Integrated Genetic Approaches in Therapeutic Discovery for Rare Diseases

OBJECTIVE: Comprehensive clinical characterization of congenital titinopathy to facilitate diagnosis and management of this important emerging disorder. METHODS: Using massively parallel sequencing we identified 30 patients from 27 families with 2 pathogenic nonsense, frameshift and/or splice site TTN mutations in trans. We then undertook a detailed analysis of the clinical, histopathological and imaging features of these patients. RESULTS: All patients had prenatal or early onset hypotonia and/or congenital contractures. None had ophthalmoplegia. Scoliosis and respiratory insufficiency typically developed early and progressed rapidly, whereas limb weakness was often slowly progressive, and usually did not prevent independent walking. Cardiac involvement was present in 46% of patients. Relatives of 2 patients had dilated cardiomyopathy. Creatine kinase levels were normal to moderately elevated. Increased fiber size variation, internalized nuclei and cores were common histopathological abnormalities. Cap-like regions, whorled or ring fibers, and mitochondrial accumulations were also observed. Muscle magnetic resonance imaging showed gluteal, hamstring and calf muscle involvement. Western blot analysis showed a near-normal sized titin protein in all samples. The presence of 2 mutations predicted to impact both N2BA and N2B cardiac isoforms appeared to be associated with greatest risk of cardiac involvement. One-third of patients had 1 mutation predicted to impact exons present in fetal skeletal muscle, but not included within the mature skeletal muscle isoform transcript. This strongly suggests developmental isoforms are involved in the pathogenesis of this congenital/early onset disorder. INTERPRETATION: This detailed clinical reference dataset will greatly facilitate diagnostic confirmation and management of patients, and has provided important insights into disease pathogenesis. Ann Neurol 2018;83:1105-1124.

As the deadline for the millennium development goals approaches, it has become clear that the goals linked to maternal and newborn health are the least likely to be achieved by 2015. It is therefore critical to ensure that all possible data, tools and methods are fully exploited to help address this gap. Among the methods that are under-used, mapping has always represented a powerful way to 'tell the story' of a health problem in an easily understood way. In addition to this, the advanced analytical methods and models now being embedded into Geographic Information Systems allow a more in-depth analysis of the causes behind adverse maternal and newborn health (MNH) outcomes. This paper examines the current state of the art in mapping the geography of MNH as a starting point to unleashing the potential of these under-used approaches. Using a rapid literature review and the description of the work currently in progress, this paper allows the identification of methods in use and describes a framework for methodological approaches to inform improved decision-making. The paper is aimed at health metrics and geography of health specialists, the MNH community, as well as policy-makers in developing countries and international donor agencies.

OBJECTIVE: Limb-girdle muscular dystophy 2A (LGMD2A, OMIM) is a slowly progressive myopathy caused by the deficiency in calpain 3, a calcium-dependent cysteine protease of the skeletal muscle. METHODS: In this study, we carried out an observational study of clinical manifestations and disease progression in genetically confirmed LGMD2A patients for up to 4 years. A total of 85 patients, aged 14-65 years, were recruited in three centers located in metropolitan France, the Basque country, and the Reunion Island. They were followed up every 6 months for 2 years and a subgroup was assessed annually thereafter for two more years. Data collected for all patients included clinical history, blood parameters, muscle strength assessed by manual muscle testing (MMT) and quantitative muscle testing, functional scores, and pulmonary and cardiac functions. In addition, CT scans of the lower limbs were performed in a subgroup of patients. RESULTS: Our study confirms the clinical description of a slowly progressive disorder with onset in the first or second decade of life with some degree of variability related to gender and mutation type. The null mutations lead to a more severe phenotype while compound heterozygote patients are the least affected. Muscle weakness is remarkably symmetrical and predominant in the axial muscles of the trunk and proximal muscles of the lower limb. There was a high correlation between the weakness at individual muscle level as assessed by MMT and the loss of density in CT scan analysis. INTERPRETATION: All the generated data will help to determine the endpoints for further clinical studies.

Recombinant adeno-associated virus (rAAV) is currently the best vector for gene delivery into the skeletal muscle. However, the 5-kb packaging size of this virus is a major obstacle for large gene transfer. This past decade, many different strategies were developed to circumvent this issue (concatemerization-splicing, overlapping vectors, hybrid dual or fragmented AAV). Loss of function mutations in the DYSF gene whose coding sequence is 6.2kb lead to progressive muscular dystrophies (LGMD2B: OMIM_253601; MM: OMIM_254130; DMAT: OMIM_606768). In this study, we compared large gene transfer techniques to deliver the DYSF gene into the skeletal muscle. After rAAV8s intramuscular injection into dysferlin deficient mice, we showed that the overlap strategy is the most effective approach to reconstitute a full-length messenger. After systemic administration, the level of dysferlin obtained on different muscles corresponded to 0.5- to 2-fold compared to the normal level. We further demonstrated that the overlapping vector set was efficient to correct the histopathology, resistance to eccentric contractions and whole body force in the dysferlin deficient mice. Altogether, these data indicate that using overlapping vectors could be a promising approach for a potential clinical treatment of dysferlinopathies.

The development of medical approaches requires preclinical and clinical trials for assessment of therapeutic efficacy. Such evaluation entails the use of biomarkers, which provide information on the response to the therapeutic intervention. One newly-proposed class of biomarkers is the microRNA (miRNA) molecules. In muscular dystrophies (MD), the dysregulation of miRNAs was initially observed in muscle biopsy and later extended to plasma samples, suggesting that they may be of interest as biomarkers. First, we demonstrated that dystromiRs dysregulation occurs in MD with either preserved or disrupted expression of the dystrophin-associated glycoprotein complex, supporting the utilization of dystromiRs as generic biomarkers in MD. Then, we aimed at evaluation of the capacity of miRNAs as monitoring biomarkers for experimental therapeutic approach in MD. To this end, we took advantage of our previously characterized gene therapy approach in a mouse model for α-sarcoglycanopathy. We identified a dose-response correlation between the expression of miRNAs on both muscle tissue and blood serum and the therapeutic benefit as evaluated by a set of new and classically-used evaluation methods. This study supports the utility of profiling circulating miRNAs for the evaluation of therapeutic outcome in medical approaches for MD.

Limb Girdle Muscular Dystrophies type 2I (LGMD2I), a recessive autosomal muscular dystrophy, is caused by mutations in the Fukutin Related Protein (FKRP) gene. It has been proposed that FKRP, a ribitol-5-phosphate transferase, is a participant in α-dystroglycan (αDG) glycosylation, which is important to ensure the cell/matrix anchor of muscle fibers. A LGMD2I knock-in mouse model was generated to express the most frequent mutation (L276I) encountered in patients. The expression of FKRP was not altered neither at transcriptional nor at translational levels, but its function was impacted since abnormal glycosylation of αDG was observed. Skeletal muscles were functionally impaired from 2 months of age and a moderate dystrophic pattern was evident starting from 6 months of age. Gene transfer with a rAAV2/9 vector expressing Fkrp restored biochemical defects, corrected the histological abnormalities and improved the resistance to eccentric stress in the mouse model. However, injection of high doses of the vector induced a decrease of αDG glycosylation and laminin binding, even in WT animals. Finally, intravenous injection of the rAAV-Fkrp vector into a dystroglycanopathy mouse model due to Fukutin (Fktn) knock-out indicated a dose-dependent toxicity. These data suggest requirement for a control of FKRP expression in muscles.

BACKGROUND AND PURPOSE: The aim was to determine the genetic background of unknown muscular dystrophy in five French families. METHODS: Twelve patients with limb girdle muscular dystrophy or distal myopathy were clinically evaluated. Gene mutations were identified using targeted exon sequencing and mutated DNAJB6 was tested in vitro. RESULTS: Five patients presented with distal lower limb weakness whilst others had proximal presentation with a variable rate of progression starting at the mean age of 38.5 years. Two novel mutations (c.284A>T, p.Asn95Ile, two families; and c.293_295delATG, p.Asp98del, one family) as well as the previously reported c.279C>G (p.Phe93Leu, two families) mutation in DNAJB6 were identified. All showed a reduced capacity to prevent protein aggregation. CONCLUSIONS: The mutational and phenotypical spectrum of DNAJB6-caused muscle disease is larger than previously reported, including also dysphagia. The originally reported c.279C>G (p.Phe93Leu) mutation is now identified in four different populations and appears to be a mutational hotspot. Our report confirms that some DNAJB6 mutations cause distal-onset myopathy and hence DNAJB6 defects should be considered broadly in dominant muscular dystrophy families.

Abstract The identification of a dysferlin‐deficient animal model that accurately displays both the physiological and behavior aspects of human dysferlinopathy is critical for the evaluation of potential therapeutics. Disease progression in dysferlin‐deficient mice is relatively mild, compared to the debilitating human disease which manifests in impairment of particular motor functions. Since there are no other known models of dysferlinopathy in other species, locomotor proficiency and muscular anatomy through MRI (both lower leg and hip region) were evaluated in dysferlin‐deficient B6.A‐ Dysf prmd /GeneJ (Bla/J) mice to define disease parameters for therapeutic assessment. Despite the early and progressive gluteal muscle dystrophy and significant fatty acid accumulation, the emergence of significant motor function deficits was apparent at approximately 1 year of age for standard motor challenges including the rotarod, a marble bury test, grip strength, and swimming speed. Earlier observations of decreased performance for Bla/J mice were evident during extended monitoring of overall exploration and rearing activity. Comprehensive treadmill gait analyses of the Bla/J model indicated significant differences in paw placement angles and stance in relation to speed and platform slope. At 18 months of age, there was no significant difference in the life expectancy of Bla/J mice compared to wild type. Consistent with progressive volume loss and fatty acid accumulation in the hip region observed by MRI , mass measurement of individual muscles confirmed gluteal and psoas muscles were the only muscles demonstrating a significant decrease in muscle mass, which is analogous to hip‐girdle weakness observed in human dysferlin‐deficient patients. Collectively, this longitudinal analysis identifies consistent disease parameters that can be indicators of efficacy in studies developing treatments for human dysferlin deficiency.

Duchenne Muscular Dystrophy (DMD) is a lethal muscle disorder, caused by mutations in the DMD gene and affects approximately 1:5000-6000 male births. In this report, we identified dysregulation of members of the Dlk1-Dio3 miRNA cluster in muscle biopsies of the GRMD dog model. Of these, we selected miR-379 for a detailed investigation because its expression is high in the muscle, and is known to be responsive to glucocorticoid, a class of anti-inflammatory drugs commonly used in DMD patients. Bioinformatics analysis predicts that miR-379 targets EIF4G2, a translational factor, which is involved in the control of mitochondrial metabolic maturation. We confirmed in myoblasts that EIF4G2 is a direct target of miR-379, and identified the DAPIT mitochondrial protein as a translational target of EIF4G2. Knocking down DAPIT in skeletal myotubes resulted in reduced ATP synthesis and myogenic differentiation. We also demonstrated that this pathway is GC-responsive since treating mice with dexamethasone resulted in reduced muscle expression of miR-379 and increased expression of EIF4G2 and DAPIT. Furthermore, miR-379 seric level, which is also elevated in the plasma of DMD patients in comparison with age-matched controls, is reduced by GC treatment. Thus, this newly identified pathway may link GC treatment to a mitochondrial response in DMD.

Limb-girdle muscular dystrophy type 2D (LGMD2D) is a rare autosomal-recessive disease, affecting striated muscle, due to mutation of SGCA, the gene coding for α-sarcoglycan. Nowadays, more than 50 different SGCA missense mutations have been reported. They are supposed to impact folding and trafficking of α-sarcoglycan because the defective polypeptide, although potentially functional, is recognized and disposed of by the quality control of the cell. The secondary reduction of α-sarcoglycan partners, β-, γ- and δ-sarcoglycan, disrupts a key membrane complex that, associated to dystrophin, contributes to assure sarcolemma stability during muscle contraction. The complex deficiency is responsible for muscle wasting and the development of a severe form of dystrophy. Here, we show that the application of small molecules developed to rescue ΔF508-CFTR trafficking, and known as CFTR correctors, also improved the maturation of several α-sarcoglycan mutants that were consequently rescued at the plasma membrane. Remarkably, in myotubes from a patient with LGMD2D, treatment with CFTR correctors induced the proper re-localization of the whole sarcoglycan complex, with a consequent reduction of sarcolemma fragility. Although the mechanism of action of CFTR correctors on defective α-sarcoglycan needs further investigation, this is the first report showing a quantitative and functional recovery of the sarcoglycan-complex in human pathologic samples, upon small molecule treatment. It represents the proof of principle of a pharmacological strategy that acts on the sarcoglycan maturation process and we believe it has a great potential to develop as a cure for most of the patients with LGMD2D.

The giant protein titin is the third most abundant protein in striated muscle. Mutations in its gene are responsible for diseases affecting the cardiac and/or the skeletal muscle. Titin has been reported to be expressed in multiple isoforms with considerable variability in the I-band, ensuring the modulation of the passive mechanical properties of the sarcomere. In the M-line, only the penultimate Mex5 exon coding for the specific is7 domain has been reported to be subjected to alternative splicing. Using the CRISPR-Cas9 editing technology, we generated a mouse model where we stably prevent the expression of alternative spliced variant(s) carrying the corresponding domain. Interestingly, the suppression of the domain induces a phenotype mostly in tissues usually expressing the isoform that has been suppressed, indicating that it fulfills (a) specific function(s) in these tissues allowing a perfect adaptation of the M-line to physiological demands of different muscles.

AIMS: Previously, detection of ANO5 protein has been complicated by unspecific antibodies, most of which have not identified the correct protein. The aims of the study were to specify ANO5 protein expression in human skeletal muscle, and to investigate if the ANO5 protein levels are affected by different ANO5 mutations in anoctaminopathy patients. METHODS: Four different antibodies were tested for ANO5 specificity. A sample preparation method compatible with membrane proteins, combined with tissue fractionation was used to determine ANO5 expression in cell cultures expressing ANO5, in normal muscles and eight patient biopsies with six different ANO5 mutations in homozygous or compound heterozygous states, and in other dystrophies. RESULTS: Only one specific monoclonal N-terminal ANO5 antibody was efficient in detecting the protein, showing that ANO5 is expressed as a single 107 kD polypeptide in human skeletal muscle. The truncating mutations c.191dupA and c.1261C>T were found to abolish ANO5 expression, whereas the studied point mutations had variable effects; however, all the ANO5 mutations resulted in clearly reduced ANO5 expression in the patient muscle membrane fraction. Attempts to detect ANO5 using immunohistochemistry were not yet successful. CONCLUSIONS: The data presented here indicate that the ANO5 protein expression is decreased in ANO5-mutated muscular dystrophy and that most of the non-truncating pathogenic ANO5 mutations likely destabilize the protein and cause its degradation. The method described here allows direct analysis of human ANO5 protein, which can be used in diagnostics, for evaluating the pathogenicity of the potentially harmful ANO5 variants of uncertain significance.

Abstract Maple syrup urine disease (MSUD) is a rare recessively inherited metabolic disorder causing accumulation of branched chain amino acids leading to neonatal death, if untreated. Treatment for MSUD represents an unmet need because the current treatment with life-long low-protein diet is challenging to maintain, and despite treatment the risk of acute decompensations and neuropsychiatric symptoms remains. Here, based on significant liver contribution to the catabolism of the branched chain amino acid leucine, we develop a liver-directed adeno-associated virus (AAV8) gene therapy for MSUD. We establish and characterize the Bckdha ( branched chain keto acid dehydrogenase a ) −/− mouse that exhibits a lethal neonatal phenotype mimicking human MSUD. Animals were treated at P0 with intravenous human BCKDHA AAV8 vectors under the control of either a ubiquitous or a liver-specific promoter. BCKDHA gene transfer rescued the lethal phenotype. While the use of a ubiquitous promoter fully and sustainably rescued the disease (long-term survival, normal phenotype and correction of biochemical abnormalities), liver-specific expression of BCKDHA led to partial, though sustained rescue. Here we show efficacy of gene therapy for MSUD demonstrating its potential for clinical translation.

X-linked myotubular myopathy (XLMTM) is a severe congenital disorder in male infants that leads to generalized skeletal muscle weakness and is frequently associated with fatal respiratory failure. XLMTM is caused by loss-of-function mutations in the MTM1 gene, which encodes myotubularin, the founder member of a family of 15 homologous proteins in mammals. We recently demonstrated the therapeutic efficacy of intravenous delivery of rAAV vectors expressing MTM1 in animal models of myotubular myopathy. Here, we tested whether the closest homologues of MTM1, MTMR1, and MTMR2 (the latter being implicated in Charcot-Marie-Tooth neuropathy type 4B1) are functionally redundant and could represent a therapeutic target for XLMTM. Serotype 9 recombinant AAV vectors encoding either MTM1, MTMR1, or MTMR2 were injected into the tibialis anterior muscle of Mtm1-deficient knockout mice. Two weeks after vector delivery, a therapeutic effect was observed with Mtm1 and Mtmr2, but not Mtmr1; with Mtm1 being the most efficacious transgene. Furthermore, intravenous administration of a single dose of the rAAV9-Mtmr2 vector in XLMTM mice improved the motor activity and muscle strength and prolonged survival throughout a 3-month study. These results indicate that strategies aiming at increasing MTMR2 expression levels in skeletal muscle may be beneficial in the treatment of myotubular myopathy.

For many companies, Business Process Re-engineering (BPR) is becoming a mean to attaining a competitive advantage. But BPR is too often related to specific processes while it should ensure a global performance by covering multi-processes. For that purpose, methodologies are required to perform an integrated Business Multi-Processes Reengineering. GRAI approach has been developped for more than 10 years and gradually improved through a lot of industrial experiments, each of them followed by a research step. This approach allows to perform such an integrated Business Multi-Processes Reengineering. In this paper we describe first the new requirements for BPR and then the GRAI approach. Finally, we present a recent application of the GRAI approach for the re-engineering of a workshop in the most important french company in the domain of defense industry.

Genetic variants in Fukutin-related protein (FKRP), an essential enzyme of the glycosylation pathway of α-dystroglycan, can lead to pathologies with different severities affecting the eye, brain, and muscle tissues. Here, we generate an in vitro cellular system to characterize the cellular localization as well as the functional potential of the most common FKRP patient missense mutations. We observe a differential retention in the endoplasmic reticulum (ER), the indication of misfolded proteins. We find data supporting that mutant protein able to overcome this ER-retention through overexpression present functional levels comparable to the wild-type. We also identify a specific region in FKRP protein localized between residues 300 and 321 in which genetic variants found in patients lead to correctly localized proteins but which are nevertheless functionally impaired or catalytically dead in our model, indicating that this particular region might be important for the enzymatic activity of FKRP within the Golgi. Our system thus allows the functional testing of patient-specific mutant proteins and the identification of candidate mutants to be further explored with the aim of finding pharmacological treatments targeting the protein quality control system.

INTRODUCTION: We report the genetic analysis of a large series of 76 Algerian patients from 65 unrelated families who presented with early onset severe muscular dystrophy and a clinical phenotype resembling limb-girdle muscular dystrophy type 2C. METHODS: To define the genetic basis of the diseases in these families, we undertook a series of analyses of the γ-sarcoglycan (SGCG) and DMD genes. RESULTS: Fifteen families were shown to carry SGCG variants. Only 2 kinds of causative mutations were identified in the population, mostly in the homozygous state: the well-known c.525delT and the previously described c.87dupT. In the DMD gene, 12 distinctive patterns of deletion were identified, mostly affecting the dystrophin central region. CONCLUSIONS: Our data suggest that a simple molecular screen consisting of 2 allele-specific polymerase chain reactions (PCRs) and a set of 3 multiplex PCRs can diagnose half of the patients who present with progressive muscular dystrophy in the developing nation of Algeria. Muscle Nerve 56: 129-135, 2017.

Congenital titinopathies are myopathies with variable phenotypes and inheritance modes. Here, we fully characterized, using an integrated approach (deep phenotyping, muscle morphology, mRNA and protein evaluation in muscle biopsies), two siblings with congenital multicore myopathy harboring three TTN variants predicted to affect titin stability and titin-myosin interactions. Muscle biopsies showed multicores, type 1 fiber uniformity and sarcomeric structure disruption with some thick filament loss. Immunohistochemistry and Western blotting revealed a marked reduction of fast myosin heavy chain isoforms. This is the first observation of a titinopathy suggesting that titin defect leads to secondary loss of fast myosin heavy chain isoforms.

Abstract Maple syrup urine disease (MSUD) is rare autosomal recessive metabolic disorder caused by the dysfunction of the mitochondrial branched‐chain 2‐ketoacid dehydrogenase (BCKD) enzyme complex leading to massive accumulation of branched‐chain amino acids and 2‐keto acids. MSUD management, based on a life‐long strict protein restriction with nontoxic amino acids oral supplementation represents an unmet need as it is associated with a poor quality of life, and does not fully protect from acute life‐threatening decompensations or long‐term neuropsychiatric complications. Orthotopic liver transplantation is a beneficial therapeutic option, which shows that restoration of only a fraction of whole‐body BCKD enzyme activity is therapeutic. MSUD is thus an ideal target for gene therapy. We and others have tested AAV gene therapy in mice for two of the three genes involved in MSUD, BCKDHA and DBT . In this study, we developed a similar approach for the third MSUD gene, BCKDHB . We performed the first characterization of a Bckdhb −/− mouse model, which recapitulates the severe human phenotype of MSUD with early‐neonatal symptoms leading to death during the first week of life with massive accumulation of MSUD biomarkers. Based on our previous experience in Bckdha −/− mice, we designed a transgene carrying the human BCKDHB gene under the control of a ubiquitous EF1α promoter, encapsidated in an AAV8 capsid. Injection in neonatal Bckdhb −/− mice at 10 14 vg/kg achieved long‐term rescue of the severe MSUD phenotype of Bckdhb −/− mice. These data further validate the efficacy of gene therapy for MSUD opening perspectives towards clinical translation.

gene encoding alpha-sarcoglycan (α-SG). Here, we report the results of a mechanistic screening ascertaining the molecular mechanisms involved in the degradation of the most prevalent misfolded R77C-α-SG protein. We performed a combinatorial study to identify drugs potentializing the effect of a low dose of the proteasome inhibitor bortezomib on the R77C-α-SG degradation inhibition. Analysis of the screening associated to artificial intelligence-based predictive ADMET characterization of the hits led to identification of the HDAC inhibitor givinostat as potential therapeutical candidate. Functional characterization revealed that givinostat effect was related to autophagic pathway inhibition, unveiling new theories concerning degradation pathways of misfolded SG proteins. Beyond the identification of a new therapeutic option for LGMD R3 patients, our results shed light on the potential repurposing of givinostat for the treatment of other genetic diseases sharing similar protein degradation defects such as LGMD R5 and cystic fibrosis.