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There is considerable variability in disease progression for patients with amyotrophic lateral sclerosis (ALS) including the age of disease onset, site of disease onset, and survival time. There is growing evidence that short structural variations (SSVs) residing in frequently overlooked genomic regions can contribute to complex disease mechanisms and can explain, in part, the phenotypic variability in ALS patients. Here, we discuss SSVs recently characterized by our laboratory and how these discoveries integrate into the current literature on ALS, particularly in the context of application to future clinical trials. These markers may help to identify and differentiate patients for clinical trials that have a similar ALS disease mechanism(s), thereby reducing the impact of participant heterogeneity. As evidence accumulates for the genetic markers discovered in SQSTM1, SCAF4, and STMN2, we hope to improve the outcomes of future ALS clinical trials.
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Esclerosis Amiotrófica Lateral , Esclerosis Amiotrófica Lateral/diagnóstico , Esclerosis Amiotrófica Lateral/genética , Progresión de la Enfermedad , Marcadores Genéticos/genética , Humanos , Factores de Empalme Serina-ArgininaRESUMEN
Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease classified as both a neurodegenerative and neuromuscular disorder. With a complex aetiology and no current cure for ALS, broadening the understanding of disease pathology and therapeutic avenues is required to progress with patient care. Alpha-synuclein (αSyn) is a hallmark for disease in neurodegenerative disorders, such as Parkinson's disease, Lewy body dementia, and multiple system atrophy. A growing body of evidence now suggests that αSyn may also play a pathological role in ALS, with αSyn-positive Lewy bodies co-aggregating alongside known ALS pathogenic proteins, such as SOD1 and TDP-43. This review endeavours to capture the scope of literature regarding the aetiology and development of ALS and its commonalities with "synucleinopathy disorders". We will discuss the involvement of αSyn in ALS and motor neuron disease pathology, and the current theories and strategies for therapeutics in ALS treatment, as well as those targeting αSyn for synucleinopathies, with a core focus on small molecule RNA technologies.
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Esclerosis Amiotrófica Lateral , Enfermedad por Cuerpos de Lewy , Atrofia de Múltiples Sistemas , Sinucleinopatías , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/terapia , Humanos , Cuerpos de Lewy/metabolismo , Enfermedad por Cuerpos de Lewy/patología , Atrofia de Múltiples Sistemas/patología , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
BACKGROUND: Several studies have demonstrated a lower apolipoprotein E4 (APOE ε4) allele frequency in African-Americans, but yet an increased age-related prevalence of AD. An algorithm for prevention clinical trials incorporating TOMM40'523 (Translocase of Outer Mitochondria Membrane) and APOE depends on accurate TOMM40'523-APOE haplotypes. METHODS: We have compared the APOE and TOMM40'523 phased haplotype frequencies of a 9.5 kb TOMM40/APOE genomic region in West African, Caucasian, and African-American cohorts. RESULTS: African-American haplotype frequency scans of poly-T lengths connected in phase with either APOE ε4 or APOE ε3 differ from both West Africans and Caucasians and represent admixture of several distinct West African and Caucasian haplotypes. A new West African TOMM40'523 haplotype, with APOE ε4 connected to a short TOMM40'523 allele, is observed in African-Americans but not Caucasians. CONCLUSION: These data have therapeutic implications for the age of onset risk algorithm estimates and the design of a prevention trial for African-Americans or other mixed ethnic populations.
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Apolipoproteínas E/genética , Población Negra/genética , Proteínas de Transporte de Membrana/genética , Población Blanca/genética , África Occidental , Estudios de Cohortes , Femenino , Frecuencia de los Genes , Haplotipos , Humanos , Masculino , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Poli T/genética , Estados UnidosRESUMEN
Pharmacogenetics (PGx) is the study and application of how interindividual differences in our genomes can influence drug responses. By evaluating individuals' genetic variability in genes related to drug metabolism, PGx testing has the capabilities to individualise primary care and build a safer drug prescription model than the current "one-size-fits-all" approach. In particular, the use of PGx testing in psychiatry has shown promising evidence in improving drug efficacy as well as reducing toxicity and adverse drug reactions. Despite randomised controlled trials demonstrating an evidence base for its use, there are still numerous barriers impeding its implementation. This review paper will discuss the management of mental health conditions with PGx-guided treatment with a strong focus on youth mental illness. PGx testing in clinical practice, the concerns for its implementation in youth psychiatry, and some of the barriers inhibiting its integration in clinical healthcare will also be discussed. Overall, this paper provides a comprehensive review of the current state of knowledge and application for PGx in psychiatry and summarises the capabilities of genetic information to personalising medicine for the treatment of mental ill-health in youth.
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Neurofilament heavy (NEFH) is one of the critical proteins required for the formation of the neuronal cytoskeleton and polymorphisms in NEFH are reported as a rare cause of sporadic ALS (sALS). In the current study, a candidate tetranucleotide (TTTA) repeat variant in NEFH was selected using an in-silico short structural variant (SSV) evaluation algorithm and investigated in two cohorts of North American sALS patients, both separately and combined (Duke cohort n = 138, Coriell cohort n = 333; combined cohort n = 471), compared to a group of healthy controls from the Coriell Institute biobank (n = 496). Stratification according to site of disease onset revealed that the 9 TTTA allele was associated with reduced disease risk, specifically confined to spinal-onset sALS patients in the Duke cohort (p = 0.001). Furthermore, carriage of the 10 TTTA allele was associated with a 2.7 year later age of disease onset in the larger combined sALS cohort (p = 0.02). These results suggest that the 9 and 10 TTTA motif length may have a protective advantage for potentially lowering the risk of sALS and delaying the age of disease onset, however, these results need to be replicated in larger multicenter and multi-ethnic cohorts.
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Esclerosis Amiotrófica Lateral , Predisposición Genética a la Enfermedad , Proteínas de Neurofilamentos/genética , Esclerosis Amiotrófica Lateral/epidemiología , Esclerosis Amiotrófica Lateral/genética , Humanos , Filamentos Intermedios , Mutación , Polimorfismo GenéticoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective, early degeneration of motor neurons in the brain and spinal cord. Motor neurons have long axonal projections, which rely on the integrity of neuronal cytoskeleton and mitochondria to regulate energy requirements for maintaining axonal stability, anterograde and retrograde transport, and signaling between neurons. The formation of protein aggregates which contain cytoskeletal proteins, and mitochondrial dysfunction both have devastating effects on the function of neurons and are shared pathological features across several neurodegenerative conditions, including ALS, Alzheimer's disease, Parkinson's disease, Huntington's disease and Charcot-Marie-Tooth disease. Furthermore, it is becoming increasingly clear that cytoskeletal integrity and mitochondrial function are intricately linked. Therefore, dysregulations of the cytoskeletal network and mitochondrial homeostasis and localization, may be common pathways in the initial steps of neurodegeneration. Here we review and discuss known contributors, including variants in genetic loci and aberrant protein activities, which modify cytoskeletal integrity, axonal transport and mitochondrial localization in ALS and have overlapping features with other neurodegenerative diseases. Additionally, we explore some emerging pathways that may contribute to this disruption in ALS.
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Esclerosis Amiotrófica Lateral , Enfermedades Neurodegenerativas , Esclerosis Amiotrófica Lateral/patología , Citoesqueleto/metabolismo , Citoesqueleto/patología , Humanos , Mitocondrias/metabolismo , Neuronas Motoras/patología , Enfermedades Neurodegenerativas/metabolismoRESUMEN
The translocase of outer mitochondrial membrane 40 (TOMM40) '523' polymorphism has previously been associated with age of Alzheimer's disease onset and cognitive functioning in non-pathological ageing, but has not been explored as a candidate risk marker for cognitive decline in Parkinson's disease (PD). Therefore, this longitudinal study investigated the role of the '523' variant in cognitive decline in a patient cohort from the Parkinson's Progression Markers Initiative. As such, a group of 368 people with PD were assessed annually for cognitive performance using multiple neuropsychological protocols, and were genotyped for the TOMM40 '523' variant using whole-genome sequencing data. Covariate-adjusted generalised linear mixed models were utilised to examine the relationship between TOMM40 '523' allele lengths and cognitive scores, while taking into account the APOE ε genotype. Cognitive scores declined over the 5-year study period and were lower in males than in females. When accounting for APOE ε4, the TOMM40 '523' variant was not robustly associated with overall cognitive performance. However, in APOE ε3/ε3 carriers, who accounted for ~60% of the whole cohort, carriage of shorter '523' alleles was associated with more severe cognitive decline in both sexes, while carriage of the longer alleles in females were associated with better preservation of global cognition and a number of cognitive sub-domains, and with a delay in progression to dementia. The findings indicate that when taken in conjunction with the APOE genotype, TOMM40 '523' allele length is a significant independent determinant and marker for the trajectory of cognitive decline and risk of dementia in PD.
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Abnormal mitochondrial function is a key process in the pathogenesis of Parkinson's disease (PD). The central pore-forming protein TOM40 of the mitochondria is encoded by the translocase of outer mitochondrial membrane 40 homologue gene (TOMM40). The highly variant '523' poly-T repeat is associated with age-related cognitive decline and age of onset in Alzheimer's disease, but whether it plays a role in modifying the risk or clinical course of PD it yet to be elucidated. The TOMM40 '523' allele length was determined in 634 people with PD and 422 healthy controls from an Australian cohort and the Parkinson's Progression Markers Initiative (PPMI) cohort, using polymerase chain reaction or whole genome sequencing analysis. Genotype and allele frequencies of TOMM40 '523' and APOE ε did not differ significantly between the cohorts. Analyses revealed TOMM40 '523' allele groups were not associated with disease risk, while considering APOE ε genotype. Regression analyses revealed the TOMM40 S/S genotype was associated with a significantly later age of symptom onset in the PPMI PD cohort, but not after correction for covariates, or in the Australian cohort. Whilst variation in the TOMM40 '523' polymorphism was not associated with PD risk, the possibility that it may be a modifying factor for age of symptom onset warrants further investigation in other PD populations.
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Apolipoproteínas E/genética , Disfunción Cognitiva/genética , Predisposición Genética a la Enfermedad , Proteínas de Transporte de Membrana/genética , Enfermedad de Parkinson/genética , Edad de Inicio , Anciano , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Australia/epidemiología , Disfunción Cognitiva/patología , Estudios de Cohortes , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética , Humanos , Masculino , Persona de Mediana Edad , Mitocondrias/genética , Mitocondrias/patología , Membranas Mitocondriales/metabolismo , Membranas Mitocondriales/patología , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Enfermedad de Parkinson/epidemiología , Enfermedad de Parkinson/patología , Polimorfismo Genético , Factores de RiesgoRESUMEN
OBJECTIVE: There is a critical need to establish genetic markers that explain the complex phenotypes and pathogenicity of ALS. This study identified a polymorphism in the Stathmin-2 gene and investigated its association with sporadic ALS (sALS) disease risk, age-of onset and survival duration. METHODS: The candidate CA repeat was systematically analyzed using PCR, Sanger sequencing and high throughput capillary separation for genotyping. Stathmin-2 expression was investigated using RT-PCR in patient olfactory neurosphere-derived (ONS) cells and RNA sequencing in laser-captured spinal motor neurons. RESULTS: In a case-control analysis of a combined North American sALS cohort (n = 321) and population control group (n = 332), long/long CA genotypes were significantly associated with disease risk (p = 0.042), and most strongly when one allele was a 24 CA repeat (p = 0.0023). In addition, longer CA allele length was associated with earlier age-of-onset (p = 0.039), and shorter survival duration in bulbar-onset cases (p = 0.006). In an Australian longitudinal sALS cohort (n = 67), ALS functional rating scale scores were significantly lower in carriers of the long/long genotype (p = 0.034). Stathmin-2 mRNA expression was reduced in sporadic patient ONS cells. Additionally, sALS patients and controls exhibited variable expression of Stathmin-2 mRNA according to CA genotype in laser-captured spinal motor neurons. CONCLUSIONS: We report a novel non-coding CA repeat in Stathmin-2 which is associated with sALS disease risk and has disease modifying effects. The potential value of this variant as a disease marker and tool for cohort enrichment in clinical trials warrants further investigation.
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Research is increasingly focusing on gut inflammation as a contributor to Parkinson's disease (PD). Such gut inflammation is proposed to arise from a complex interaction between various genetic, environmental, and lifestyle factors, however these factors are under-characterized. This study investigated the association between PD and single-nucleotide polymorphisms (SNPs) in genes responsible for binding of bacterial metabolites and intestinal homeostasis, which have been implicated in intestinal infections or inflammatory bowel disease. A case-control analysis was performed utilizing the following cohorts: (i) patients from the Australian Parkinson's Disease Registry (APDR) (n = 212); (ii) a Caucasian subset of the Parkinson's Progression Markers Initiative (PPMI) cohort (n = 376); (iii) a combined control group (n = 404). The following SNPs were analyzed: PGLYRP2 rs892145, PGLYRP4 rs10888557, TLR1 rs4833095, TLR2 rs3804099, TLR4 rs7873784, CD14 rs2569190, MUC1 rs4072037, MUC2 rs11825977, CLDN2 rs12008279 and rs12014762, and CLDN4 rs8629. PD risk was significantly associated with PGLYRP4 rs10888557 genotype in both cohorts. PGLYRP2 rs892145 and TLR1 rs4833095 were also associated with disease risk in the APDR cohort, and TLR2 rs3804099 and MUC2 rs11825977 genotypes in the PPMI cohort. Interactive risk effects between PGLYRP2/PGLYRP4 and PGLYRP4/TLR2 were evident in the APDR and PPMI cohorts, respectively. In the APDR cohort, the PGLYRP4 GC genotype was significantly associated with age of symptom onset, independently of gender, toxin exposure or smoking status. This study demonstrates that genetic variation in the bacterial receptor PGLYRP4 may modulate risk and age-of-onset in idiopathic PD, while variants in PGLYRP2, TLR1/2, and MUC2 may also influence PD risk. Overall, this study provides evidence to support the role of dysregulated host-microbiome signaling and gut inflammation in PD, and further investigation of these SNPs and proteins may help identify people at risk of developing PD or increase understanding of early disease mechanisms.
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OBJECTIVE: As structural variations may underpin susceptibility to complex neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), the objective of this study was to investigate a structural variant (SV) within sequestosome 1 (SQSTM1). METHODS: A candidate insertion/deletion variant within intron 5 of the SQSTM1 gene was identified using a previously established SV evaluation algorithm and chosen according to its subsequent theoretical effect on gene expression. The variant was systematically assessed through PCR, polyacrylamide gel fractionation, Sanger sequencing, and reverse transcriptase PCR. RESULTS: A reliable and robust assay confirmed the polymorphic nature of this variant and that the variant may influence SQSTM1 transcript levels. In a North American cohort of patients with familial ALS (fALS) and sporadic ALS (sALS) (n = 403) and age-matched healthy controls (n = 562), we subsequently showed that the SQSTM1 variant is associated with fALS (p = 0.0036), particularly in familial superoxide dismutase 1 mutation positive patients (p = 0.0005), but not with patients with sALS (p = 0.97). CONCLUSIONS: This disease association highlights the importance and implications of further investigation into SVs that may provide new targets for cohort stratification and therapeutic development.
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OBJECTIVE: To test the hypothesis that rs573116164 will have disease-modifying effects in patients with superoxide dismutase 1 (SOD1) familial amyotrophic lateral sclerosis (fALS), we characterized rs573116164 within a cohort of 190 patients with fALS and 560 healthy age-matched controls to assess the variant for association with various measures of disease. METHODS: Using a previously described bioinformatics evaluation algorithm, a polymorphic short structural variant associated with SOD1 was identified according to its theoretical effect on gene expression. An 12-18 poly-T repeat (rs573116164) within the 3' untranslated region of serine and arginine rich proteins-related carboxy terminal domain associated factor 4 (SCAF4), a gene that is adjacent to SOD1, was assessed for disease association and influence on survival and age at onset in an fALS cohort using PCR, Sanger sequencing, and capillary separation techniques for allele detection. RESULTS: In a North American cohort of predominantly SOD1 fALS patients (n =190) and age-matched healthy controls (n = 560), we showed that carriage of an 18T SCAF4 allele was associated with disease within this cohort (odds ratio [OR] 6.6; 95% confidence interval [CI] 3.9-11.2; p = 4.0e-11), but also within non-SOD1 cases (n = 27; OR 5.3; 95% CI 1.9-14.5; p = 0.0014). This finding suggests genetically SOD1-independent effects of SCAF4 on fALS susceptibility. Furthermore, carriage of an 18T allele was associated with a 26-month reduction in survival time (95% CI 6.6-40.8; p = 0.014), but did not affect age at onset of disease. CONCLUSIONS: The findings in this fALS cohort suggest that rs573116164 could have SOD1-independent and broader relevance in ALS, warranting further investigation in other fALS and sporadic ALS cohorts, as well as studies of functional effects of the 18T variant on gene expression.
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The scientific landscape surrounding amyotrophic lateral sclerosis (ALS) continues to shift as the number of genes associated with the disease risk and pathogenesis, and the cellular processes involved, continues to grow. Despite decades of intense research and over 50 potentially causative or disease-modifying genes identified, etiology remains unexplained and treatment options remain limited for the majority of ALS patients. Various factors have contributed to the slow progress in understanding and developing therapeutics for this disease. Here, we review the genetic basis of ALS, highlighting factors that have contributed to the elusiveness of genetic heritability. The most commonly mutated ALS-linked genes are reviewed with an emphasis on disease-causing mechanisms. The cellular processes involved in ALS pathogenesis are discussed, with evidence implicating their involvement in ALS summarized. Past and present therapeutic strategies and the benefits and limitations of the model systems available to ALS researchers are discussed with future directions for research that may lead to effective treatment strategies outlined.
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The interaction between the gut microbiota and alpha-synuclein (αSyn) aggregation in Parkinson's disease (PD) is receiving increasing attention. The objective of this study was to investigate gut microbiota, and effects of an inflammatory lipopolysaccharide (LPS) trigger in a human αSyn over-expressing mouse model of PD (Thy1-αSyn). Stool samples from patients with confirmed PD and Thy1-αSyn mice were analyzed using 16S ribosomal RNA sequencing. Compared to healthy controls, the relative abundance of mucin-degrading Verrucomicrobiae and LPS-producing Gammaproteobacteria were greater in PD patients. In mice, the abundance of Gammaproteobacteria was negligible in both Thy1-αSyn and wild-type (WT) animals, while Verrucomicrobiae were reduced in Thy1-αSyn mice. The effect of LPS on intestinal barrier function was investigated in vitro using intestinal epithelial (IEC-6) cells, and in vivo via administration of LPS in drinking water to Thy1-αSyn mice. Acute exposure to LPS in vitro resulted in a reduction and altered distribution of the tight junction markers ZO-1 and e-Cadherin around the cell membrane in IEC-6 cells, as shown by immunohistochemistry. LPS administration in Thy1-αSyn mice resulted in the emergence of early motor manifestations at 10 weeks, compared to untreated mice who were still asymptomatic at this age. This study reaffirms that an altered microbiome exists in patients with PD, and supports the notion of a proinflammatory gut microbiome environment as a trigger for PD pathogenesis.
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INTRODUCTION: In this article we discuss several human neurological diseases and their relationship to specific highly polymorphic small structural variants (SVs). Unlike genome-wide association analysis (GWAS), this methodology is not a genome screen to define new possibly associated genes, requiring statistical corrections for a million association tests. SVs provide local mapping information at a specific locus. Used with phylogenetic analysis, the specific association of length variants can be mapped and recognized. AREAS COVERED: This experimental strategy provides identification of DNA variants, particularly variable length Simple Sequence Repeats (SSRs or STRs or microsatellites) that provide specific local association data at the SV locus. Phylogenetic analysis that includes the specific appearance of different length SV variations can differentiate specific phenotypic risks in a population such as age of onset related to variable length polymorphisms and risk of phenotypic variations associated with several adjacent structural variations (SVs). We focus on data for three recent examples associated with Alzheimer's disease, Levy Bodies, and Parkinson's disease. EXPERT OPINION: SVs are understudied, but have led directly to mechanism of pathogenesis studies involving the regulation of gene expression. The identification of specific length polymorphisms associated with clinical disease has led to translational advances and new drug discovery.
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Enfermedad de Alzheimer/genética , Enfermedad por Cuerpos de Lewy/genética , Enfermedad de Parkinson/genética , Enfermedad de Alzheimer/fisiopatología , Animales , Exones , Regulación de la Expresión Génica/genética , Variación Genética , Estudio de Asociación del Genoma Completo , Humanos , Enfermedad por Cuerpos de Lewy/fisiopatología , Repeticiones de Microsatélite , Enfermedad de Parkinson/fisiopatología , Filogenia , Polimorfismo de Nucleótido Simple , PronósticoRESUMEN
There has been a decline in the number of new drugs registered over the past decade and regulatory concerns for safety as well as payer concerns for efficacy have focused attention on stratified medicine. Integration of pharmacogenetics into the drug development pipeline will contribute to the development of new stratified drugs. We describe here the concept of pipeline pharmacogenetics and its application throughout the phases of drug discovery. Pipeline pharmacogenetics enables the evaluation of the genetic contribution to safety potentially lowering barriers to registration as well as providing rationale for efficacy and enabling co-development of genetic in vitro diagnostics.
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Diseño de Fármacos , Industria Farmacéutica/tendencias , Farmacogenética , Animales , Ensayos Clínicos como Asunto/métodos , Evaluación Preclínica de Medicamentos/métodos , Humanos , Vigilancia de Productos Comercializados/métodosRESUMEN
Mutations in the leucine-rich repeat kinase-2 gene (LRRK2) are responsible for some forms of familial as well as sporadic Parkinson's disease (PD). The purpose of this study was to examine the frequency of a single pathogenic mutation (6055G > A) in the kinase domain of this gene in United States and Tunisian familial PD and to compare clinical characteristics between patients with and without the mutation. Standardized case report forms were used for clinical and demographic data collection. We investigated the frequency of the most common substitution of LRRK2 (G2019S, 6055G>A) and its impact on epidemiological and phenotypic features. The frequency of mutations in Tunisian families was 42% (38/91) and in U.S. families 2.6% (1/39), with the unique opportunity to compare homozygous (n = 23) and heterozygous (n = 109) Tunisian carriers of G2019S substitutions. Individuals with G2019S substitutions had an older age at onset but few other differences compared with families negative for the substitution. Patients with LRRK2 mutations had typical clinical features of PD. Comparisons between individuals with heterozygous and homozygous LRRK2 mutations suggested that gene dosage was not correlated with phenotypic differences; however, the estimated penetrance was greater in homozygotes across all age groups.
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Salud de la Familia , Glicina/genética , Enfermedad de Parkinson/epidemiología , Enfermedad de Parkinson/genética , Proteínas Serina-Treonina Quinasas/genética , Serina/genética , Adulto , Anciano , Anciano de 80 o más Años , Comparación Transcultural , Análisis Mutacional de ADN , Femenino , Pruebas Genéticas/métodos , Genotipo , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Masculino , Persona de Mediana Edad , Mutación/genética , América del Norte/epidemiología , América del Norte/etnología , Túnez/epidemiología , Túnez/etnología , Población BlancaRESUMEN
Nemaline myopathy is a human neuromuscular disorder associated with muscle weakness, Z-line accumulations (rods), and myofibrillar disorganization. Disease-causing mutations have been identified in genes encoding muscle thin filament proteins: actin, nebulin, slow troponin T, betaTropomyosin, and alphaTropomyosin(slow). Skeletal muscle expresses three tropomyosin (Tm) isoforms from separate genes: alphaTm(fast)(alphaTm, TPM1), betaTm (TPM2), and alphaTm(slow) (gammaTm, TPM3). In this article, we show that the level of betaTm, but not alphaTm(fast) protein, is reduced in human patients with mutations in alphaTm(slow) and in a transgenic mouse model of alphaTm(slow)(Met9Arg) nemaline myopathy. A postnatal time course of Tm expression in muscles of the mice indicated that the onset of alphaTm(slow)(Met9Arg) expression coincides with the decline of betaTm. Reduction of betaTm levels is independent of the degree of pathology (rods) within a muscle and is detected before the onset of muscle weakness. Thus, reduction in the level of betaTm represents an early clinical diagnostic marker for alphaTm(slow)-based mutations. Examinations of tropomyosin dimer formation using either recombinant proteins or sarcomeric extracts show that the mutation reduces the formation of the preferred alpha/beta heterodimer. We suggest this perturbation of tropomyosin isoform levels and dimer preference alters sarcomeric thin filament dynamics and contributes to muscle weakness in nemaline myopathy.
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Regulación de la Expresión Génica/fisiología , Músculo Esquelético/metabolismo , Mutación , Miopatías Nemalínicas/metabolismo , Tropomiosina/metabolismo , Animales , Animales Recién Nacidos , Arginina/genética , Northern Blotting/métodos , Western Blotting/métodos , Análisis Mutacional de ADN/métodos , Dimerización , Modelos Animales de Enfermedad , Humanos , Metionina/genética , Ratones , Ratones Transgénicos , Miopatías Nemalínicas/genética , Regiones Promotoras Genéticas/genética , Estructura Secundaria de Proteína/fisiología , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Factores de Tiempo , Tropomiosina/clasificación , Tropomiosina/genéticaRESUMEN
We have previously reported a Met9Arg mutation in the human skeletal muscle alpha tropomyosin gene (TPM3) associated with autosomal dominant nemaline myopathy [Nat. Genet. 9 (1995) 75]. We describe here the generation of wild-type (Wt-tpm3) and Met9Arg (M9R-tpm3) mutant human skeletal muscle slow alpha tropomyosin using the Baculovirus expression vector system (BEVS). This system produces correct posttranslationally modified recombinant tropomyosin proteins in insect cells. We show that the interactions of Wt-tpm3 with actin and tropomyosin are comparable to those of fast alpha tropomyosin isolated from chicken striated muscle. However, the recombinant M9R-tpm3 is at least 100 times less effective at binding actin than Wt-tpm3. This paper represents the first study of this mutation directly on the human isoform of tropomyosin that is involved in nemaline myopathy. It also represents the first time that human tpm3 has been produced using BEVS. This system can now be used to accurately demonstrate the effect of this (and other disease-associated tropomyosin mutations) on the interactions of tpm3 with the other protein components of the muscle thin filament, including those responsible for differing forms of nemaline myopathy.
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Baculoviridae/genética , Miopatías Nemalínicas/genética , Tropomiosina/genética , Tropomiosina/metabolismo , Actinas/metabolismo , Animales , Baculoviridae/metabolismo , Línea Celular , Vectores Genéticos , Humanos , Punto Isoeléctrico , Espectrometría de Masas , Mutación , Miopatías Nemalínicas/metabolismo , Isoformas de Proteínas , Proteínas Recombinantes/metabolismo , Tropomiosina/química , Tropomiosina/aislamiento & purificaciónRESUMEN
Direct protein extraction from animals is the only approach available to obtain caltrin, calcium transport inhibitor. Here we report the expression and purification of caltrin, previously shown to hinder the influx of calcium into epididymal spermatozoa. Cloning of the caltrin gene into the pCDNA3.1 V5/His-TOPO vector and the subsequent ligation of the caltrin-His sequence into the transfer vector pBacPAK9 allowed the expression of recombinant caltrin using the baculovirus expression vector system (BEVS). Recombinant His-tagged caltrin was purified utilising both nickel (II)-nitrilotriacetic acid (Ni(2+)-NTA) and cobalt (II)-carboxymethylaspartate (Co(2+)-CmAsp) immobilised metal affinity chromatography (IMAC). Using the BEVS, caltrin-His was identified in the supernatant and in the cell lysate, suggesting that caltrin is a secreted protein. Based on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot results, purified recombinant caltrin-His was ascertained to be approximately 14.5kDa. Purification under the Co(2+) system yielded significantly purer protein samples when compared to the Ni(2+) system. Furthermore, Co(2+) was observed to bind the recombinant caltrin-His protein with higher efficiency and specificity and to yield a higher total protein concentration. Collectively, our results indicate that the Co(2+) system would be a better approach for purifying caltrin-His proteins than the Ni(2+).