). Here we present a patient with CMT in whom whole exome sequencing identified the mutation p.Lys57Glu in the GNB4 gene (NM_021629.3:c.169A>G). The patient, now 41 years old, is a sporadic case in the family. At the age of 35 he presented with severe disability (CMT neuropathy score 29), profound muscle atrophies, pes cavus and scoliosis. Previously, the patient was tested for PMP22 duplications/deletions and later also with 64 CMT gene panel, with no causal variant found. Subsequently, whole exome sequencing was performed. The p.Lys57Glu in the GNB4 gene was identified as the most probable causal variant, the mutation is not present in the patient's parents, neither in his unaffected sister, therefore we assume that the mutation arose de novo. Taken together, these findings support the causal and pathogenic character of the variant. Our report provides important evidence that GNB4 should become an established CMT gene and our findings confirm the original publication by Soong et al. (2013).

Improving diagnosis of inherited peripheral neuropathies through gene panel analysis.

BACKGROUND: Inherited peripheral neuropathies (IPN) are the most common inherited neurological condition. It represents a highly heterogeneous group, both clinically and genetically. Targeted disease specific gene panel massively parallel sequencing (MPS) seems to be a useful tool in diagnosis of disorders with high genetic heterogeneity. METHODS: In our study, we have designed, validated and updated our own custom gene panel of all known genes associated with IPN. One hundred and ninety-eight patients have been tested so far. Only patients in whom mutations in more common causes or relevant genes have already been excluded were enrolled. Five consecutive panel designs were prepared according to recent literature search, the last one covering ninety-three genes. Each patient was tested only once. All data were evaluated with at least two different pipelines. RESULTS: In summary, causative mutation has been found in fifty-one patients (26 %). The results were inconclusive in thirty-one (16 %) patients. No variants of likely significance to IPN were found in one hundred and sixteen (58 %) patients. CONCLUSION: MPS gene panel enables testing of all known IPN causes at once with high coverage and at an affordable cost making it truly a method of choice also in IPN. Gene panel testing results in several interesting results and findings.

Evaluation of muscle strength and manual dexterity in patients with Charcot-Marie-Tooth disease.

STUDY DESIGN: Matched pair study. INTRODUCTION: Differences in hand-muscle strength/dexterity between dominant (DH) and non-dominant (NDH) hand in Charcot-Marie-Tooth disease (CMT) are not well understood. PURPOSE OF THE STUDY: To compare muscle strength/dexterity between DH and NDH and to correlate manual dexterity, strength and sensory function. PATIENTS AND METHODS: Thirty CMT patients were studied using functional muscle testing (FMT) and strength (dynamometry), dexterity (the Nine Hole Peg Test [NHPT]), and Jebsen-Taylor Hand Function [JTT]), and sensory function (the Nottingham Sensory Assessment [NSA]). RESULTS: Scores were worse for DH than NDH on FMT (p = 0.043) and NHPT (p = 0.014) but not on JTT (p = 0.098), handgrip strength (p = 0.710) or tripod pinch (p = 0.645). NSA did not correlate significantly with any tests (p's0.05). CONCLUSIONS: In CMT disease, DH appears more impaired than NDH in terms of function and dexterity. Greater muscle weakness in DH may also emerge as CMT progresses. LEVEL OF EVIDENCE: 3b.

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.

Mechano-transduction effect of shockwaves in the treatment of lumbar facet joint pain: comparative effectiveness evaluation of shockwave therapy, steroid injections and radiofrequency medial branch neurotomy.

OBJECTIVE: Lumbar facet joints (FJ) is a common source of low back pain and contributes approximmately on one third of chronic low back pain. Medial branch radiofrequency neurotomy is considered as a gold standard in the treatment of facet joint pain. Corticosteroid injections have also presented effect in FJ pain. As an interventional procedures, they carry not-negligible risk of possible complications including infection, damage to nerve root or medial branch structures. Shockwave therapy (SWT) is a non-invasive method for treatment of various musculoskeletal disorders. Its effect is based on transduction of mechanical energy, transferred to cascade of various biochemical processes in target tissue. Its efficacy was proved in the treatment of different painful conditions. The efficacy of SWT was not yet studied in FJ pain. Aim of our work was to compare the efficacy of SWT against interventional treatment procedures - radiofrequency neurotomy and corticosteroid FJ injections. METHODS: A retrospective study was done on 62 selected patients with unilateral chronic lumbar facet pain. There were 32 women and 30 men, divided into SWT group, corticosteroid injections group radiofrequency group. Nociceptive and neuropathic pain intensity and severity of pain were measured. RESULTS: Shockwave therapy had shown better longterm results compared to FJ injections group and little inferior efficacy compared to RMBN. We did not observe any adverse effects and complications in SWT group. Moreover, in SWT and RMBN groups, significant longterm improvement in daily activities limitation, was observed. CONCLUSIONS: SWT appears to be a safe and perspective option in the treatment of FJ pain with negligible side effects.

Autosomal recessive limb-girdle muscular dystrophies in the Czech Republic.

BACKGROUND: Autosomal recessive limb-girdle muscular dystrophies (LGMD2) include a number of disorders with heterogeneous etiology that cause predominantly weakness and wasting of the shoulder and pelvic girdle muscles. In this study, we determined the frequency of LGMD subtypes within a cohort of Czech LGMD2 patients using mutational analysis of the CAPN3, FKRP, SGCA, and ANO5 genes. METHODS: PCR-sequencing analysis; sequence capture and targeted resequencing. RESULTS: Mutations of the CAPN3 gene are the most common cause of LGMD2, and mutations in this gene were identified in 71 patients in a set of 218 Czech probands with a suspicion of LGMD2. Totally, we detected 37 different mutations of which 12 have been described only in Czech LGMD2A patients. The mutation c.550delA is the most frequent among our LGMD2A probands and was detected in 47.1% of CAPN3 mutant alleles. The frequency of particular forms of LGMD2 was 32.6% for LGMD2A (71 probands), 4.1% for LGMD2I (9 probands), 2.8% for LGMD2D (6 probands), and 1.4% for LGMD2L (3 probands).Further, we present the first results of a new approach established in the Czech Republic for diagnosis of neuromuscular diseases: sequence capture and targeted resequencing. Using this approach, we identified patients with mutations in the DYSF and SGCB genes. CONCLUSIONS: We characterised a cohort of Czech LGMD2 patients on the basis of mutation analysis of genes associated with the most common forms of LGMD2 in the European population and subsequently compared the occurrence of particular forms of LGMD2 among countries on the basis of our results and published studies.