RESUMEN
With the recent advances in next-generation sequencing (NGS), mitochondrial whole-genome sequencing has begun to be applied to the field of the forensic biology as an alternative to the traditional Sanger-type sequencing (STS). However, experimental workflows, commercial solutions, and output data analysis must be strictly validated before being implemented into the forensic laboratory. In this study, we performed an internal validation for an NGS-based typing of the entire mitochondrial genome using the Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific) on the Ion S5 sequencer (Thermo Fisher Scientific). Concordance, repeatability, reproducibility, sensitivity, and heteroplasmy detection analyses were assessed using the 2800 M and 9947A standard control DNA as well as typical casework specimens, and results were compared with conventional Sanger sequencing and another NGS sequencer in a different laboratory. We discuss the strengths and limitations of this approach, highlighting some issues regarding noise thresholds and heteroplasmy detection, and suggesting solutions to mitigate these effects and improve overall data interpretation. Results confirmed that the Precision ID Whole mtDNA Genome Panel is highly reproducible and sensitive, yielding useful full mitochondrial DNA sequences also from challenging DNA specimens, thus providing further support for its use in forensic practice.
Asunto(s)
Genoma Mitocondrial , ADN Mitocondrial/genética , Haplotipos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN/métodosRESUMEN
Serum biomarkers that might detect clinical progression are currently lacking for Spinal and bulbar muscular atrophy (SBMA), thus limiting the effectiveness of possible future pharmacological trials. Elevation of cardiac troponin T (cTnT) unrelated to myocardial damage in a motor neuron (MN) disease as amyotrophic lateral sclerosis (ALS) was associated to disease severity. We enrolled 47 SBMA patients and 5 Spinal muscular atrophy (SMA) type 3 adult patients as control group; each SBMA patient was evaluated at baseline and at one-year follow-up visit. Demographic and clinical data including functional scores (SBMAFRS) were collected; serum was collected as standard of care and tested for cardiac troponins. Levels of cTnT but not cTnI were increased in SBMA with respect to reference values; unlike other neuromuscular diseases, SMA patients had overall normal cTnT values. Median cTnT concentrations did not change after one year and values were correlated to motor function, particularly with lower limb subdomain, at baseline only. Variations of cTnT and of SBMAFRS were unrelated. The cautiously promising results of cTnT as potential biomarker should undergo a more extensive clinical validation, including studies with longer follow-up period. When evaluating SBMA patients for a potential cardiac damage cTnI testing should be coupled or preferred to cTnT.
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Esclerosis Amiotrófica Lateral , Atrofia Bulboespinal Ligada al X , Atrofia Muscular Espinal , Enfermedades Neuromusculares , Atrofias Musculares Espinales de la Infancia , Adulto , Humanos , Troponina T , Atrofia Muscular Espinal/diagnóstico , BiomarcadoresRESUMEN
Duchenne muscular dystrophy (DMD) is one of the most frequent and severe childhood muscle diseases. Its pathophysiology is multifaceted and still incompletely understood, but we and others have previously shown that oxidative stress plays an important role. In particular, we have demonstrated that inhibition of mitochondrial monoamine oxidases could improve some functional and biohumoral markers of the pathology. In the present study we report the use of dystrophic mdx mice to evaluate the efficacy of a dual monoamine oxidase B (MAO-B)/semicarbazide-sensitive amine oxidase (SSAO) inhibitor, PXS-5131, in reducing inflammation and fibrosis and improving muscle function. We found that a one-month treatment starting at three months of age was able to decrease reactive oxygen species (ROS) production, fibrosis, and inflammatory infiltrate in the tibialis anterior (TA) and diaphragm muscles. Importantly, we also observed a marked improvement in the capacity of the gastrocnemius muscle to maintain its force when challenged with eccentric contractions. Upon performing a bulk RNA-seq analysis, PXS-5131 treatment affected the expression of genes involved in inflammatory processes and tissue remodeling. We also studied the effect of prolonged treatment in older dystrophic mice, and found that a three-month administration of PXS-5131 was able to greatly reduce the progression of fibrosis not only in the diaphragm but also in the heart. Taken together, these results suggest that PXS-5131 is an effective inhibitor of fibrosis and inflammation in dystrophic muscles, a finding that could open a new therapeutic avenue for DMD patients.
RESUMEN
Human genetic variation (polymorphisms) in genes coding proteins involved in the absorption, distribution, metabolism, and elimination (ADME) of drugs can have a strong effect on drug exposure and downstream efficacy and safety outcomes. Vamorolone, a dissociative steroidal anti-inflammatory drug for treating Duchenne muscular dystrophy (DMD), primarily undergoes oxidation by CYP3A4 and CYP3A5 and glucuronidation by UDP-glucuronosyltransferases. This work assesses the pharmacokinetics (PKs) of vamorolone and sources of interindividual variability (IIV) in 81 steroid-naïve boys with DMD aged 4 to <7 years old considering the genetic polymorphisms of CYPS3A4 (CYP3A4*22, CYP3A4*1B), CYP3A5 (CYP3A5*3), and UGT1A1 (UGT1A1*60) utilizing population PK modeling. A one-compartment model with zero-order absorption (Tk0, duration of absorption), linear clearance (CL/F), and volume (V/F) describes the plasma PK data for boys with DMD receiving a wide range of vamorolone doses (0.25-6 mg/kg/day). The typical CL/F and V/F values of vamorolone were 35.8 L/h and 119 L, with modest IIV. The population Tk0 was 3.14 h yielding an average zero-order absorption rate (k0) of 1.16 mg/kg/h with similar absorption kinetics across subjects at the same vamorolone dose (i.e., no IIV on Tk0). The covariate analysis showed that none of the genetic covariates had any significant impact on the PKs of vamorolone in boys with DMD. Thus, the PKs of vamorolone is very consistent in these young boys with DMD.
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Citocromo P-450 CYP3A , Glucuronosiltransferasa , Distrofia Muscular de Duchenne , Humanos , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/genética , Masculino , Preescolar , Niño , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Polimorfismo Genético , Modelos Biológicos , Farmacogenética , PregnadienodiolesRESUMEN
Background: Dilated cardiomyopathy (DCM) is a major complication of, and leading cause of mortality in Duchenne muscular dystrophy (DMD). Its severity, age at onset, and rate of progression display wide variability, whose molecular bases have been scarcely elucidated. Potential DCM-modifying factors include glucocorticoid (GC) and cardiological treatments, DMD mutation type and location, and variants in other genes. Methods and Results: We retrospectively collected 3138 echocardiographic measurements of left ventricular ejection fraction (EF), shortening fraction (SF), and end-diastolic volume (EDV) from 819 DMD participants, 541 from an Italian multicentric cohort and 278 from the Cooperative International Neuromuscular Group Duchenne Natural History Study (CINRG-DNHS). Using generalized estimating equation (GEE) models, we estimated the yearly rate of decrease of EF (-0.80%) and SF (-0.41%), while EDV increase was not significantly associated with age. Utilizing a multivariate generalized estimating equation (GEE) model we observed that mutations preserving the expression of the C-terminal Dp71 isoform of dystrophin were correlated with decreased EDV (-11.01âmL/m2, pâ=â0.03) while for dp116 were correlated with decreased EF (-4.14%, pâ=â<0.001). The rs10880 genotype in the LTBP4 gene, previously shown to prolong ambulation, was also associated with increased EF and decreased EDV (+3.29%, pâ=â0.002, and -10.62âmL/m2, pâ=â0.008) with a recessive model. Conclusions: We quantitatively describe the progression of systolic dysfunction progression in DMD, confirm the effect of distal dystrophin isoform expression on the dystrophin-deficient heart, and identify a strong effect of LTBP4 genotype of DCM in DMD.
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Cardiomiopatías , Distrofia Muscular de Duchenne , Humanos , Distrofina/genética , Distrofina/metabolismo , Haplotipos , Estudios Retrospectivos , Volumen Sistólico , Función Ventricular Izquierda , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/complicaciones , Cardiomiopatías/etiología , Cardiomiopatías/genética , Isoformas de Proteínas/genética , Proteínas de Unión a TGF-beta Latente/genéticaRESUMEN
OBJECTIVE: To validate an Italian version of the Rasch-Built Overall ALS Disability Scale (ROADS) in a broad population of patients and assess its longitudinal performance over time. METHODS: 270 ALS patients referring to the Motor Neuron Disease Clinic of the University of Padova and Modena (Italy) accepted to compile the Italian version of the ROADS and results were correlated with the ALSFRSr and ALSAQ-40 scores, FVC values, and creatinine or albumin blood levels. To verify test-retest reliability, patients were asked to fill in a second copy of the scale within 5-7 days. Thirty-nine patients compiled a further copy of questionnaire during the follow up visit (after 133 days on average) which allowed us a longitudinal assessment of the scale. RESULTS: We found a good external construct validity between ROADS and either ALSFRS-R (correlation coefficient = 0.85) or ALSAQ-40 (correlation coefficient = - 0.84). Test-retest reliability was excellent with a concordance-correlation coefficient of 0.93. Yet, we observed a significant correlation between changes over time of the ROADS normalised sum score (- 2.18 point loss per month) and those of both the ALSFRS-R (positive correlation; Rho = 0.64, p ≤ 0.0001) or the ALSAQ-40 (negative correlation; Rho = - 0.60, p = 0.014). CONCLUSIONS: The Italian version of ROADS proved to be a reliable marker to monitor overall disability in ALS patients. Further studies are necessary to assess its longitudinal performance.
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Esclerosis Amiotrófica Lateral , Enfermedad de la Neurona Motora , Humanos , Esclerosis Amiotrófica Lateral/diagnóstico , Reproducibilidad de los Resultados , Actividades Cotidianas , Encuestas y Cuestionarios , ItaliaRESUMEN
Serum creatinine has been indicated as a potential marker of motor function in SBMA and results form previous longitudinal studies pointed to its decline over time. This is a longitudinal retrospective study investigating creatinine changes over a 36-month-period in 73 patients with SBMA. Severity and progression of the disease was assessed according to serum creatine kinase (CK) values, manual muscle testing (MMT), SBMA functional rating scale (SBMAFRS) score, 6-min-walk test (6MWT) value, and spirometry (forced vital capacity, fVC%) obtained at the baseline and at each of the annual follow-up visits. Baseline serum creatinine concentrations positively correlated with 6MWT, the MMT megascore score of both the upper (ULM) and lower (LLM) limbs and SBMAFRS. No correlation was found with CK or fVC% values. Similar correlation results were achieved at all the subsequent time points. Longitudinal assessments conducted by the generalized estimating equations (GEE) method returned significant changes for SBMAFRS (- 1.41 points per year, p < 0.001), ULM and LLM (- 0.69, p = 0.01; and - 1.07, p < 0.001, respectively), 6MWT (- 47 m, p < 0.001) but not for creatinine (- 0.82, p > 0.05). We also observed that creatinine levels at baseline did not correlate with changes in the other measures from baseline at each annual visit. Our data do not support a role for serum creatinine as sensitive biomarker of disease progression, and possibily prognosis, in SBMA.
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Atrofia Bulboespinal Ligada al X , Atrofia Muscular Espinal , Humanos , Creatinina , Estudios Retrospectivos , Biomarcadores , Progresión de la EnfermedadRESUMEN
Mutations in the RYR1 gene, encoding ryanodine receptor 1 (RyR1), are a well-known cause of Central Core Disease (CCD) and Multi-minicore Disease (MmD). We screened a cohort of 153 patients carrying an histopathological diagnosis of core myopathy (cores and minicores) for RYR1 mutation. At least one RYR1 mutation was identified in 69 of them and these patients were further studied. Clinical and histopathological features were collected. Clinical phenotype was highly heterogeneous ranging from asymptomatic or paucisymptomatic hyperCKemia to severe muscle weakness and skeletal deformity with loss of ambulation. Sixty-eight RYR1 mutations, generally missense, were identified, of which 16 were novel. The combined analysis of the clinical presentation, disease progression and the structural bioinformatic analyses of RYR1 allowed to associate some phenotypes to mutations in specific domains. In addition, this study highlighted the structural bioinformatics potential in the prediction of the pathogenicity of RYR1 mutations. Further improvement in the comprehension of genotype-phenotype relationship of core myopathies can be expected in the next future: the actual lack of the human RyR1 crystal structure paired with the presence of large intrinsically disordered regions in RyR1, and the frequent presence of more than one RYR1 mutation in core myopathy patients, require designing novel investigation strategies to completely address RyR1 mutation effect.
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Miopatías Estructurales Congénitas , Miopatía del Núcleo Central , Canal Liberador de Calcio Receptor de Rianodina , Humanos , Músculo Esquelético/patología , Mutación/genética , Miopatías Estructurales Congénitas/genética , Miopatías Estructurales Congénitas/patología , Miopatía del Núcleo Central/genética , Miopatía del Núcleo Central/patología , Canal Liberador de Calcio Receptor de Rianodina/genéticaRESUMEN
Genetic modifiers of Duchenne muscular dystrophy (DMD) are variants located in genes different from the disease-causing gene DMD, but associated with differences in disease onset, progression, or response to treatment. Modifiers described so far have been tested mainly for associations with ambulatory function, while their effect on upper limb function, which is especially relevant for quality of life and independence in non-ambulatory patients, is unknown. We tested genotypes at several known modifier loci (SPP1, LTBP4, CD40, ACTN3) for association with Performance Upper Limb version 1.2 score in an Italian multicenter cohort, and with Brooke scale score in the Cooperative International Neuromuscular Group Duchenne Natural History Study (CINRG-DNHS), using generalized estimating equation (GEE) models of longitudinally collected data, with age and glucocorticoid treatment as covariates. CD40 rs1883832, previously linked to earlier loss of ambulation, emerged as a modifier of upper limb function, negatively affecting shoulder and distal domains of PUL (p = 0.023 and 0.018, respectively) in the Italian cohort, as well as of Brooke score (p = 0.018) in the CINRG-DNHS. These findings will be useful for the design and interpretation of clinical trials in DMD, especially for non-ambulatory populations.
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Distrofia Muscular de Duchenne , Actinina/genética , Estudios de Cohortes , Genotipo , Humanos , Distrofia Muscular de Duchenne/genética , Calidad de Vida , Extremidad SuperiorRESUMEN
Peripherin (PRPH), a type III intermediate filament, assembles with neurofilaments in neurons of the peripheral nervous system, including lower motor neurons (LMN). To evaluate the role of PRPH in LMN degeneration, we assessed PRPH and neurofilament light chain (NfL) in cerebrospinal fluid (CSF) and serum of 91 patients with motor neuron diseases (MND) and 69 controls. Overall, we found PRPH to be more concentrated in serum than in CSF. Serum PRPH resulted significantly increased in MND patients but it was unrelated to CSF-NfL or survival in the amyotrophic lateral sclerosis (ALS) subset. PRPH might represent a marker of LMN involvement.
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Enfermedad de la Neurona Motora/metabolismo , Proteínas de Neurofilamentos/metabolismo , Periferinas/metabolismo , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Humanos , Estudios Longitudinales , Enfermedad de la Neurona Motora/sangre , Enfermedad de la Neurona Motora/líquido cefalorraquídeo , Proteínas de Neurofilamentos/sangre , Proteínas de Neurofilamentos/líquido cefalorraquídeo , Periferinas/sangre , Periferinas/líquido cefalorraquídeo , Estudios RetrospectivosRESUMEN
OBJECTIVE: Respiratory insufficiency is a major complication of Duchenne muscular dystrophy (DMD). Its progression shows considerable interindividual variability, which has been less thoroughly characterized and understood than in skeletal muscle. We collected pulmonary function testing (PFT) data from a large retrospective cohort followed at Centers collaborating in the Italian DMD Network. Furthermore, we analyzed PFT associations with different DMD mutation types, and with genetic variants in SPP1, LTBP4, CD40, and ACTN3, known to modify skeletal muscle weakness in DMD. Genetic association findings were independently validated in the Cooperative International Neuromuscular Research Group Duchenne Natural History Study (CINRG-DNHS). METHODS AND RESULTS: Generalized estimating equation analysis of 1852 PFTs from 327 Italian DMD patients, over an average follow-up time of 4.5 years, estimated that forced vital capacity (FVC) declined yearly by -4.2%, forced expiratory volume in 1 sec by -5.0%, and peak expiratory flow (PEF) by -2.9%. Glucocorticoid (GC) treatment was associated with higher values of all PFT measures (approximately + 15% across disease stages). Mutations situated 3' of DMD intron 44, thus predicted to alter the expression of short dystrophin isoforms, were associated with lower (approximately -6%) PFT values, a finding independently validated in the CINRG-DNHS. Deletions amenable to skipping of exon 51 and 53 were independently associated with worse PFT outcomes. A meta-analysis of the two cohorts identified detrimental effects of SPP1 rs28357094 and CD40 rs1883832 minor alleles on both FVC and PEF. INTERPRETATION: These findings support GC efficacy in delaying respiratory insufficiency, and will be useful for the design and interpretation of clinical trials focused on respiratory endpoints in DMD.