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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.

A variable-length poly-T variant in intron 6 of the TOMM40 gene, rs10524523, is associated with risk and age-of-onset of sporadic (late-onset) Alzheimer's disease. In Caucasians, the three predominant alleles at this locus are Short (S), Long (L) or Very long (VL). On an APOE e3/3 background, the S/VL and VL/VL genotypes are more protective than S/S. The '523 poly-T has regulatory properties, in that the VL poly-T results in higher expression than the S poly-T in luciferase expression systems. The aim of the current work was to identify effects on cellular bioenergetics of increased TOM40 protein expression. MitoTracker Green fluorescence and autophagic vesicle staining was the same in control and over-expressing cells, but TOM40 over-expression was associated with increased expression of TOM20, a preprotein receptor of the TOM complex, the mitochondrial chaperone HSPA9, and PDHE1a, and increased activities of the oxidative phosphorylation complexes I and IV and of the TCA member α-ketoglutaric acid dehydrogenase. Consistent with the complex I findings, respiration was more sensitive to inhibition by rotenone in control cells than in the TOM40 over-expressing cells. In the absence of inhibitors, total cellular ATP, the mitochondrial membrane potential, and respiration were elevated in the over-expressing cells. Spare respiratory capacity was greater in the TOM40 over-expressing cells than in the controls. TOM40 over-expression blocked Ab-elicited decreases in the mitochondrial membrane potential, cellular ATP levels, and cellular viability in the control cells. These data suggest elevated expression of TOM40 may be protective of mitochondrial function.

Introduction Dementia is one of the major health threats to our aging society, and Alzheimer's disease (AD) is the leading cause. In Japan, ∼15% of the elderly population has dementia. The apolipoprotein E ( APOE ) genotype and a polymorphism (rs10524523) in the translocase of outer mitochondrial membrane 40 ( TOMM40 ) gene have been associated with the age of onset of AD. However, differences in allele frequencies of these markers in different ethnic populations are not well known. Methods Whole blood samples were collected from 300 Japanese subjects, and genomic DNA was extracted to determine APOE alleles and TOMM40 rs10524523 genotypes. Results Our results indicated that the APOE e3– TOMM40 ′523 short haplotype is less frequent in Japanese subjects than in Caucasians, whereas the APOE e3– TOMM40 ′523 long and APOE e3– TOMM40 ′523 very long haplotypes are more frequent in Japanese subjects than in Caucasians. We also showed that the APOE e4– TOMM40 ′523 short haplotype, which was noted to be frequently observed in African Americans, was also found in the Japanese population, although it is extremely rare in the Caucasian population. Discussion A biomarker risk assignment algorithm, using a combination of APOE, TOMM40 ′523 genotype, and age, has been developed to assign near-term risk for developing the onset of mild cognitive impairment due to AD and is being used as an enrichment tool in an ongoing delay-of-onset clinical trial. Understanding the characterization of APOE and TOMM40 allele frequencies in the Japanese population is the first step in developing a risk algorithm for AD research and clinical applications for AD prevention in Japan.

Introduction Family history (FH) of Alzheimer's disease (AD) affects mitochondrial function and may modulate effects of translocase of the outer mitochondrial membrane 40 kDa ( TOMM40 ) rs10524523 (‘523) poly-T length on memory decline. Methods For 912 nonapolipoprotein e4 middle-aged adults and 365 aged adults across the AD spectrum, linear mixed models gauged FH and TOMM40 ‘523 interactions on memory and global cognition between baseline and up to 10 years later. A cerebrospinal fluid mitochondrial function biomarker was also assessed. Results For FH negative participants, gene-dose preservation of memory and global cognition was seen for "very long" versus "short" carriers. For FH positive, an opposite gene-dose decline was seen for very long versus short carriers. Maternal FH was a stronger predictor in aged, but not middle-aged, participants. Similar gene-dose effects were seen for the mitochondrial biomarker aspartate aminotransferase. Discussion These results may clarify conflicting findings on TOMM40 poly-T length and AD-related decline.

Short structural variants (SSVs) are short genomic variants (

The methodology of Genome-Wide Association Screening (GWAS) has been applied for more than a decade. Translation to clinical utility has been limited, especially in Alzheimer's Disease (AD). It has become standard practice in the analyses of more than two dozen AD GWAS studies to exclude the apolipoprotein E (APOE) region because of its extraordinary statistical support, unique thus far in complex human diseases. New genes associated with AD are proposed frequently based on SNPs associated with odds ratio (OR)

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.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.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.

Introduction A straightforward, reproducible blood-based test that predicts age-dependent risk of Alzheimer's disease (AD) could be used as an enrichment tool for clinical development of therapies. This study evaluated the prognostic performance of a genetics-based biomarker risk algorithm (GBRA) established on a combination of apolipoprotein E (APOE)/translocase of outer mitochondrial membrane 40 homolog (TOMM40) genotypes and age, then compare it to cerebrospinal fluid (CSF) biomarkers, neuroimaging, and neurocognitive tests using data from two independent AD cohorts. Methods The GBRA was developed using data from the prospective Joseph and Kathleen Bryan, Alzheimer's Disease Research Center study (n = 407; 86 conversion events [mild cognitive impairment {MCI} or late-onset Alzheimer's disease {LOAD}]). The performance of the algorithm was tested using data from the Alzheimer's Disease Neuroimaging Initiative study (n = 660; 457 individuals categorized as MCI or LOAD). Results The positive predictive values and negative predictive values of the GBRA are in the range of 70%–80%. The relatively high odds ratio (approximately 3–5) and significant net reclassification index scores comparing the GBRA to a version based on APOE and age alone support the value of the GBRA in risk prediction for MCI due to LOAD. Performance of the GBRA compares favorably with CSF and imaging (functional magnetic resonance imaging) biomarkers. In addition, the GBRA “high” and “low” AD-risk categorizations correlated well with pathologic CSF biomarker levels, positron emission tomography amyloid burden, and neurocognitive scores. Discussion Unlike dynamic markers (i.e., imaging, protein, or lipid markers) that may be influenced by factors unrelated to disease, genomic DNA is easily collected, stable, and the technical methods for measurement are robust, inexpensive, and widely available. The performance characteristics of the GBRA support its use as a pharmacogenetic enrichment tool for LOAD delay-of-onset clinical trials and merit further evaluation for its clinical utility in evaluating therapeutic efficacy.

ObjectiveTo 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.MethodsUsing 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.ResultsIn 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.ConclusionsThe 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.

If we are to find treatments to postpone, reduce the risk of, or completely prevent the clinical onset of Alzheimer disease (AD), we need faster methods to evaluate promising preclinical AD treatments, new ways to work together in support of common goals, and a determination to expedite the initiation and performance of preclinical AD trials. In this article, we note some of the current challenges, opportunities and emerging strategies in preclinical AD treatment. We describe the Collaboration for Alzheimer's Prevention (CAP)-a convening, harmonizing and consensus-building initiative to help stakeholders advance AD prevention research with rigour, care and maximal impact-and we demonstrate the impact of CAP on the goals and design of new preclinical AD trials.

Introduction Dementia is one of the major health threats to our aging society, and Alzheimer's disease (AD) is the leading cause. In Japan, ∼15% of the elderly population has dementia. The apolipoprotein E (APOE) genotype and a polymorphism (rs10524523) in the translocase of outer mitochondrial membrane 40 (TOMM40) gene have been associated with the age of onset of AD. However, differences in allele frequencies of these markers in different ethnic populations are not well known. Methods Whole blood samples were collected from 300 Japanese subjects, and genomic DNA was extracted to determine APOE alleles and TOMM40 rs10524523 genotypes. Results Our results indicated that the APOE ε3–TOMM40′523 short haplotype is less frequent in Japanese subjects than in Caucasians, whereas the APOE ε3–TOMM40′523 long and APOE ε3–TOMM40′523 very long haplotypes are more frequent in Japanese subjects than in Caucasians. We also showed that the APOE ε4–TOMM40′523 short haplotype, which was noted to be frequently observed in African Americans, was also found in the Japanese population, although it is extremely rare in the Caucasian population. Discussion A biomarker risk assignment algorithm, using a combination of APOE, TOMM40′523 genotype, and age, has been developed to assign near-term risk for developing the onset of mild cognitive impairment due to AD and is being used as an enrichment tool in an ongoing delay-of-onset clinical trial. Understanding the characterization of APOE and TOMM40 allele frequencies in the Japanese population is the first step in developing a risk algorithm for AD research and clinical applications for AD prevention in Japan., Highlights • Linkage between the translocase of outer mitochondrial membrane 40 (TOMM40′523) and apolipoprotein E (APOE) allele differs depending on the population. • The APOE ε3–TOMM40′523 short haplotype is less frequent in the Japanese population than in Caucasian ones. • The APOE ε3–TOMM40′523 long and very long haplotypes are more frequent in the Japanese population than in Caucasian ones.

It is hypothesized that retrotransposons have played a fundamental role in primate evolution and that enhanced neurologic retrotransposon activity in humans may underlie the origin of higher cognitive function. As a potential consequence of this enhanced activity, it is likely that neurons are susceptible to deleterious retrotransposon pathways that can disrupt mitochondrial function. An example is observed in the TOMM40 gene, encoding a β-barrel protein critical for mitochondrial preprotein transport. Primate-specific Alu retrotransposons have repeatedly inserted into TOMM40 introns, and at least one variant associated with late-onset Alzheimer's disease originated from an Alu insertion event. We provide evidence of enriched Alu content in mitochondrial genes and postulate that Alus can disrupt mitochondrial populations in neurons, thereby setting the stage for progressive neurologic dysfunction. This Alu neurodegeneration hypothesis is compatible with decades of research and offers a plausible mechanism for the disruption of neuronal mitochondrial homeostasis, ultimately cascading into neurodegenerative disease.

The variable length poly-T, rs10524523 ('523') located within the TOMM40 gene, was recently associated with several phenotypes of cognitive function. The short (S) allele is associated with later AD onset age and better cognitive performance, compared to the longer alleles (long and very-long (VL)). There is strong linkage disequilibrium between variants in the TOMM40 and APOE genes. In this study, we investigated the effect of '523' on cognitive performance in a sample of cognitively normal Jewish elderly with type 2 diabetes, a group at particularly high risk for cognitive impairment. Using a MANCOVA procedure, we compared homozygous carriers of the S/S allele (N=179) to carriers of the VL/VL allele (N=152), controlling for demographic and cardiovascular covariates. The S/S group performed better than the VL/VL group (p=0.048), specifically in the executive function (p=0.04) and episodic memory (p=0.050) domains. These results suggest that previous findings of an association of the TOMM40 short allele with better cognitive performance, independently from the APOE variant status, are pertinent to elderly with diabetes.

Background We investigated the genomic region spanning the Translocase of the Outer Mitochondrial Membrane 40-kD ( TOMM40 ) and Apolipoprotein E ( APOE ) genes, that has been associated with the risk and age of onset of late-onset Alzheimer's disease (LOAD) to determine whether a highly polymorphic, intronic poly-T within this region (rs10524523; hereafter, 523) affects expression of the APOE and TOMM40 genes. Alleles of this locus are classified as S, short; L, long; and VL, very long based on the number of T residues. Methods We evaluated differences in APOE messenger RNA (mRNA) and TOMM40 mRNA levels as a function of the 523 genotype in two brain regions from APOE e3/e3 white autopsy-confirmed LOAD cases and normal controls. We further investigated the effect of the 523 locus in its native genomic context using a luciferase expression system. Results The expression of both genes was significantly increased with disease. Mean expression of APOE and TOMM40 mRNA levels were higher in VL homozygotes compared with S homozygotes in the temporal and occipital cortexes from normal and LOAD cases. Results of a luciferase reporter system were consistent with the human brain mRNA analysis; the 523 VL poly-T resulted in significantly higher expression than the S poly-T. Although the effect of poly-T length on reporter expression was the same in HepG2 hepatoma and SH-SY5Y neuroblastoma cells, the magnitude of the effect was greater in the neuroblastoma than in the hepatoma cells, which implies tissue-specific modulation of the 523 poly-T. Conclusions These results suggest that the 523 locus may contribute to LOAD susceptibility by modulating the expression of TOMM40 and/or APOE transcription.

Structural variants (SVs) include all insertions, deletions, and rearrangements in the genome, with several common types of nucleotide repeats including single sequence repeats, short tandem repeats, and insertion‐deletion length variants. Polyallelic SVs provide highly informative markers for association studies with well‐phenotyped cohorts. SVs can influence gene regulation by affecting epigenetics, transcription, splicing, and/or translation.1 Accurate assays of polyallelic SV loci are required to define the range and allele frequency of variable length alleles.2

A polyT repeat in an intronic polymorphism (rs10524523) in the TOMM40 gene, which encodes an outer mitochondrial membrane translocase involved in the transport of amyloid-β and other proteins into mitochondria, has been implicated in Alzheimer's disease and APOE-TOMM40 genotypes have been shown to modify disease risk and age at onset of symptoms. Because of the similarities between Alzheimer's disease and sporadic inclusion body myositis (s-IBM), and the importance of amyloid-β and mitochondrial changes in s-IBM, we investigated whether variation in poly-T repeat lengths in rs10524523 also influence susceptibility and age at onset in a cohort of 90 Caucasian s-IBM patients (55 males; age 69.1 ± 9.6). In carriers of APOE ε3/ε3 or ε3/ε4, genotypes with a very long (VL) poly-T repeat were under-represented in s-IBM compared to controls and were associated with a later age at symptom onset, suggesting that these genotypes may be protective. Our study is the first to suggest that polymorphisms in genes controlling mitochondrial function can influence susceptibility to s-IBM and have disease modifying effects. However, further studies in other s-IBM populations are needed to confirm these findings, as well as expression studies of different TOMM40 alleles in muscle tissue.

Chromosome 19q13.32 is a gene rich region, and has been implicated in multiple human phenotypes in adulthood including lipids traits, Alzheimer's disease, and longevity. Peroxisome Proliferator Activated Receptor Gamma (PPARγ) is a ligand-activated nuclear transcription factor that plays a role in human complex traits that are also genetically associated with the chromosome 19q13.32 region. Here, we study the effects of PPARγ on the regional expression regulation of the genes clustered within chromosome 19q13.32, specifically TOMM40, APOE, and APOC1, applying two complementary approaches. Using the short hairpin RNA (shRNA) method in the HepG2 cell-line we knocked down PPARγ expression and measured the effect on mRNA expression. We discovered PPARγ knock down increased the levels of TOMM40-, APOE-, and APOC1-mRNAs, with the highest increase in expression observed for APOE-mRNA. To complement the PPARγ knockdown findings we also examined the effects of low doses of PPARγ agonists (nM range) on mRNA expression of these genes. Low (nM) concentrations of pioglitazone (Pio) decreased transcription of TOMM40, APOE, and APOC1 genes, with the lowest mRNA levels for each gene observed at 1.5nM. Similar to the effect of PPARγ knockdown, the strongest response to pioglitazone was also observed for APOE-mRNA, and rosiglitazone (Rosi), another PPARγ agonist, produced results that were consistent with these. In conclusion, our results further established a role for PPARγ in regional transcriptional regulation of chr19q13.32, underpinning the association between PPARγ, the chr19q13.32 genes cluster, and human complex traits and disease.

Cognitive decline is a feared aspect of growing old. It is a major contributor to lower quality of life and loss of independence in old age. We investigated the genetic contribution to individual differences in nonpathological cognitive ageing in five cohorts of older adults. We undertook a genome-wide association analysis using 549 692 single-nucleotide polymorphisms (SNPs) in 3511 unrelated adults in the Cognitive Ageing Genetics in England and Scotland (CAGES) project. These individuals have detailed longitudinal cognitive data from which phenotypes measuring each individual's cognitive changes were constructed. One SNP - rs2075650, located in TOMM40 (translocase of the outer mitochondrial membrane 40 homolog) - had a genome-wide significant association with cognitive ageing (P=2.5 × 10 -8). This result was replicated in a meta-analysis of three independent Swedish cohorts (P=2.41 × 10 -6). An Apolipoprotein E (APOE) haplotype (adjacent to TOMM40), previously associated with cognitive ageing, had a significant effect on cognitive ageing in the CAGES sample (P=2.18 × 10 -8; females, P=1.66 × 10 -11; males, P=0.01). Fine SNP mapping of the TOMM40/APOE region identified both APOE (rs429358; P=3.66 × 10 -11) and TOMM40 (rs11556505; P=2.45 × 10 -8) as loci that were associated with cognitive ageing. Imputation and conditional analyses in the discovery and replication cohorts strongly suggest that this effect is due to APOE (rs429358). Functional genomic analysis indicated that SNPs in the TOMM40/APOE region have a functional, regulatory non-protein-coding effect. The APOE region is significantly associated with nonpathological cognitive ageing. The identity and mechanism of one or multiple causal variants remain unclear. © 2014 Macmillan Publishers Limited.

The Duchenne muscular dystrophy (DMD) locus has been localized to the short arm of the human X chromosome (Xp21) by detection of structural abnormalities and by genetic linkage studies. A library highly enriched for human DNA from Xp21 was constructed using DNA isolated from a male patient who had a visible deletion and three X-linked disorders (DMD, retinitis pigmentosa and chronic granulomatous disease). Seven cloned DNA probes from this library and the probe 754 (refs 5, 8) are used in the present study to screen for deletions in the DNA isolated from 57 unrelated males with DMD. Five of these DMD males are shown to exhibit deletions for one of the cloned DNA segments and at least 38 kb of surrounding DNA. In addition, two subclones from the same region detect four restriction fragment length polymorphisms which exhibit no obligate recombination with DMD in 34 meiotic events. These new DNA segments will complement the existing Xp21 probes for use in carrier detection and prenatal diagnosis of DMD. Elucidation of the end points of the five deletions will help delineate the extent of the DMD locus and ultimately lead to an understanding of the specific sequences involved in DMD.

Clinical trials for Alzheimer's disease are now focusing on the earliest stages of the disease with the goal of delaying dementia onset. There is great utility in using genetic variants to identify individuals at high age-dependent risk when the goal is to begin treatment before the development of any cognitive symptoms. Genetic variants identified through large-scale genome-wide association studies have not substantially improved the accuracy provided by APOE genotype to identify people at high risk of late-onset Alzheimer's disease (LOAD). We describe novel approaches, focused on molecular phylogenetics, to finding genetic variants that predict age at LOAD onset with sufficient accuracy and precision to be useful. We highlight the discovery of a polymorphism in TOMM40 that, in addition to APOE, may improve risk prediction and review how TOMM40 genetic variants may impact the develop of LOAD independently from APOE. The analysis methods described in this review may be useful for other genetically complex human diseases.

The first and most firmly established genetic risk factor for sporadic late onset Alzheimer’s disease (LOAD) is the e4 allele of the apolipoprotein E (APOE) gene [1]. Carrying the APOEe4 variant significantly increases the lifetime risk for LOAD, with the number of copies present indicative of level of risk [1,2] and is associated with lower age of clinical disease onset [1,3–6]. Furthermore, genome-wide association studies (GWAS) for sporadic LOAD confirmed that APOE is the major susceptibility genomic region for the disease and reported significant associations with markers within the APOE linkage disequilibrium (LD) locus (contains APOE, TOMM40 and APOC1 genes). The strongest association signal (by wide margin) in these studies was found at the APOE LD region and no other LOAD-association in the human genome remotely approached the same level of significance [7–10]. However, the molecular mechanism underlying the reported genetic LOAD-associations with APOE LD region in general and APOEe4 haplotype in particular has yet to be discovered.