CLCN1 Research Articles

Becker congenital myotonia in black African with molecular findings

BackgroundCongenital myotonia is a congenital disorder that affects skeletal muscles with myotonia. Affected muscles show stiffness and pain sometimes. The two major types of myotonia congenita are known as Thomsen disease and Becker disease. These conditions are distinguished by the severity of their symptoms and their patterns of inheritance. The causative factor is mutations in CLCN1 gene. Myotonia congenita is rarely reported in black especially in black African.Case presentationThis is a case report of Becker Congenital Myotonia in a 36-year-old male from Benin. The symptoms arose at the age of 7 years with regular and progressive course and muscles pains. Electromyogram, blood sampling, laboratory investigations and muscles biopsy confirm the diagnostic with molecular finding.ConclusionThe authors report a case of Becker congenital myotonia in a black African with molecular confirmation. Mexiletine was used as symptomatic agent with good results.

Renal Expression of CLC-5 and Megalin/Cubilin in Dent-1 Disease With Nonsense Mutations of CLCN5 Gene.

The study aims to explore the clinicopathological features and whether the nonsense mutations of CLCN5 gene have effect on the renal expression of CLC-5 protein and megalin/cubilin complex in children with Dent-1 disease. The clinicopathological features and genetic examination of three patients with Dent-1 disease were investigated. The expression of CLC-5 and megalin/cubilin complex in renal tissues was detected by using immunohistochemistry method. Urinary albumin, α1-microglobulin, β2-microglobulin, retinol binding protein, and calcium levels were measured by immunonephelometry. Urinary calcium and low molecular weight proteinuria (LMWP) were enhanced in three patients, and two presented with nephrotic range proteinuria. Focal glomerular obsolescence, minor tubulointerstitial injury, and focal calcification in corticomedullary junction were found in one patient. Nonsense mutations of CLCN5 gene from their mothers were identified in all three patients with Dent-1 disease; however, the expression of CLC-5 protein was not decreased in renal tubular cells. As the receptor complex of albumin and LMWP reabsorption, the expression of megalin/cubilin in the brush border of proximal tubules was decreased in Dent-1 patients. Even if the renal CLC-5 protein is expressed normally, the reduced expression of megalin/cubilin in the brush border of renal proximal tubules may be helpful to understand the physiopathology of Dent-1 disease with nonsense mutations of CLCN5 gene.

Chloroquine increases osteoclast activity in vitro but does not improve the osteopetrotic bone phenotype of ADO2 mice.

Autosomal Dominant Osteopetrosis type II (ADO2) is a bone disease of impaired osteoclastic bone resorption that usually results from heterozygous missense mutations in the chloride channel 7 (CLCN7) gene. We created mouse models of ADO2 by introducing a knock-in (p.G213R) mutation in the Clcn7 gene, which is analogous to one of the common mutations (G215R) found in humans. The mutation leads to severe osteopetrosis and lethality in homozygous mice but produces substantial phenotypic variability in heterozygous mice on different genetic backgrounds that phenocopy the human disease of ADO2. ADO2 is an osteoclast-intrinsic disease, and lysosomal enzymes and proteins are critical for osteoclast activity. Chloroquine (CQ) is known to affect lysosomal trafficking, intracellular signaling and the lysosomal and vesicular pH, suggesting it might improve ADO2 osteoclast function. We tested this hypothesis in cell culture studies using osteoclasts derived from wild-type (WT or ADO2+/+) and ADO2 heterozygous (ADO2+/-) mice and found that CQ and its metabolite desethylchloroquine (DCQ), significantly increased ADO2+/- osteoclasts bone resorption activity in vitro, whereas bone resorption of ADO2+/+ osteoclasts was increased only by DCQ. In addition, we exploited our unique animal model of ADO2 on 129 background to identify the effect of CQ for the treatment of ADO2. Female ADO2 mice at 8weeks of age were treated with 5 doses of CQ (1, 2.5, 5, 7.5 and 10mg/kgBW/day) via drinking water for 6months. Bone mineral density and bone micro-architecture were analyzed by longitudinal in vivo DXA and micro-CT at baseline, 3 and 6months. Serum bone biomarkers (CTX, TRAP and P1NP) were also analyzed at these time points. CQ treatment at the doses tested failed to produce any significant changes of aBMD, BMC (whole body, femur and spine) and trabecular BV/TV (distal femur) in ADO2 mice compared to the control group (water only). Further, levels of bone biomarkers were not significantly changed due to CQ treatment in these mice. Our findings indicate that while CQ increased osteoclast activity in vitro, it did not improve the osteopetrotic bone phenotypes in ADO2 heterozygous mice.

Myotonia Congenita: Clinical Characteristic and Mutation Spectrum of CLCN1 in Chinese Patients.

Background: CLCN1-related myotonia congenita (MC) is one of the most common forms of non-dystrophic myotonia, in which muscle relaxation is delayed after voluntary or evoked contraction. However, there is limited data of clinical and molecular spectrum of MC patients in China.Patients and Methods: Five patients with myotonia congenita due to mutations in CLCN1 gene were enrolled, which were identified through trio-whole-exome sequencing or panel-based next-generation sequencing test. The clinical presentation, laboratory data, electrophysiological tests, muscular pathology feature, and genetic results were collected and reviewed. We also searched all previously reported cases of MC patients with genetic diagnosis in Chinese populations, and their data were reviewed.Results: The median onset age of five patients was 3.0 years old, ranging from 1.0 to 5.0 years old, while the median age of admit was 5.0 years old, ranging from 3.5 to 8.8 years old. Five patients complained of muscle stiffness when rising from chairs or starting to climb stairs (5/5, 100.0%), four patients complained of delayed relaxation of their hands after forceful grip (4/5, 80.0%), all of which improved with exercise (warm-up phenomenon) (5/5, 100%). Electromyogram was conducted in five patients, which all revealed myotonic change (100%). Genetic tests revealed nine potential disease-causing variants in CLCN1 gene, including two novel variants: c.962T>A (p.V321E) and c.1250A>T (p.E417V). Literature review showed that 43 MC Chinese patients with genetic diagnosis have been reported till now (including our five patients). Forty-seven variants in CLCN1 gene were found, which consisted of 33 missense variants, 6 nonsense variants, 5 frame-shift variants, and 3 splicing variants. Variants in exon 8, 15, 12, and 16 were most prevalent, while the most common variants were c.892G>A (p.A298T) (n = 9), c.139C>T (p.R47W) (n = 3), c.1205C>T(p.A402V) (n = 3), c.1657A>T (p.I553F) (n = 3), c.1679T>C (p.M560T) (n = 3), c.350A>G (p.D117G) (n = 2), c.762C>G (p.C254W) (n = 2), c.782A>G (P.Y261C) (n = 2), and c.1277C>A (p.T426N) (n = 2).Conclusion: Our results reported five CLCN1-related MC patients, which expanded the clinical and genetic spectrum of MC patients in China. Based on literature review, 43MC Chinese patients with genetic diagnosis have been reported till now, and variants in exon eight were most prevalent in Chinese MC patients while c.892G>A (p.A298T) was probably a founder mutation.

Teaching Video NeuroImage: Carbamazepine Improves Gait Initiation in Autosomal Recessive Myotonia Congenita.

A 60-year-old man presented with muscle stiffness. He had tended to fall since childhood. His parents were first cousins, and his relatives had no symptoms. Examination showed myotonia in ocular, hand, and limb muscles, followed by improvement with repeated activity (warm-up phenomenon; Video 1). Cold exposure did not aggravate myotonia. His lower legs showed muscle hypertrophy. Genetic tests showed a homozygous p.M560T mutation in the CLCN1 gene, which encodes skeletal muscle chloride channel 1, consistent with autosomal recessive myotonia congenita (Becker disease).1 Although treatment with mexiletine or phenytoin did not improve his symptoms, carbamazepine (350 mg/d) did, especially the walking disturbance (Video 2). Although the warm-up phenomenon is sometimes also observed in sodium channel myotonia,2 it is characteristic of myotonia congenita and an important clue for the diagnosis.

CHANNELOPATHIES AND RELATED DISORDERS: EP.220 Repetitive nerve stimulation decrement as a discrimination tool for hereditary myotonic syndromes

Hereditary myotonic syndromes (HMS) are a genetically heterogeneous group of muscle chloride or sodium channelopathies (primary or secondary) with myotonia as the primary symptom. Myotonia is caused by delayed skeletal muscle relaxation, the severity of which varies and overlaps between genetic variants. The possibility of applying EMG for differential diagnosis of patients with Myotonia as Main (sole) disease Symptom (MMS) is an option for optimization of molecular genetic testing of HMS. The aim of this study is to compare repetitive nerve stimulation (RNS) at various frequencies in genotyped patients with MMS for optimizing EMG use with DNA analysis. 74 (44.6%) of 166 genotyped HMS patients with MMS at disease onset or during follow-ups (regardless of myotonia severity) were included in the study. MMS genetic causes were found in 51 (68.9%) patients: two recessive mutations in CLCN1 gene (2/CLCN1), 13 (17.6%) in DMPK gene, 5 (6.8%) in SCN4A gene, 3 (4.1%) with the combination of a heterozygous mutation in CLCN1 and ZNF9 genes (CLCN1/ZNF9), 2 (2.7%) with the combination of a heterozygous mutation in CLCN1 and SCN4A genes (CLCN1/SCN4A). For 45 patients, 28 healthy volunteers, and 5 heterozygous carriers of the p.R894* (Carry-894) mutation in CLCN1 gene, the short exercise test (SET) and repetitive nerve stimulation (RNS) 10, 30, and 50-Hz were performed. Based on SET pattern I in 1 (2.2%) patient with 2/CLCN1; pattern II in 6 (13.3%) patients with 2/CLCN1, 2 (4.4%) with DMPK and 1 (2.2%) with CLCN1/SCN4A; normal pattern III in all others including remaining 35 (82.2%) patients were detected. RNS 10, 30, or 50-Hz, M-wave amplitude decrement >10% was detected in 21 (46.5%), 38 (84.4%) and 42 (93.3%) patients, respectively. RNS 50-Hz decrement was detected in all patients with 2/CLCL1, DMPK, and CLCN1/SCN4A amounting 80.1±17.4%, 42.3±24.3%, and 85.0±0% accordingly. In 3 (60%) patients with SCN4A decrement 23.9±13.7%, 2 (66.7%) with CLCN1/ZNF9 decrement 37.2±2.2%, and 4 (80%) with Carry-894 decrement 36.1±14.7% were detected. Our study confirmed that the highest decrement at RNS 50-Hz in MMS patients, regardless of myotonia severity, correlates with mutations 2/CLCN1 or CLCN1/SCN4A; intermediate decrement correlats with mutations DMPK, CLCN1/ZNF9 or SCN4A. The 50 Hz RNS study can help in differential diagnosis and optimization of the molecular genetic testing in patients with НMS.

Establishment of an induced pluripotent stem cell line (NCKDi003-A) from a patient with X-linked Dent disease (X-Dent) carrying the hemizygote mutation p. T277P (c. 829A > C) in the CLCN5 gene

Dent disease (DD) is a rare X-linked proximal tubulopathy associated with low molecular weight proteinuria (LMWP), hypercalciuria, nephrolithiasis and phosphoruria, which may progress to chronic kidney disease (CKD). About 60% of cases are caused by the mutation in CLCN5 gene. Recently, we identified a mutation in the sequence of homodimer of CLCN5 gene in a patient with DD. The Peripheral Blood Mononuclear Cells (PBMCs) of the patient were obtained and a line of induced pluripotent stem cells (iPSCs) was successfully generated. The iPSC line will be useful for further study of the pathogenesis and drug screening for DD.

Regulation of ClC-2 Chloride Channel Proteostasis by Molecular Chaperones: Correction of Leukodystrophy-Associated Defect.

The ClC-2 channel plays a critical role in maintaining ion homeostasis in the brain and the testis. Loss-of-function mutations in the ClC-2-encoding human CLCN2 gene are linked to the white matter disease leukodystrophy. Clcn2-deficient mice display neuronal myelin vacuolation and testicular degeneration. Leukodystrophy-causing ClC-2 mutant channels are associated with anomalous proteostasis manifesting enhanced endoplasmic reticulum (ER)-associated degradation. The molecular nature of the ER quality control system for ClC-2 protein remains elusive. In mouse testicular tissues and Leydig cells, we demonstrated that endogenous ClC-2 co-existed in the same protein complex with the molecular chaperones heat shock protein 90β (Hsp90β) and heat shock cognate protein (Hsc70), as well as the associated co-chaperones Hsp70/Hsp90 organizing protein (HOP), activator of Hsp90 ATPase homolog 1 (Aha1), and FK506-binding protein 8 (FKBP8). Further biochemical analyses revealed that the Hsp90β-Hsc70 chaperone/co-chaperone system promoted mouse and human ClC-2 protein biogenesis. FKBP8 additionally facilitated membrane trafficking of ClC-2 channels. Interestingly, treatment with the Hsp90-targeting small molecule 17-allylamino-17-demethoxygeldanamycin (17-AAG) substantially boosted ClC-2 protein expression. Also, 17-AAG effectively increased both total and cell surface protein levels of leukodystrophy-causing loss-of-function ClC-2 mutant channels. Our findings highlight the therapeutic potential of 17-AAG in correcting anomalous ClC-2 proteostasis associated with leukodystrophy.

Exercise test for patients with new-onset paroxysmal kinesigenic dyskinesia.

The pathogenesis of primary paroxysmal kinesigenic dyskinesia (PKD) remains unclear, and channelopathy is a possibility. In a pilot study, we found that PKD patients had abnormal exercise test (ET) results. To investigate the ET performances in patients affected by PKD, and the role of the channelopathies in the pathogenesis of PKD, we compared the ET results of PKD patients, control subjects, and hypokalemic periodic paralysis (HoPP) patients, and we analyzed ET changes in 32 PKD patients before and after treatment. Forty-four PKD patients underwent genetic testing for the PRRT2, SCN4A, and CLCN1 genes. Sixteen of 59 (27%) patients had abnormal ET results in the PKD group, while 28 of 35 (80%) patients had abnormal ET results in the HoPP group. Compared with the control group, the PKD group showed a significant decrease in the compound muscle action potential (CMAP) amplitude and area after the long ET (LET), while the HoPP group showed not only greater decreases in the CMAP amplitude and area after the LET but also greater increases in the CMAP amplitude and area immediately after the LET. The ET parameters before and after treatment were not significantly different. Nine of 44 PKD patients carried PRRT2 mutations, but the gene abnormalities were unrelated to any ET parameter. The PKD group demonstrated an abnormal LET result by electromyography (EMG), and this abnormality did not seem to correlate with the PRRT2 variant or sodium channel blocker therapy.

Diversity of functional alterations of the ClC-5 exchanger in the region of the proton glutamate in patients with Dent disease 1.

Mutations in the CLCN5 gene encoding the 2Cl- /1H+ exchanger ClC-5 are associated with Dent disease 1, an inherited renal disorder characterized by low-molecular-weight (LMW) proteinuria and hypercalciuria. In the kidney, ClC-5 is mostly localized in proximal tubule cells,where it is thought to play a key role in the endocytosis of LMW proteins. Here, we investigated the consequences of eight previously reported pathogenic missense mutations of ClC-5 surrounding the "proton glutamate" that serves as a crucial H+ -binding site for the exchanger. A complete loss of function was observed for a group of mutants that were either retained in the endoplasmic reticulum of HEK293T cells or unstainable at plasma membrane due to proteasomal degradation. In contrast, the currents measured for thesecond group of mutations in Xenopus laevis oocytes were reduced. Molecular dynamics simulations performed on a ClC-5 homology model demonstrated that such mutations mightalter ClC-5 protonation by interfering with the water pathway. Analysis of clinical data from patients harboring these mutations demonstrated no phenotype/genotype correlation. This study reveals that mutations clustered in a crucial region of ClC-5 have diverse molecular consequences in patients with Dent disease 1, ranging from altered expression to defects in transport.

Functional analysis of the F337C mutation in the CLCN1 gene associated with dominant myotonia congenita reveals an alteration of the macroscopic conductance and voltage dependence.

ABSTRACTBackground Myotonia congenita (MC) is a common channelopathy affecting skeletal muscle and which is due to pathogenic variants within the CLCN1 gene. Various alterations in the function of the channel have been reported and we here illustrate a novel one.MethodsA patient presenting the symptoms of myotonia congenita was shown to bear a new heterozygous missense variant in exon 9 of the CLCN1 gene (c.1010 T > G, p.(Phe337Cys)). Confocal imaging and patch clamp recordings of transiently transfected HEK293 cells were used to functionally analyze the effect of this variant on channel properties.ResultsConfocal imaging showed that the F337C mutant incorporated as well as the WT channel into the plasma membrane. However, in patch clamp, we observed a smaller conductance for F337C at −80 mV. We also found a marked reduction of the fast gating component in the mutant channels, as well as an overall reduced voltage dependence.ConclusionTo our knowledge, this is the first report of a mixed alteration in the biophysical properties of hClC‐1 consisting of a reduced conductance at resting potential and an almost abolished voltage dependence.

Clinical manifestation and genetic findings in three boys with low molecular Weight Proteinuria - three case reports for exploring Dent Disease and Fanconi syndrome

BackgroundDent disease is an X-linked form of progressive renal disease. This rare disorder was characterized by hypercalciuria, low molecular weight (LMW) proteinuria and proximal tubular dysfunction, caused by pathogenic variants in CLCN5 (Dent disease 1) or OCRL (Dent disease 2) genes. Fanconi syndrome is a consequence of decreased water and solute resorption in the proximal tubule of the kidney. Fanconi syndrome caused by proximal tubular dysfunction such as Dent disease might occur in early stage of the disease.Case presentationThree cases reported in this study were 3-, 10- and 14-year-old boys, and proteinuria was the first impression in all the cases. All the boys presented with LMW proteinuria and elevated urine albumin-to-creatinine ratio (ACR). Case 1 revealed a pathogenic variant in exon 11 of CLCN5 gene [NM_001127899; c.1444delG] and a nonsense mutation at nucleotide 1509 [p.L503*], and he was diagnosed as Dent disease 1. Case 2 carried a deletion of exon 3 and 4 of OCRL1 gene [NM_000276.4; c.120-238delG…A] and a nonsense mutation at nucleotide 171 in exon 5 [p.E57*], and this boy was diagnosed as Dent disease 2. Genetic analysis of Case 3 showed a missense mutation located in exon 2 of HNF4A gene [EF591040.1; c.253C > T; p.R85W] which is responsible for Fanconi syndrome. All of three pathogenic variants were not registered in GenBank.ConclusionsUrine protein electrophoresis should be performed for patients with proteinuria. When patients have LMW proteinuria and/or hypercalciuria, definite diagnosis and identification of Dent disease and Fanconi syndrome requires further genetic analyses.

Diabetic Cidp-Like Neuropathy in a Pazient With Becker’s Myotonia

Becker’s disease (BM) is an autosomal recessive congenital myotonia, characterized by impaired muscle relaxation after voluntary contraction and variable degrees of muscle weakness. BM is caused by mutations in CLCN1 gene on chromosome 7q35 encoding the major skeletal muscle chloride channel CLC-1. Although neuropathy has been associated with myotonic dystrophy, to date no case of neuropathy during the course of BM has been reported. Herein we describe the case of a patient with BM and diabetes who progressive gait disturbance, distal paresthesias, muscle weakness. Neurophysiological assessment and diagnostic lumbar puncture revealed the presence of a chronic inflammatory demyelinating neuropathy. The case here presented developed a diabetic chronic demyelinating inflammatory neuropathy resembling CIDP during the course of BM. The most consistent hypothesis explaining such an unusual association is that the inflammatory neuropathy is related to the concomitant occurrence of diabetes.

A Novel Mutation in the CLCN1 Gene Causing Autosomal Recessive Myotonia Congenita in Siblings.

A Novel Mutation in the CLCN1 Gene Causing Autosomal Recessive Myotonia Congenita in Siblings.

Next-generation sequencing application to investigate skeletal muscle channelopathies in a large cohort of Italian patients

Non-dystrophic myotonias and periodic paralyses are a heterogeneous group of disabling diseases classified as skeletal muscle channelopathies. Their genetic characterization is essential for prognostic and therapeutic purposes; however, several genes are involved. Sanger-based sequencing of a single gene is time-consuming, often expensive; thus, we designed a next-generation sequencing panel of 56 putative candidate genes for skeletal muscle channelopathies, codifying for proteins involved in excitability, excitation-contraction coupling, and metabolism of muscle fibres. We analyzed a large cohort of 109 Italian patients with a suspect of NDM or PP by next-generation sequencing. We identified 24 patients mutated in CLCN1 gene, 15 in SCN4A, 3 in both CLCN1 and SCN4A, 1 in ATP2A1, 1 in KCNA1 and 1 in CASQ1. Eight were novel mutations: p.G395Cfs*32, p.L843P, p.V829M, p.E258E and c.1471+4delTCAAGAC in CLCN1, p.K1302R in SCN4A, p.L208P in ATP2A1 and c.280–1G>C in CASQ1 genes. This study demonstrated the utility of targeted next generation sequencing approach in molecular diagnosis of skeletal muscle channelopathies and the importance of the collaboration between clinicians and molecular geneticists and additional methods for unclear variants to make a conclusive diagnosis.

Small molecules restore the function of mutant CLC5 associated with Dent disease.

Dent disease type 1 is caused by mutations in the CLCN5 gene that encodes CLC5, a 2Cl−/H+ exchanger. The CLC5 mutants that have been functionally analysed constitute three major classes based on protein expression, cellular localization and channel function. We tested two small molecules, 4‐phenylbutyrate (4PBA) and its analogue 2‐naphthoxyacetic acid (2‐NOAA), for their effect on mutant CLC5 function and expression by whole‐cell patch‐clamp and Western blot, respectively. The expression and function of non‐Class I CLC5 mutants that have reduced function could be restored by either treatment. Cell viability was reduced in cells treated with 2‐NOAA. 4PBA is a FDA‐approved drug for the treatment of urea cycle disorders and offers a potential therapy for Dent disease.

Pathobiologic Mechanisms of Neurodegeneration in Osteopetrosis Derived From Structural and Functional Analysis of 14 ClC-7 Mutants.

ClC-7 is a chloride-proton antiporter of the CLC protein family. In complex with its accessory protein Ostm-1, ClC-7 localizes to lysosomes and to the osteoclasts' ruffled border, where it plays a critical role in acidifying the resorption lacuna during bone resorption. Gene inactivation in mice causes severe osteopetrosis, neurodegeneration, and lysosomal storage disease. Mutations in the human CLCN7 gene are associated with diverse forms of osteopetrosis. The functional evaluation of ClC-7 variants might be informative with respect to their pathogenicity, but the cellular localization of the protein hampers this analysis. Here we investigated the functional effects of 13 CLCN7 mutations identified in 13 new patients with severe or mild osteopetrosis and a known ADO2 mutation. We mapped the mutated amino acid residues in the homology model of ClC-7 protein, assessed the lysosomal colocalization of ClC-7 mutants and Ostm1 through confocal microscopy, and performed patch-clamp recordings on plasma-membrane-targeted mutant ClC-7. Finally, we analyzed these results together with the patients' clinical features and suggested a correlation between the lack of ClC-7/Ostm1 in lysosomes and severe neurodegeneration. © 2020 American Society for Bone and Mineral Research (ASBMR).

Hereditary myotonia in American Bulldog associated with a novel frameshift mutation in the CLCN1 gene

Hereditary myotonia (HM) is a genetic disorder that occurs due to mutations in the chloride channel and results in delayed relaxation of the skeletal muscles. HM has been described in 12 dog breeds, and in five of them, molecular studies of this disorder were performed and mutations in the CLCN1 gene were described. In this study, an affected American Bulldog with HM clinically characterized by muscle hypertrophy, myotonic discharges, and nondystrophic myotonia with a “warm-up” phenomenon was evaluated, and the candidate canine CLCN1 gene was sequenced. The molecular analysis revealed a frameshift mutation NM_001003124.2:c.436_437insCTCT that resulted in a frameshift and a premature stop codon NP_001003124.1:pTyr146SerfsTer49 . Two aberrant alternative CLCN1 transcripts were observed in an affected dog, the expected transcript with the 4 bp insertion, NM_001003124.2:r.436_437insctct, and an unexpected transcript containing parts of intron 6 in addition to the insertion in exon 4, NM_001003124.2:[r.436_437insctct;r.774_775ins79]. In conclusion, the frameshift mutation in the CLCN1 gene is associated with autosomal recessive HM in American Bulldog and this study constitutes the first description of the disease in this breed.

Recessive myotonia congenita caused by a homozygous splice site variant in CLCN1 gene: a case report

BackgroundMyotonia congenita is a rare neuromuscular disease, which is characterized by a delay in muscle relaxation after evoked or voluntary contraction. Myotonia congenita can be inherited in a dominant (Thomsen disease) and recessive form (Becker disease) and both are caused by pathogenic variants in the CLCN1 gene. Noncanonical splice site variants are often classified as variants of uncertain significance, due to insufficient accuracy of splice-predicting tools. Functional analysis using minigene plasmids is widely used in such cases. Moreover, functional analysis is very useful in investigation of the disease pathogenesis, which is necessary for development of future therapeutic approaches. To our knowledge only one noncanonical splice site variant in the CLCN1 gene was functionally characterized to date. We further contribute to this field by evaluation the molecular mechanism of splicing alteration caused by the c.1582 + 5G > A in a homozygous state.Case presentationWe report a clinical case of an affected 6-y.o boy with athletic appearance due to muscle hypertrophy, calf muscle stiffness, cramping and various myotonic signs in a consanguineous family with no history of neuromuscular disorders. The neurological examination showed percussion-activated myotonia in the hands and legs. Plasma creatine kinase enzyme and transaminases levels were normal. Electromyography at the time of examination shows myotonic runs in the upper and lower extremities.ConclusionsFunctional analysis of the variant in a minigene system showed alteration of splicing leading to loss of function, thereby confirming that the variant is pathogenic.

Identification of Somatic Mutations in CLCN2 in Aldosterone-Producing Adenomas.

Somatic mutations driving aldosterone production have been identified in approximately 90% of aldosterone-producing adenomas (APAs) using an aldosterone synthase (CYP11B2) immunohistochemistry (IHC)-guided DNA sequencing approach. In the present study, using CYP11B2-guided whole-exome sequencing (WES) and targeted amplicon sequencing, we detected 2 somatic variants in CLCN2 in 2 APAs that were negative for currently known aldosterone-driver mutations. The CLCN2 gene encodes the voltage-gated chloride channel ClC-2. CLCN2 germline variants have previously been shown to cause familial hyperaldosteronism type II. Somatic mutations in CLCN2 were identified in 2 of 115 APAs, resulting in a prevalence of 1.74%. One of the CLCN2 somatic mutations (c.G71A,p.G24D) was identical to a previously described germline variant causing early-onset PA, but was present only as a somatic mutation. The second CLCN2 mutation, which affects the same region of the gene, has not been reported previously (c.64-2_74del). These findings prove that WES of CYP11B2-guided mutation-negative APAs can help determine rarer genetic causes of sporadic PA.