Proteomic characterization of neuromelanin granules isolated from human substantia nigra by laser-microdissection.

Neuromelanin is a complex polymer pigment found primarily in the dopaminergic neurons of human substantia nigra. Neuromelanin pigment is stored in granules including a protein matrix and lipid droplets. Neuromelanin granules are yet only partially characterised regarding their structure and function. To clarify the exact function of neuromelanin granules in humans, their enrichment and in-depth characterization from human substantia nigra is necessary. Previously published global proteome studies of neuromelanin granules in human substantia nigra required high tissue amounts. Due to the limited availability of human brain tissue we established a new method based on laser microdissection combined with mass spectrometry for the isolation and analysis of neuromelanin granules. With this method it is possible for the first time to isolate a sufficient amount of neuromelanin granules for global proteomics analysis from ten 10 µm tissue sections. In total 1,000 proteins were identified associated with neuromelanin granules. More than 68% of those proteins were also identified in previously performed studies. Our results confirm and further extend previously described findings, supporting the connection of neuromelanin granules to iron homeostasis and lysosomes or endosomes. Hence, this method is suitable for the donor specific enrichment and proteomic analysis of neuromelanin granules.

From T-tubule to sarcolemma: damage-induced dysferlin translocation in early myogenesis.

The dysferlin gene is mutated in limb-girdle muscular dystrophy type 2B, Miyoshi myopathy, and distal anterior compartment myopathy. In mature skeletal muscle, dysferlin is located predominantly at the sarcolemma, where it plays a role in membrane fusion and repair. To investigate the role of dysferlin during early muscle differentiation, its localization was studied at high resolution in a muscle cell line. This demonstrated that dysferlin is not expressed at the plasmalemma of myotubes but mostly localizes to the T-tubule network. However, dysferlin translocated to the site of injury and toward the plasma membrane in a Ca2+-dependent fashion in response to a newly designed in vitro wounding assay. This reaction was specific to the full-length protein, as heterologously expressed deletion mutants of distinct C2 domains of dysferlin did not show this response. These results shed light on the dynamics of muscle membrane repair and are highly indicative of a specific role of dysferlin in this process in early myogenesis.

Identification of an X-chromosomal locus and haplotype modulating the phenotype of a mitochondrial DNA disorder.

Mitochondrial DNA (mtDNA) mutations are a major cause of human disease. A large number of different molecular defects ultimately compromise oxidative phosphorylation, but it is not clear why the same biochemical defect can cause diverse clinical phenotypes. There is emerging evidence that nuclear genes modulate the phenotype of primary mtDNA disorders. Here, we define an X-chromosomal haplotype that interacts with specific MTND mutations to cause visual failure in the most common mtDNA disease, Leber hereditary optic neuropathy. This effect is independent of the mtDNA genetic background and explains the variable penetrance and sex bias that characterizes this disorder.

Mitochondrial DNA and survival after sepsis: a prospective study.

BACKGROUND: Human genome evolution has been shaped by infectious disease. Although most genetic studies have focused on the immune system, recovery after sepsis is directly related to physiological reserve that is critically dependent on mitochondrial function. We investigated whether haplogroup H, the most common type of mitochondrial DNA (mtDNA) in Europe, contributes to the subtle genetic variation in survival after sepsis. METHODS: In a prospective study, we included 150 individuals who were sequentially admitted to the intensive care unit in a hospital in Newcastle upon Tyne, UK. After clinical data were obtained, patients underwent mtDNA haplotyping by analysis with PCR and restriction fragment length polymorphism. As endpoints, we used death during the 6-month period or survival at 6 months. FINDINGS: Follow-up was complete for all study participants, although the haplotype of two patients could not be reliably determined. On admission to the intensive care unit, the frequency of mtDNA haplogroup H in study patients did not differ between study patients admitted with severe sepsis and 542 age-matched controls from the northeast of England. MtDNA haplogroup H was a strong independent predictor of outcome during severe sepsis, conferring a 2.12-fold (95% CI 1.02-4.43) increased chance of survival at 180 days compared with individuals without the haplogroup H. INTERPRETATION: Although haplogroup H is the most recent addition to the group of European mtDNA, paradoxically it is also the most common. Increased survival after sepsis provides one explanation for this observation. MtDNA haplotyping offers a new means of risk stratification of patients with severe infections, which suggests new avenues for therapeutic intervention.

Minimum prevalence of spinocerebellar ataxia 17 in the north east of England.

OBJECTIVE: To determine the minimum prevalence of spinocerebellar ataxia type 17 (SCA17) in the north east of England. PATIENTS AND METHODS: A defined region containing 2,516,500 individuals with 192 families with undiagnosed ataxia, 90 patients with a Huntington's disease-like phenotype and 292 controls. The number of (CAG/CAA)(n) repeats in the SCA17/TBP gene was determined by fluorescent PCR and sequenced in affected individuals. RESULTS: The mean repeat size for 584 control alleles was 34 (S.D.=3.58), ranging from 25 to 40. Two index cases had larger alleles with repeat lengths greater than the control range. Affected family members presented in adult life with ataxia followed by extrapyramidal features and cognitive impairment. In one family 44 repeats were associated with a younger age of onset than has been previously described. CONCLUSIONS: The minimum prevalence of SCA17 in the north east of England was 0.16/100,000 (upper 95% confidence interval 0.31/100,000).

A polymorphism at codon 31 of gene p21 is not associated with primary open angle glaucoma in Caucasians.

BACKGROUND: Primary open angle glaucoma (POAG) is considered to be a neurodegenerative optic neuropathy, in which cell death occurs by apoptosis. p21, is an important protective component of the apoptotic pathway, regulating cellular arrest in the presence of DNA damage. An unstable or altered p21 protein could modify the cellular response to genomic injury and abolish the effect of p21. A previous study on a Chinese cohort suggested that the p21 codon 31 polymorphism may alter the state of apoptosis in glaucomatous optic neuropathy, failing to protect the ganglion cells. The aim of this study was to test the hypothesis that a p21 codon 31 polymorphism is associated with POAG on a Caucasian cohort. METHODS: 140 POAG patients and a control group of 73 healthy individuals were included in the study. All the subjects were of Caucasian origin. Genomic DNA was amplified by polymerase chain reaction, followed by enzymatic restriction fragment length polymorphism technique (PCR-RFLP). Patients and controls were genotyped for a single nucleotide polymorphism (C/A transversion) in the third base of codon 31 of p21, which leads to a serine (Ser)/arginine (Arg) substitution. RESULTS: The distribution of the genotypes in the POAG patients showed 128 (91.4%) Ser homozygotes, 10 (7.1%) Ser/Arg heterozygotes and 2 (1.5%) Arg homozygotes. In the control cohort, there were 61 (83.6%) Ser homozygotes and 12 (16.4%) Ser/Arg heterozygotes. No Arg homozygotes were present amongst the control group. Both the allelic and genotypic frequencies of the Ser or Arg residues at codon 31 were not significantly different between POAG patients and controls (Fisher's exact test, P = 0.20 for alleles and P = 0.0561 for genotypes). CONCLUSION: This study suggests that the p21 codon 31 polymorphism does not contribute to the risk of POAG in the Caucasian population.

Mitochondrial DNA haplogroup cluster UKJT reduces the risk of PD.

There is increasing evidence that genetic variants of mitochondrial DNA have an important role in the cause of idiopathic Parkinson's disease. We determined the mitochondrial DNA haplogroup of 455 Parkinson's disease cases, 185 Alzheimer's disease cases, and 447 healthy English control subjects. The UKJT haplogroup cluster was associated with a 22% reduction in population-attributable risk for Parkinson's disease. There was no association between individual haplogroups or the UKJT cluster and Alzheimer's disease, confirming that the association with Parkinson's disease was disease specific and not a general effect seen in all neurodegenerative diseases.

Co-segregation and heteroplasmy of two coding-region mtDNA mutations within a matrilineal pedigree.

The ENG1 Leber's hereditary optic neuropathy (LHON) family spans six generations and comprises more than 90 maternally related individuals. In this pedigree, the G:A LHON mutation at nucleotide position 11778 shows a complex pattern of segregation in which it is homoplasmic mutant in two branches, homoplasmic wildtype in another, and heteroplasmic in a fourth branch. In addition, there is co-segregation of the 11778 mutant allele and of a G:A silent polymorphism at nucleotide position 5471 in 18 of 19 family members. This co-segregation indicates that the two substitutions arose either simultaneously, or nearly so, in the same "founder" mtDNA molecule. However, the highly divergent mitochondrial allele ratios in the one family member suggest that there has been a complex origin and segregation "history" of these two substitutions. Taking all of the results into consideration, the evidence supports sequential single mutations at sites 5471 and 11778, in close temporal proximity, with subsequent segregation of the intermediate mutational genotype to high levels in one branch of the ENG1 LHON family. In other branches, either the double wildtype or double mutant genotype has become essentially homoplasmic.

Apolipoprotein E promoter polymorphisms do not have a major influence on the risk of developing primary open angle glaucoma.

PURPOSE: Primary open angle glaucoma (POAG) is a major cause of late onset visual failure of unknown etiology. Recent genetic association studies have implicated the apolipoprotein E (APOE) gene in the pathophysiology of primary open angle glaucoma, but there have been conflicting findings. METHODS: To resolve this issue we studied 140 cases and 73 controls that were carefully phenotyped, and used a logistic regression model to simultaneously analyze the effect of apolipoprotein E genotype and functional polymorphisms in the apolipoprotein E gene promoter while controlling for potentially confounding variables. RESULTS: We found no evidence of an association between the apolipoprotein E promoter region polymorphisms and primary open angle glaucoma. CONCLUSIONS: Apolipoprotein E promoter polymorphisms are unlikely to have a major impact on the pathophysiology of primary open angle glaucoma.

Molecular epidemiology of spinocerebellar ataxia type 6.

We performed a population-based clinical and molecular genetic study of spinocerebellar ataxia type 6 (SCA6) in the northeast of England. The minimum point prevalence of SCA6 was 1.59 in 100,000 (95% confidence interval [CI], 1.04-2.14), and the number of individuals who either had SCA6 or are at risk of developing SCA6 was at least 5.21 in 100,000 (95% CI, 4.31-6.10), or 1 in 19,210. Microsatellite analysis of the CACNA1A gene indicated a founder effect for SCA6 within this region.

The role of apolipoprotein E gene polymorphisms in primary open-angle glaucoma.

OBJECTIVE: To test the hypothesis that genetic polymorphisms of the apolipoprotein E (APOE) gene are associated with primary open-angle glaucoma (POAG), based on the association between neurodegenerative diseases and the APOE genotype. METHODS: Genomic DNA was examined from an unrelated cohort of 137 POAG patients and 75 control subjects from the ophthalmology department of the Royal Victoria Infirmary. The APOE allele frequency (epsilon2, epsilon3, and epsilon4 alleles) was studied by polymerase chain reaction amplification of the related locus (19q13.2), enzymatic digestion of the products, gel electrophoresis, and imaging under UV illumination. For statistical analysis, we used a logistic regression model that included intraocular pressure as a continuous variable to study the possible correlation between POAG and APOE allele frequency. RESULTS: Logistic regression analysis showed no statistically significant association between the frequency of the APOE allele and POAG for the population studied, irrespective of the IOP (epsilon2 odds ratio, 0.82; 95% confidence interval, 0.12-5.79 [P =.84]; epsilon3 odds ratio, 0.39; 95% confidence interval, 0.10-1.49 [P =.17]; and epsilon4 odds ratio, 3.84; 95% confidence interval, 0.80-18.49 [P =.09]). CONCLUSION: In our cohort, the APOE genotype does not constitute a risk factor for developing POAG, even in patients with normal-tension glaucoma.Clinical Relevance Apolipoprotein E polymorphisms do not appear to be contributory to POAG.