C283Y mutation and other C‐terminal nucleotide changes in the γ‐sarcoglycan gene in the Bulgarian gypsy population

Sarcoglycanopathies, affecting the dystrophin‐associated sarcoglycan (SG) complex, are a heterogeneous group of neuromuscular disorders. A subgroup of these disorders, limb‐girdle muscular dystrophy type 2C (LGMD2C) is an autosomal recessive disorder, clinically manifested as an early onset, severe Duchenne‐like muscular dystrophy. LGMD2C is caused by mutations in the γ‐SG gene, localized on 13q12. Recently, a number of mutations have been described in that gene, among which C283Y, a “private” Gypsy mutation (eight codons before the 3′ end of the gene) is detected. In this article, we report on a single‐strand conformation polymorphism (SSCP) method for fast C283Y mutation detection, using direct dry blood spot amplification. The method permits a large number of samples to be easily screened. To check heterozygote carriers of C283Y mutation among Gypsy population in Bulgaria, the SSCP analysis was applied on 400 Gypsy newborns from northeast Bulgaria. Our results show 2.25% of heterozygosity, which means that 1 in 50 Gypsies carries the mutation. Moreover, new SSCP migration patterns were detected that revealed two polymorphisms still unavailable in the literature. One of these changes was 984G→A, leading to substitution of conserved serine at position 287 with asparagine and the second one is 1049C→G at the 3′ UTR (untranslated region). The present data could help the understanding the role of these sequences for the protein function. Hum Mutat 14:40–44, 1999. © 1999 Wiley‐Liss, Inc.     

Correlations between clinical severity, genotype and muscle pathology in limb girdle muscular dystrophy type 2A

Background

Limb girdle muscular dystrophy type 2A (LGMD2A) is characterised by wide variability in clinical features and rate of progression. Patients with two null mutations usually have a rapid course, but in the remaining cases (two missense mutations or compound heterozygote mutations) prognosis is uncertain.

Methods

We conducted what is to our knowledge the first systematic histopathological, biochemical and molecular investigation of 24 LGMD2A patients, subdivided according to rapid or slow disease progression, to determine if some parameters could correlate with disease progression.

Results

We found that muscle histopathology score and the extent of regenerating and degenerating fibres could be correlated with the rate of disease course when the biochemical and molecular data do not offer sufficient information. Comparison of clinical and muscle histopathological data between LGMD2A and four other types of LGMD (LGMD2B–E) also gave another important and novel result. We found that LGMD2A has significantly lower levels of dystrophic features (ie degenerating and regenerating fibres) and higher levels of chronic changes (ie lobulated fibres) compared with other LGMDs, particularly LGMD2B. These results might explain the observation that atrophic muscle involvement seems to be a clinical feature peculiar to LGMD2A patients.

Conclusions

Distinguishing patterns of muscle histopathological changes in LGMD2A might reflect the effects of a disease‐specific pathogenetic mechanism and provide clues complementary to genetic data.     

The genetic profile of dysferlinopathy in a cohort of 209 cases: Genotype–phenotype relationship and a hotspot on the inner DysF domain

Dysferlinopathy is a group of autosomal recessive muscular dystrophies caused by variants in the dysferlin gene (DYSF), with variable proximal and distal muscle involvement. We performed DYSF gene analyses of 200 cases suspected of having dysferlinopathy (Cohort 1), and identified diagnostic variants in 129/200 cases, including 19 novel variants. To achieve a comprehensive genetic profile of dysferlinopathy, we analyzed the variant data from 209 affected cases from unrelated 209 families, including 80 previously diagnosed and 129 newly diagnosed cases (Cohort 2). Among the 90 types of variants identified in 209 cases, the NM_003494.3: c.2997G>T; p.Trp999Cys, was the most frequent (96/420; 22.9%), followed by c.1566C>G; p.Tyr522* (45/420; 10.7%) on an allele base. p.Trp999Cys was found in 70/209 cases (33.5%), including 20/104 cases (19.2%) with the Miyoshi muscular phenotype and 43/82 cases (52.4%) with the limb‐girdle phenotype. In the analysis of missense variants, p.Trp992Arg, p.Trp999Arg, p.Trp999Cys, p.Ser1000Phe, p.Arg1040Trp, and p.Arg1046His were located in the inner DysF domain, representing in 113/160 missense variants (70.6%). This large cohort highlighted the frequent missense variants located in the inner DysF domain as a hotspot for missense variants among our cohort of 209 cases (>95%, Japanese) and hinted at their potential as targets for future therapeutic strategies.     

Functional consequences of an LMNA mutation associated with a new cardiac and non-cardiac phenotype

Heritable dilated cardiomyopathy is a genetically highly heterogeneous disease. To date 17 different chromosomal loci have been described for autosomal dominant forms of dilated cardiomyopathy with or without additional clinical manifestations. Among the 10 mutated genes associated with dilated cardiomyopathy, the lamin A/C (LMNA) gene has been reported in forms associated with conduction-system disease with or without skeletal muscle myopathy. For the first time, we report here a French family affected with a new phenotype composed of an autosomal dominant severe dilated cardiomyopathy with conduction defects or atrial/ventricular arrhythmias, and a specific quadriceps muscle myopathy. In all previously reported cases with both cardiac and neuromuscular involvement, neuromuscular disorders preceded cardiac abnormalities. The screening of the coding sequence of the LMNA gene on all family members was performed and we identified a missense mutation (R377H) in the lamin A/C gene that cosegregated with the disease in the family. Cell transfection experiments showed that the R377H mutation leads to mislocalization of both lamin and emerin. These results were obtained in both muscular (C2C12) and non-muscular cells (COS-7). This new phenotype points out the wide spectrum of neuromuscular and cardiac manifestations associated with lamin A/C mutations, with the functional consequence of this mutation seemingly associated with a disorganization of the lamina.     

LAMA2 gene mutation update: Toward a more comprehensive picture of the laminin‐α2 variome and its related phenotypes

Congenital muscular dystrophy type 1A (MDC1A) is one of the main subtypes of early‐onset muscle disease, caused by disease‐associated variants in the laminin‐α2 (LAMA2) gene. MDC1A usually presents as a severe neonatal hypotonia and failure to thrive. Muscle weakness compromises normal motor development, leading to the inability to sit unsupported or to walk independently. The phenotype associated with LAMA2 defects has been expanded to include milder and atypical cases, being now collectively known as LAMA2‐related muscular dystrophies (LAMA2‐MD). Through an international multicenter collaborative effort, 61 new LAMA2 disease‐associated variants were identified in 86 patients, representing the largest number of patients and new disease‐causing variants in a single report. The collaborative variant collection was supported by the LOVD‐powered LAMA2 gene variant database ( https://www.LOVD.nl/LAMA2 ), updated as part of this work. As of December 2017, the database contains 486 unique LAMA2 variants (309 disease‐associated), obtained from direct submissions and literature reports. Database content was systematically reviewed and further insights concerning LAMA2‐MD are presented. We focus on the impact of missense changes, especially the c.2461A > C (p.Thr821Pro) variant and its association with late‐onset LAMA2‐MD. Finally, we report diagnostically challenging cases, highlighting the relevance of modern genetic analysis in the characterization of clinically heterogeneous muscle diseases.     

Genetic and clinical findings in a Chinese cohort of patients with collagen VI‐related myopathies

Collagen VI‐related myopathy, caused by pathogenic variants in the genes encoding collagen VI, represents a clinical continuum from Ullrich congenital muscular dystrophy (UCMD) to Bethlem myopathy (BM). Clinical data of 60 probands and their family members were collected and muscle biopsies of 26 patients were analyzed. COL6A1, COL6A2 and COL6A3 exons were analyzed by direct sequencing or next generation sequencing (NGS). Sixty patients were characterized by delayed motor milestones, muscle weakness, skin and joint changes with 40 UCMD and 20 BM. Muscle with biopsies revealed dystrophic changes and showed completely deficiency of collagen VI or sarcolemma specific collagen VI deficiency. We identified 62 different pathogenic variants in these 60 patients, with 34 were first reported while 28 were previously known; 72 allelic pathogenic variants in COL6A1 (25/72, 34.7%), COL6A2 (33/72, 45.8%) and COL6A3 (14/72, 19.4%). We also found somatic mosaic variant in the parent of 1 proband by personal genome machine amplicon deep sequencing for mosaicism. Here we provide clinical, histological and genetic evidence of collagen VI‐related myopathy in 60 Chinese patients. NGS is a valuable approach for diagnosis and accurate diagnosis provides useful information for genetic counseling of related families.     

A Clinical and Molecular Analysis of Branchio‐Oculo‐Facial Syndrome Patients in Russia Revealed New Mutations in TFAP2A

Branchio‐oculo‐facial syndrome (BOFS, OMIM# 113620) is a rare autosomal dominant disorder characterised by branchial cleft sinus defects, ocular anomalies and facial dysmorphisms, including lip or palate cleft or pseudocleft, and is associated with mutations in the TFAP2A gene. Here, we performed clinical analysis and mutation diagnostics in seven BOFS patients in Russia. The phenotypic presentation of BOFS observed in three patients showed high heterogeneity, including variation in its main clinical manifestations (linear loci of cervical cutaneous aplasia, ocular anomalies and orofacial cleft). In certain other cases, isolated ocular anomalies, or an orofacial cleft with accessory BOFS symptoms, were observed. In five BOFS patients, conductive hearing loss was diagnosed. Direct sequencing of the coding region of the TFAP2A gene revealed missense mutations in four BOFS patients. One patient was observed to have a previously described mutation (p.Arg251Gly), while three patients from two families were found to have novel mutations: p.Arg213Ser and p.Val210Asp. These novel mutations were not present in healthy members of the same family and therefore should be classified as de novo.     

Ficolin‐A/2, acting as a new regulator of macrophage polarization,mediates the inflammatory response in experimental mouse colitis

Human ficolin‐2 (FCN‐2) and mouse ficolin‐A (FCN‐A, a ficolin‐2‐like molecule in mouse) are activators of the lectin complement pathway, present in normal plasma and usually associated with infectious diseases, but little is known about the role of FCN‐A/2 in inflammatory bowel disease (IBD). In our present study, we found that patients with IBD exhibited much higher serum FCN‐2 levels than healthy controls. In the dextran sulphate sodium‐induced acute colitis mouse model, FCN‐A knockout mice showed much milder disease symptoms with less histological damage, lower expression levels of pro‐inflammatory cytokines [interleukin‐6 (IL‐6), IL‐1β and tumour necrosis factor‐α (TNF‐α)], chemokines (CXCL1/2/10 and CCL4) and higher levels of the anti‐inflammatory cytokine IL‐10 compared with wild‐type mice. We demonstrated that FCN‐A/2 exacerbated the inflammatory pathogenesis of IBD by stimulating M1 polarization through the TLR4/MyD88/MAPK/NF‐κB signalling pathway in macrophages. Hence, our data suggest that FCN‐A/2 may be used as a novel therapeutic target for IBD.     

Additive hypothermic effects of the 5‐HT1A receptor agonist 8‐OH‐DPAT and the dopamine D2/3 receptor agonist 7‐OH‐DPAT in the rat

The objective of this study was to examine possible interactions between serotonergic and dopaminergic agents lowering core temperature via stimulation of 5‐HT_1A and dopamine (DA) D₂ receptors, respectively. The effects of the 5‐HT_1A receptor agonist (±)‐8‐hydroxy‐2‐(di‐n‐propylamino)tetralin HBr (8‐OH‐DPAT) and the DA D_2/3 receptor agonist 7‐OH‐DPAT on core temperature was monitored in adult male Wistar rats, approximately 300 g body weight. The temperature probe was connected to a PC‐assisted temperature instrument, and an automated printer device was activated when the temperature reading had stabilized (±0.1 °C) for 10 s. As expected, 7‐OH‐DPAT [0.5 and 2.0 μmol kg^–1 subcutaneous (s.c.)] as well as 8‐OH‐DPAT (0.15–2.4 μmol kg^–1 s.c.), produced a dose‐dependent hypothermia. When combined, there were additive effects of the two compounds, although the effects of 7‐OH‐DPAT were attenuated by 8‐OH‐DPAT at the higher doses (0.6–2.4 μmol kg^–1), in all probability because of emerging DA D₂ receptor blocking properties of the latter compound.     

A role for gangliosides and β1‐integrin in the motility of olfactory ensheathing glia

Olfactory ensheathing glia (OEG) are found in the olfactory mucosa, nerve and bulb, and provide in vivo ensheathment for the unmyelinated olfactory axons within the central and peripheral nervous system domains. OEG cells are able to migrate long distances within the neuropil of the central nervous system. Because gangliosides such as 9‐O‐acetyl GD3 have crucial regulatory roles in neuronal migration during development, we analyzed whether OEG in organotypical cultures are revealed by anti‐9‐O‐acetyl GD3 and/or gangliosides are recognized by the A2B5 antibody (G‐A2B5), and whether these gangliosides are involved in OEG migration. Our results showed that all OEG migrating out of a section of olfactory bulb onto a laminin substrate bound to the 9‐O‐acetyl GD3 and A2B5 antibodies, and that 2′,3′‐cyclic nucleotide phosphodiesterase (CNPase) colocalized with 9‐O‐acetyl GD3 and with G‐A2B5. Additionally, we showed that the immune blockade of 9‐O‐acetyl GD3 or G‐A2B5 reduced the migration of OEG on laminin, and that 9‐O‐acetyl GD3 and G‐A2B5 colocalized with the β1‐integrin subunit. We also confirmed the phenotype of in‐vitro‐grown OEG cells derived from adult rats, showing that they express CNPase, and also α‐smooth muscle actin, which is not expressed by Schwann cells. Our data showed that the gangliosides 9‐O‐acetyl GD3 and G‐A2B5 participate in the migratory activity of OEG cells, and that the β1‐integrin subunit colocalizes with these gangliosides. These results suggest a new role for β1‐integrin and gangliosides in the polarized migration of OEG cells, and provide new information on the molecules controlling OEG motility and behavior.     

Novel KCNQ2 and KCNQ3 Mutations in a Large Cohort of Families with Benign Neonatal Epilepsy: First Evidence for an Altered Channel Regulation by Syntaxin‐1A

Mutations in the KCNQ2 and KCNQ3 genes encoding for K_v7.2 (KCNQ2; Q2) and K_v7.3 (KCNQ3; Q3) voltage‐dependent K⁺ channel subunits, respectively, cause neonatal epilepsies with wide phenotypic heterogeneity. In addition to benign familial neonatal epilepsy (BFNE), KCNQ2 mutations have been recently found in families with one or more family members with a severe outcome, including drug‐resistant seizures with psychomotor retardation, electroencephalogram (EEG) suppression‐burst pattern (Ohtahara syndrome), and distinct neuroradiological features, a condition that was named “KCNQ2 encephalopathy.” In the present article, we describe clinical, genetic, and functional data from 17 patients/families whose electroclinical presentation was consistent with the diagnosis of BFNE. Sixteen different heterozygous mutations were found in KCNQ2, including 10 substitutions, three insertions/deletions and three large deletions. One substitution was found in KCNQ3. Most of these mutations were novel, except for four KCNQ2 substitutions that were shown to be recurrent. Electrophysiological studies in mammalian cells revealed that homomeric or heteromeric KCNQ2 and/or KCNQ3 channels carrying mutant subunits with newly found substitutions displayed reduced current densities. In addition, we describe, for the first time, that some mutations impair channel regulation by syntaxin‐1A, highlighting a novel pathogenetic mechanism for KCNQ2‐related epilepsies.     

miR‐155 overexpression is followed by downregulation of its target gene,NFE2L2, and altered pattern of VEGFA expression in the liver of melanoma B16‐bearing mice at the premetastatic stage

MicroRNAs are involved in the control of tumour progression and in metastatic cascade dynamics. However, the role of microRNAs in distant organ reorganization at the premetastatic stage is less clear, although the process of premetastatic niche formation is a crucial event according to modern concepts of tumour dissemination. The role of the present study was to investigate the expression levels of miR‐155, miR‐21, miR‐205 and miR‐let7b, as well as that of their target genes, in target organs of melanoma metastasis at the premetastatic stage. The expression levels of both the pro‐oncogenic miR‐155 and the tumour suppressive miR‐205 were found to be altered in the premetastatic liver of melanoma B16‐bearing mice. Bioinformatics analysis identified the target genes of miR‐155 to be nuclear factor, erythroid 2 like 2 (NFE2L2), secretogranin II, miR‐205, semaphorin 5A and vascular endothelial growth factor A (VEGFA). Among those, the redox status regulatory factor NFE2L2 was downregulated, which corresponded to increased levels of miR‐155. Due to the ability of pro‐oxidative events to initiate angiogenesis, VEGFA levels were evaluated in the premetastatic liver by immunohistochemistry, which revealed increased VEGFA expression in the central parts of the organ and diminished expression in the periphery. Taken together, these findings may support the concept of functional organ reorganization due to melanoma progression.     

Mendelian Disorders of Cornification Caused by Defects in Intracellular Calcium Pumps: Mutation Update and Database for Variants in ATP2A2 and ATP2C1 Associated with Darier Disease and Hailey–Hailey Disease

The two disorders of cornification associated with mutations in genes coding for intracellular calcium pumps are Darier disease (DD) and Hailey–Hailey disease (HHD). DD is caused by mutations in the ATP2A2 gene, whereas the ATP2C1 gene is associated with HHD. Both are inherited as autosomal‐dominant traits. DD is mainly defined by warty papules in seborrheic and flexural areas, whereas the major symptoms of HHD are vesicles and erosions in flexural skin. Both phenotypes are highly variable. In 12%–40% of DD patients and 12%–55% of HHD patients, no mutations in ATP2A2 or ATP2C1 are found. We provide a comprehensive review of clinical variability in DD and HHD and a review of all reported mutations in ATP2A2 and ATP2C1. Having the entire spectrum of ATP2A2 and ATP2C1 variants allows us to address the question of a genotype–phenotype correlation, which has not been settled unequivocally in DD and HHD. We created a database for all mutations in ATP2A2 and ATP2C1 using the Leiden Open Variation Database (LOVD v3.0), for variants reported in the literature and future inclusions. This data may be of use as a reference tool in further research on treatment of DD and HHD.