Splicing modulation mediated by small nuclear RNAs as therapeutic approaches for muscular dystrophies

Splice-modulation therapy aiming at correcting genetic defects by molecular manipulation of the premessenger RNA is a promising novel therapeutic approach for genetic diseases. In recent years, these new RNA based strategies, mostly mediated by antisense oligonucleotides (AO), have demonstrated encouraging results for muscular dystrophies, a heterogeneous group of genetic disorders characterized by muscle weakness and wasting. In particular, the clinical evaluation of antisense-mediated exon-skipping for the treatment of Duchenne muscular dystrophy has shown convincing data and therefore raised hopes and expectations for neuromuscular disorders therapy. However, AO-mediated splicing modulation still faces major hurdles such as low efficacy in specific tissues, poor cellular uptake and relatively rapid clearance from circulation, which means repeated administrations are required to achieve some therapeutic efficacy. To overcome these limitations, small nuclear RNAs (snRNA) have been used to shuttle the antisense sequences, offering the advantage of a correct subcellular localization with pre-mRNAs and the potential of a permanent correction when introduced into viral vectors. Here we review the recent progress in the development of snRNA mediated splicing modulation for muscular dystrophies, focusing on the advantages offered by this technology over classical AOs but also the challenges limiting their clinical application.     

Diagnostic protein expression in human muscle biopsies

Using immunohistochemistry in diagnosing neuromuscular diseases is meant to enhance the diagnostic yield in two ways. The first application aims at visualizing molecules which are developmentally, neurally, and/or immunologically regulated and not expressed by normal muscle. They are upregulated in pathological conditions and may help assign a given muscular biopsy to one of the main diagnostic entities (muscular dystrophies, inflammatory myopathy, neurogenic atrophy). In the past, muscle-specific molecules with a defined expression pattern during fetal myogenesis served as antigens, with the rationale that the developmental program was switched on in new fibers. Recently, myofibers in diseased muscle are thought of as targets of stimuli which are released by macrophages in muscular dystrophy, by lymphocytes in inflammatory myopathies, or by a lesioned peripheral nerve in neurogenic atrophies. This has somewhat blurred the borders between the diagnostic groups, for certain molecules, e.g. cytokines, may be upregulated after experimental necrotization, denervation, and also in inflammatory myopathies. In the second part of this review we summarise the experiences of a Centre in the North of England that specialises in the diagnosis and clinical support of patients with muscular dystrophy. Emphasis is placed on the use of protein expression to guide mutation analysis, particularly in the limb-girdle muscular dystrophies (a group of diseases that are very difficult to differentiate on clinical grounds alone). We confirm that genetic analysis is essential to corroborate the results of protein analysis in certain conditions (particularly in calpainopathy). However, we conclude that analysing biopsies for abnormal protein expression is very useful in aiding the decision between alternative diagnoses.     

Unclassifiable arrhythmic cardiomyopathy associated with Emery–Dreifuss caused by a mutation in FHL1

Emery–Dreifuss muscular dystrophy (EDMD) is a heterogeneous genetic disorder characterized by peripheral muscular weakness often associated with dilated cardiomyopathy. We characterize clinically a large family with a mutation in FHL1 gene (p.Cys255Ser). Penetrance was 44%, 100% for males and 18% for females. The heart was the main organ involved. Affected adult males had mild hypertrophy, systolic dysfunction and restriction with non‐dilated ventricles. Carriers had significant QTc prolongation. The proband presented with resuscitated cardiac arrest. There were two transplants. Pathological study of explanted heart showed fibrofatty replacement and scarring consistent with arrhythmogenic cardiomyopathy and prominent left ventricular trabeculations. Myopathic involvement was evident in all males. Females had no significant neuromuscular disease. Mutations in FHL1 cause unclassifiable cardiomyopathy with coexisting EDMD. Prognosis is poor and systolic impairment and arrhythmias are frequent. Thrombopenia and raised creatine phosphokinase should raise suspicion of an FHL‐1 disorder in X‐linked cardiomyopathy.     

Hypofunction of the sympathetic nervous system is an etiologic factor for a wide variety of chronic treatment-refractory pathologic disorders which all respond to therapy with sympathomimetic amines

The hypothesis set forth is that the basis for a great many chronic debilitating conditions that involve almost all of the physiologic systems of the body may have as the underlying cause and a common link between them, i.e., hypofunction of the sympathetic nervous system. The hypothesis considers that one of the main functions of the sympathetic nervous system is to diminish cellular permeability. Thus sympathetic hypofunction may lead to absorption of chemicals and toxins into tissues that were supposed to be impervious leading to inflammation and other adverse consequences which then cause a wide variety of symptoms. These symptoms may include pain or diminished muscular function leading to various pain syndromes or conditions related to diminished muscular function. Furthermore since the sympathetic nervous system is involved in body homeostasis and temperature regulation, sympathetic nervous system hypofunction could lead to disorders in these areas, e.g., vasomotor symptoms and edema. This defect in sympathetic nervous system has a genetic predisposition but relatives, e.g., siblings or children may manifest in a different manner which suggests some influence of external factors causing one physiological system to be more prone than another to malfunction under conditions of sympathetic hypofunction. Evidence to support this hypothesis has been provided by a large number of published anecdotes demonstrating the quick and long lasting considerable improvement in symptoms following treatment with the sympathomimetic amine dextroamphetamine sulfate (with return of symptoms if treatment is temporarily ceased thus diminishing the likelihood of spontaneous remission) despite failure to respond to a plethora of other pharmacologic agents and other therapies over many years. The physiological systems with various chronic disorders that have responded included the gastrointestinal system, skin, genitourinary system, the nervous system, the musculoskeletal system, the temperature regulation system, peripheral vasculature system, and the endocrine system. Despite the multitude of very convincing anecdotal reports showing its efficacy (and to date no reports refuting this hypothesis), there has only been one controlled study which showed the benefit of dextroamphetamine sulfate on edema and weight gain in diet-refractory patients. The flaw to date for general acceptance of this hypothesis is that most positive studies are coming from one clinical center. Furthermore, more controlled studies are needed. There has been a recent interest amongst physiologists and recent studies have been published confirming a deficiency of sympathetic nerve fibers in some of these disorders which hopefully will encourage more research into other physiologic systems leading to corroboration of this hypothesis.     

Mitral valve prolapse and aortic root dilation in adults with hypermobile Ehlers–Danlos syndrome and related disorders

Ehlers–Danlos syndromes (EDSs) are a group of inherited connective tissue disorders, and among them, classical EDS (cEDS) and hypermobile EDS (hEDS) are the most common. Mitral valve prolapse (MVP) and aortic root dilation (ARD) have previously been reported to occur at an increased frequency within cEDS and hEDS. More recently, a study performed in the pediatric population did not show increased prevalence (Ritter et al., American Journal of Medical Genetics Part A, 173(6), 1467–1472, 2017). The purpose of this study was to review a large population of individuals with cEDS, hEDS, and hypermobility spectrum disorders to determine the frequency of MVP and ARD. A retrospective chart review of 209 individuals with echocardiograms was performed. Overall, 6.4% (13/209) had MVP and 1.6% (3/189) were found to have ARD. Although the presence of MVP is higher than what has been reported in the general population, no patients had severe MVP or required surgical intervention. No patients in this cohort had an aortic root diameter requiring surgical repair. Based on the results of this study and previous studies, routine echocardiograms to assess for valvular diseases and ARD may not be necessary unless warranted by presence of symptoms or family history.     

Arterial involvement in genetic diseases

Whereas the information on the subject of arterial status is sketchy and haphazard with respect to any one genetic disorder, the number of these diseases would have precluded the provision of a critical review within the scope of this presentation. Thus, it was deemed more meaningful to approach the subject selectively. A brief summary was provided on the nature of the arterial wall and its involvement in genetic diseases either as a primary target or a secondarily affected organ, and on "affinity" of various genetic disorders for a type (elastic, muscular, or smallest), segment (proximal, distal), and layer (intimal, medial, adventitial) of the arterial tree or the arterial wall, respectively. Genetic diseases may affect arteries by "causing" (a) congenital malformations, (b) alteration of the arterial "makeup" without necessarily producing definable lesions, and (c) modification of a nongenetic arterial disease (e.g., atherosclerosis), or by "producing" (d) arterial lesions that are characteristic of (even specific for?) a given genetic disorder. A few examples were selected to illustrate (b) (tuberous sclerosis; infantile GM1-gangliosidosis), (c) Wolman's disease; familial hyperlipoproteinemias), and (d) [Hurler's disease, neurofibromatosis; Ehlers-Danlos syndrome (type IV)]. Whenever available, the results of electron microscopic studies carried out in our laboratories were included. Some of these have not been reported in the literature to date. The need for a coordinated multidisciplinary approach to the study of genetic diseases in general is stressed in closing.     

Schlafbezogene Hypoventilationen bei neuromuskul?ren Erkrankungen

Sleep-related breathing disorder is a common diagnosis in neuromuscular diseases. Many patients with neuromuscular disorders are affected by hypoventilation syndrome while sleeping. The main activator of hypoventilation is a malfunction of the diaphragm. Because of physiological muscle atony during rapid eye movement (REM) sleep breathing disorders initially occur during this sleeping state. Nocturnal hypoventilation is often only diagnosed when constrictive ventilation failure also occurs during the daytime. Hence, it is necessary to test patients with neuromuscular diseases for sleep-related breathing disorders even in the early stages of the disease. For patients with verified amyotrophic lateral sclerosis and Duchenne??s muscular dystrophy a life-prolonging effect of adequate artificial ventilation has been shown in randomized studies with a small number of cases. A subject for further research is to investigate whether in addition to life-prolonging effects a precocious adequate non-invasive breathing therapy can also influence the progression of various even non-fatal neuromuscular diseases.     

Inborn errors of metabolism underlying primary immunodeficiencies

A number of inborn errors of metabolism (IEM) have been shown to result in predominantly immunologic phenotypes, manifesting in part as inborn errors of immunity. These phenotypes are mostly caused by defects that affect the (i) quality or quantity of essential structural building blocks (e.g., nucleic acids, and amino acids), (ii) cellular energy economy (e.g., glucose metabolism), (iii) post-translational protein modification (e.g., glycosylation) or (iv) mitochondrial function. Presenting as multisystemic defects, they also affect innate or adaptive immunity, or both, and display various types of immune dysregulation. Specific and potentially curative therapies are available for some of these diseases, whereas targeted treatments capable of inducing clinical remission are available for others. We will herein review the pathogenesis, diagnosis, and treatment of primary immunodeficiencies (PIDs) due to underlying metabolic disorders.     

Duchenne型肌营养不良症的细胞治疗

Duchenne型肌营养不良症（Duchenne muscular dystrophy，DMD）表现为进行性肌肉萎缩，是一种致死性、遗传性神经肌肉疾病。尽管对肌肉萎缩的分子机理的研究取得了很大进展，但是仍然不能治愈，细胞治疗是很有前景的一种治疗方法。成肌细胞移植面临的主要限制是注射后细胞分布差，免疫排斥和细胞存活率低；骨髓干细胞移植需要解决横向分化的低效率问题；而肌源性干细胞看来能更有效地再生表达dystrophin的肌纤维。     

Unexpected allelic heterogeneity and spectrum of mutations in Fowler syndrome revealed by next‐generation exome sequencing

Protein coding genes constitute approximately 1% of the human genome but harbor 85% of the mutations with large effects on disease‐related traits. Therefore, efficient strategies for selectively sequencing complete coding regions (i.e., “whole exome”) have the potential to contribute our understanding of human diseases. We used a method for whole‐exome sequencing coupling Agilent whole‐exome capture to the Illumina DNA‐sequencing platform, and investigated two unrelated fetuses from nonconsanguineous families with Fowler Syndrome (FS), a stereotyped phenotype lethal disease. We report novel germline mutations in feline leukemia virus subgroup C cellular‐receptor‐family member 2, FLVCR2, which has recently been shown to cause FS. Using this technology, we identified three types of genetic abnormalities: point‐mutations, insertions‐deletions, and intronic splice‐site changes (first pathogenic report using this technology), in the fetuses who both were compound heterozygotes for the disease. Although revealing a high level of allelic heterogeneity and mutational spectrum in FS, this study further illustrates the successful application of whole‐exome sequencing to uncover genetic defects in rare Mendelian disorders. Of importance, we show that we can identify genes underlying rare, monogenic and recessive diseases using a limited number of patients (n=2), in the absence of shared genetic heritage and in the presence of allelic heterogeneity. Hum Mutat 31:1–6, 2010. © 2010 Wiley‐Liss, Inc.     

Genetic basis of inherited peripheral neuropathies

Progress in the elucidation of the genetic basis for inherited peripheral neuropathies has been remarkable over the last years. In particular, the molecular mechanisms underlying the autosomal dominantly inherited disorders Charcot–Marie–Tooth disease type 1A (CMT1 A), Charcot–Marie–Tooth disease type 1B (CMT1B), and hereditary neuropathy with liability to pressure palsies (HNPP) have been determined. While mutation in the gene encoding the major myelin protein, P_o has been associated with CMT1B, CMT1A and HNPP have been shown to be associated with reciprocal recombination events leading either to a large submicroscopic duplication in CMT1 A, or the corresponding DNA deletion in HNPP. Available evidence is consistent with the hypothesis that one or more genes within the relevant rearranged segment of 1.5 Mb on chromosome 17 is sensitive to gene dosage providing a novel mechanism for inherited human disorders. It is likely that the gene encoding the peripheral myelin protein PMP22 is at least one of the genes involved since the PMP22 gene maps within the CMT1A duplication (or HNPP deletion), and point mutations within it have been shown to cause a CMT phenotype in humans and comparable neuropathies in rodents (trembler and trembler^J). The mechanism(s) by which gene dosage and point mutations affecting the same gene might lead to a similar phenotype are currently unknown but recent transgenic mouse experiments suggest that similar mechanisms may also underlie other genetic diseases. © 1994 Wiley-Liss, Inc.     

Emerging pathways for hereditary axonopathies

Motor neurons are affected in a number of neurological diseases. Their unifying pathological signature is degeneration of extended projecting axons and loss of motor neurons in the prefrontal cortex and/or the spinal cord. Based on clinical criteria, hereditary forms have been traditionally divided into distinct entities, such as familial amyotrophic lateral sclerosis, hereditary motor neuropathy, spinal muscular atrophy, familial spinal paraplegia, and Charcot–Marie–Tooth disease type 2, also known as hereditary motor and sensory neuropathy II. Genetic research of the last decade has revealed remarkable heterogeneity within these disorders. Most of the identified genes to date cause disease in a classic Mendelian inheritance pattern with a high phenotypic penetrance. This rich source of molecular genetic data has already provided insight into the underlying major pathways of these diseases and should continue to do so in the future. This review attempts to cross the traditional clinical classifications in order to draw an emerging picture of common pathways between causative genes, providing a different perspective of this rapidly growing scientific field.     

Population history and its impact on medical genetics in Quebec

Knowledge of the genetic demography of Quebec is useful for gene mapping, diagnosis, treatment, community genetics and public health. The French-Canadian population of Quebec, currently about 6 million people, descends from about 8500 French settlers who arrived in Nouvelle-France between 1608 and 1759. The migrations of those settlers and their descendants led to a series of regional founder effects, reflected in the geographical distribution of genetic diseases in Quebec. This review describes elements of population history and clinical genetics pertinent to the treatment of French Canadians and other population groups from Quebec and summarizes the cardinal features of over 30 conditions reported in French Canadians. Some were discovered in French Canadians, such as autosomal recessive ataxia of the Charlevoix-Saguenay (MIM 270550), agenesis of corpus callosum and peripheral neuropathy (MIM 218000) and French-Canadian-type Leigh syndrome (MIM 220111). Other conditions are particularly frequent or have special genetic characteristics in French Canadians, including oculopharyngeal muscular dystrophy, hepatorenal tyrosinaemia, cystic fibrosis, Leber hereditary optic neuropathy and familial hypercholesterolaemia. Three genetic diseases of Quebec First Nations children are also discussed: Cree encephalitis (MIM 608505), Cree leukoencephalopathy (MIM 603896) and North American Indian childhood cirrhosis (MIM 604901).     

The Interplay of Familial and Individual Risk in Predicting Clinical Improvements in Pediatric Anxiety Disorders

Bioecological models of developmental psychopathology underscore the role of familial experiences of adversity and children’s individual-level characteristics in heightening risk for pediatric anxiety through direct, combined, and interactive effects. To date, much of the existing research dedicated to pediatric anxiety disorders has largely been examined in bioecological models of diathesis-stress using community samples. This study extends our understanding of children’s differential responsiveness to familial adversity by examining the diathesis-stress interaction of cumulative risk and children’s individual-level vulnerabilities (negative affectivity and coping efficacy) within a clinic-referred treatment study for pediatric anxiety disorders. A cumulative risk index assessing exposure to familial adversity (e.g., socioeconomic status [SES], parent psychiatric illness) and self-reported measures of children’s negative affectivity and coping efficacy were each measured at the intake of a randomized controlled clinical trial for the treatment of pediatric anxiety disorders (N = 488; 7–17 years of age). Trajectories of interviewer-rated anxiety symptoms were assessed across 12 weeks of treatment at baseline, 4 weeks, 8 weeks, and 12 weeks. Consistent with models of temperamental risk for mental health problems, negative affectivity predicted higher anxiety symptoms at intake. A significant diathesis-stress interaction between cumulative risk and coping efficacy emerged, as high risk and perceptions of lower coping efficacy attenuated declines in anxiety across 12 weeks. These patterns did not differ across treatment conditions. The results indicate that for youth experiencing high levels of stress, additional treatment efforts targeting familial stressors and coping efficacy may be important in maximizing treatment outcomes.     

Neutropenia and Primary Immunodeficiency Diseases

Primary immunodeficiency diseases (PID) are a heterogeneous group of congenital disorders of the immune system leading to recurrent infections, autoimmunity, malignancies, and hematological disorders. This review focuses specifically on inherited disorders associated with neutropenia, which may occur in isolation or as a feature of more complex immune disorders. It has been known for a long time that defined immunodeficiency syndromes, such as CD40L deficiency, WHIM syndrome, or Chédiak Higashi syndrome, may be associated with neutropenia even though the mechanisms are poorly understood. In some PID, neutropenia may result from chronic viral infection or from autoimmunity. Recently, the identification of several novel genetic defects (e.g., p14-deficiency, HAX1-deficiency, AK2-deficiency) has shed light on the pathophysiology of congenital neutropenia. This review summarizes the clinical, immunological, and genetic features of congenital neutropenia syndromes.     

Metabolische Myopathien – ein Überblick

Metabolic disorders of energy production characterise the group of rare, mainly autosomal recessively inherited metabolic muscular diseases which are often associated with multi-systemic symptoms. In this report, an update on the clinics, pathophysiology, pathomorphology and current treatment options of metabolic myopathies will be given. Beyond classic phenotypes of these disorders, one should be aware of oligosymptomatic patients who can be easily missed. The relevant gene mutations and the pathophysiology and pathomorphology they cause are now known for almost all these metabolic diseases. Establishing the correct diagnosis has become even more important since highly specific therapy options are now available for at least some of these inherited disorders, e.g. enzyme replacement therapy in Pompe disease.     

Mutation analysis of genes within the dynactin complex in a cohort of hereditary peripheral neuropathies

The cytoplasmic dynein–dynactin genes are attractive candidates for neurodegenerative disorders given their functional role in retrograde transport along neurons. The cytoplasmic dynein heavy chain (DYNC1H1) gene has been implicated in various neurodegenerative disorders, and dynactin 1 (DCTN1) genes have been implicated in a wide spectrum of disorders including motor neuron disease, Parkinson's disease, spinobulbar muscular atrophy and hereditary spastic paraplegia. However, the involvement of other dynactin genes with inherited peripheral neuropathies (IPN) namely, hereditary sensory neuropathy, hereditary motor neuropathy and Charcot–Marie–Tooth disease is under reported. We screened eight genes; DCTN1‐6 and ACTR1A and ACTR1B in 136 IPN patients using whole‐exome sequencing and high‐resolution melt (HRM) analysis. Eight non‐synonymous variants (including one novel variant) and three synonymous variants were identified. Four variants have been reported previously in other studies, however segregation analysis within family members excluded them from causing IPN in these families. No variants of disease significance were identified in this study suggesting the dynactin genes are unlikely to be a common cause of IPNs. However, with the ease of querying gene variants from exome data, these genes remain worthwhile candidates to assess unsolved IPN families for variants that may affect the function of the proteins.     

Antigen-specific immunomodulation via altered peptide ligands

An important subgroup of human cellular receptors uses peptides as signaling molecules. Modifications of these signaling peptides, usually by amino acid substitutions in crucial receptor contact sites (i.e., altered peptide ligands, APLs), is an approach for highly selective and specific modulation of the receptor function. One of the major applications of APLs is immunology, where APLs have been examined for therapeutic modulation of T cell function, both for diseases characterized by unwanted activation of T cells (e.g., autoimmune diseases) and for disorders with suboptimal T cell activation (e.g., immunotherapy of cancers and infectious disorders). APLs also occur in vivo, for example, as escape mutants of infectious agents, and play an important role in thymic positive selection. We summarize current knowledge of the basic mechanisms of the effects of APLs with special focus on T cell receptor signaling and the use of APLs for the treatment of autoimmune diseases.