Exome Sequencing of Extended Families with Alzheimer's Disease Identifies Novel Genes Implicated in Cell Immunity and Neuronal Function.

OBJECTIVE: Alzheimer's disease (AD) is a neurodegenerative disorder for which more than 20 genetic loci have been implicated to date. However, studies demonstrate not all genetic factors have been identified. Therefore, in this study we seek to identify additional rare variants and novel genes potentially contributing to AD. METHODS: Whole exome sequencing was performed on 23 multi-generational families with an average of eight affected subjects. Exome sequencing was filtered for rare, nonsynonymous and loss-of-function variants. Alterations predicted to have a functional consequence and located within either a previously reported AD gene, a linkage peak (LOD>2), or clustering in the same gene across multiple families, were prioritized. RESULTS: Rare variants were found in known AD risk genes including AKAP9, CD33, CR1, EPHA1, INPP5D, NME8, PSEN1, SORL1, TREM2 and UNC5C. Three families had five variants of interest in linkage regions with LOD>2. Genes with segregating alterations in these peaks include CD163L1 and CLECL1, two genes that have both been implicated in immunity, CTNNA1, which encodes a catenin in the cerebral cortex and MIEF1, a gene that may induce mitochondrial dysfunction and has the potential to damage neurons. Four genes were identified with alterations in more than one family include PLEKHG5, a gene that causes Charcot-Marie-Tooth disease and THBS2, which promotes synaptogenesis. CONCLUSION: Utilizing large families with a heavy burden of disease allowed for the identification of rare variants co-segregating with disease. Variants were identified in both known AD risk genes and in novel genes.

Targeted sequencing of ABCA7 identifies splicing, stop-gain and intronic risk variants for Alzheimer disease.

Several variants in the gene ABCA7 have been identified as potential causal variants for late-onset Alzheimer's disease (LOAD). In order to replicate these findings, and search for novel causal variants, we performed targeted sequencing of this gene in cohorts of non-Hispanic White (NHW) and African-American (AA) LOAD cases and controls. We sequenced the gene ABCA7 in 291 NHW LOAD cases and 103 controls. Variants were prioritized for rare, damaging variants and previously reported variants associated with LOAD, and were follow-up genotyped in 4076 NHW and 1157 AA cases and controls. We confirm three previously associated ABCA7 risk variants and extend two of these associations to other populations, an intronic variant in NHW (P=3.0×10-3) (originally reported in a Belgian population), and a splice variant originally associated in the Icelandic population, which was significantly associated in the NHW cohort (P=1.2×10-6) and nominally associated in the AA cohort (P=0.017). We also identify a 3'-UTR splice variant that segregates in four siblings of one family and is nominally associated with LOAD (P=0.040). Multiple variants in ABCA7 contribute to LOAD risk.

GWAS analysis implicates NF-κB-mediated induction of inflammatory T cells in multiple sclerosis.

To identify genes and biologically relevant pathways associated with risk to develop multiple sclerosis (MS), the Genome-Wide Association Studies noise reduction method (GWAS-NR) was applied to MS genotyping data. Regions of association were defined based on the significance of linkage disequilibrium blocks. Candidate genes were cross-referenced based on a review of current literature, with attention to molecular function and directly interacting proteins. Supplementary annotations and pathway enrichment scores were generated using The Database for Annotation, Visualization and Integrated Discovery. The candidate set of 220 MS susceptibility genes prioritized by GWAS-NR was highly enriched with genes involved in biological pathways related to positive regulation of cell, lymphocyte and leukocyte activation (P=6.1E-15, 1.2E-14 and 5.0E-14, respectively). Novel gene candidates include key regulators of NF-κB signaling and CD4+ T helper type 1 (Th1) and T helper type 17 (Th17) lineages. A large subset of MS candidate genes prioritized by GWAS-NR were found to interact in a tractable pathway regulating the NF-κB-mediated induction and infiltration of pro-inflammatory Th1/Th17 T-cell lineages, and maintenance of immune tolerance by T-regulatory cells. This mechanism provides a biological context that potentially links clinical observations in MS to the underlying genetic landscape that may confer susceptibility.

Longitudinal follow-up of late-onset Alzheimer disease families.

Historically, data for genetic studies are collected at one time point. However, for diseases with late onset or with complex phenotypes, such as Alzheimer disease (AD), restricting diagnosis to a single ascertainment contact may not be sufficient. Affection status may change over time and some initial diagnoses may be inconclusive. Follow-up provides the opportunity to resolve these complications. However, to date, previous studies have not formally demonstrated that longitudinally re-contacting families is practical or productive. To update data initially collected for linkage analysis of late-onset Alzheimer disease (LOAD), we successfully re-contacted 63 of 81 (78%) multiplex families (two to 17 years after ascertainment). Clinical status changed for 73 of the 230 (32%) non-affected participants. Additionally, expanded family history identified 20 additional affected individuals to supplement the data set. Furthermore, fostering ongoing relationships with participating families helped recruit 101 affected participants into an autopsy and tissue donation program. Despite similar presentations, discordance between clinical diagnosis and neuropathologic diagnosis was observed in 28% of those with tissue diagnoses. Most of the families were successfully re-contacted, and significant refinement and supplementation of the data was achieved. We concluded that serial contact with longitudinal evaluation of families has significant implications for genetic analyses.

Polymorphisms of the tumor suppressor gene LSAMP are associated with left main coronary artery disease.

Previous association mapping on chromosome 3q13-21 detected evidence for association at the limbic system-associated membrane protein (LSAMP) gene in individuals with late-onset coronary artery disease (CAD). LSAMP has never been implicated in the pathogenesis of CAD. We sought to thoroughly characterize the association and the gene. Non-redundant single nucleotide polymorphisms (SNPs) across the gene were examined in an initial dataset (168 cases with late-onset CAD, 149 controls). Stratification analysis on left main CAD (N = 102) revealed stronger association, which was further validated in a validation dataset (141 cases with left main CAD, 215 controls), a third control dataset (N = 255), and a family-based dataset (N = 2954). A haplotype residing in a novel alternative transcript of the LSAMP gene was significant in all independent case-control datasets (p = 0.0001 to 0.0205) and highly significant in the joint analysis (p = 0.00004). Lower expression of the novel alternative transcript was associated with the risk haplotype (p = 0.0002) and atherosclerosis burden in human aortas (p = 0.0001). Furthermore, silencing LSAMP expression in human aortic smooth muscle cells (SMCs) substantially augmented SMC proliferation (p<0.01). Therefore, the risk conferred by the LSAMP haplotype appears to be mediated by LSAMP down-regulation, which may promote SMC proliferation in the arterial wall and progression of atherosclerosis.

Gene-gene interaction between FGF20 and MAOB in Parkinson disease.

The fibroblast growth factor 20 (FGF20) and monoamine oxidase B (MAOB) genes are associated with Parkinson Disease (PD) risk and both are in the dopamine bio-pathway. Therefore, we investigated the joint effect between polymorphisms in the FGF20 and MAOB genes for evidence of interaction contributing to PD risk. Fourteen polymorphisms (eight for FGF20, six for MAOB) were genotyped in 736 families and analyzed using conditional logistic regression (CLR). Significant two-locus interactions were found in females between the polymorphisms rs1721100 of FGF20 and rs1799836 of MAOB, and between the polymorphisms rs1721082 of FGF20 and rs1799836 of MAOB. The risk alleles for each SNP identified from CLR, rs1721100 C, rs1721082 T and rs1799836 A, are consistent with previous reports. Using indicator variables for the SNP genotypes, rs1721100 GC with rs1799836 AA showed significant interaction (P = 0.021), compared with the reference group rs1721100 GG with rs1799836 GG. Using an allele-dose model for the risk alleles, rs1721100 and rs1799836 showed significant interaction (P = 0.019). We found similar interaction results between rs1721082 and rs1799836. In conclusion, variants in FGF20 and MAOB show evidence of statistical interactions, which emphasizes the importance of considering them jointly in genetic analysis of PD and illustrates potential patterns of biological interaction contributing to PD risk.

Investigation of the PARK10 gene in Parkinson disease.

Two recent association mapping studies in Parkinson disease (PD) reported three candidate genes for the PARK10 locus: EIF2B3 as a modifier of age-at-onset of PD (min P= 0.0004) and HIVEP3 as a PD risk gene (P < or = 0.006) (Oliveira et al. 2005); and LOC200008 (CDCP2) identified by the whole genome association (WGA) study of PD of Maraganore et al. (2005). However, evaluation of the on-line PD WGA results revealed two significant SNPs in HIVEP3 in the two datasets, Tier 1 and Tier 2, used by Maraganore et al. (P < or = 0.008 for Tier 1 and P=0.03 for Tier 2 dataset). Here, we revisited both the HIVEP3 and CDCP2 loci by examining 47 SNPs, mostly tagging, in an expanded PD family dataset (293 multiplex and 467 singleton families). A discordant sibpair (DSP) dataset (one DSP per family), with similar data structure as the WGA Tier 1 dataset, was also tested. We confirmed our and other previous negative findings for CDCP2. However, five significant SNPs in HIVEP3 (min P=0.004) were observed, although the two significant HIVEP3 SNPs from the PD WGA study were not significant in our datasets. Even though the sets of significant HIVEP3 markers differ between studies, these findings strongly support HIVEP3 as a candidate for PARK10. Further testing of HIVEP3 by other groups is encouraged.

Serum lipids in the GENECARD study of coronary artery disease identify quantitative trait loci and phenotypic subsets on chromosomes 3q and 5q.

Coronary artery disease (CAD) and dyslipidemia have strong genetic components. Heterogeneity complicates evaluating genetics of complex diseases such as CAD; incorporating disease-related phenotypes may help reduce heterogeneity. We hypothesized that incorporating lipoproteins in a study of CAD would increase the power to map genes, narrow linkage peaks, identify phenotypic subsets, and elucidate the contribution of established risk factors to genetic results. We performed ordered subset analysis (OSA) and quantitative trait linkage (QTL) using serum lipoproteins and microsatellite markers in 346 families with early-onset CAD. OSA defined homogeneous subsets and calculated lod scores across a chromosome after ranking families by mean lipoprotein values. QTL used variance components analysis. We found significantly increased linkage to chromosome 3q13 (LOD 5.10, p = 0.008) in families with higher HDL cholesterol, lower LDL and total cholesterol, lower triglycerides, and fewer CAD risk factors, possibly due to a concentrated non-lipoprotein-related genetic effect. OSA identified linkage on chromosome 5q34 in families with higher cholesterol, possibly representing a hereditary lipoprotein phenotype. Multiple QTLs were identified, with the strongest for: total cholesterol on chromosome 5q14 (LOD 4.3); LDL on 20p12 (LOD 3.97); HDL on 3p14 (LOD 1.65); triglycerides on 18q22 (LOD 1.43); and HDL/TC ratio on 3q27-28 (LOD 2.06). Our findings suggest the presence of etiologic heterogeneity in families with early-onset CAD, potentially due to differential effects of lipoprotein phenotypes. Candidate genes are under investigation.

Family-based case-control study of cigarette smoking and Parkinson disease.

OBJECTIVE: To determine whether people with Parkinson disease (PD) are less likely to report a history of cigarette smoking than their unaffected siblings. BACKGROUND: Previous studies reported that individuals with PD are half as likely to have smoked as those unaffected by PD. Other studies reported that smoking modified the risk of PD due to polymorphisms in the MAO-B and nNOS genes. Thus, genetic studies of PD should consider confounding or interaction with smoking history as well. The authors have collected detailed smoking histories on a family-based case-control sample ascertained for genetic studies of PD. METHODS: In a matched case-control study of 140 sibships, individuals with PD (n = 143) were compared to sibling controls (n = 168). Cigarette smoking history was collected by a structured telephone interview. Conditional logistic regression was used to examine the relationship between smoking and PD while controlling for confounding by age and sex. RESULTS: Ever smoking, current smoking, and increasing duration (in years), dose (in packs/day), and intensity (in pack-years) of smoking were significantly inversely associated with PD (p < 0.05). The association was not modified by sex, age at onset, or recency of exposure. CONCLUSIONS: Consistent with previous studies, individuals with Parkinson disease are significantly less likely to have smoked regularly than their unaffected siblings. This association was detected even though discordant sibling pairs are more likely to be overmatched for environmental exposures than unmatched case and control groups.

Apolipoprotein E controls the risk and age at onset of Parkinson disease.

BACKGROUND: Similarities between Alzheimer disease (AD) and Parkinson disease (PD) suggest a possible role for apolipoprotein E (APOE) in PD. Most previous studies seeking to establish such a link used case-control datasets and results have been inconsistent. OBJECTIVE: To investigate APOE's role in PD using family-based association analyses. METHODS: APOE functional polymorphisms were genotyped for 658 PD affected families, including 282 multiplex and 376 singleton families. The pedigree disequilibrium test (PDT) and the genotype-PDT were used to test the risk effect of APOE. The Monks-Kaplan test was used to evaluate the effect of APOE on age at onset of PD. RESULTS: APOE was significantly associated with risk of developing PD. Stratified analysis revealed that APOE was most strongly associated with families with a positive PD family history (global p = 0.003). Like AD, the APOE-4 allele increases disease risk while the APOE-3 allele decreases risk. We detected a positive association of APOE-3 (p = 0.019) and a negative association of APOE-4 (p = 0.015) with age at onset in PD. CONCLUSIONS: The APOE-4 allele increases risk and decreases age at onset of PD, an association that may not be dependent upon cognitive impairment.

No association between the APOE gene and autism.

Autism is a neurodevelopmental disorder characterized by stereotypic and repetitive behavior and interests, together with social and communicative deficiencies. The results of several genomic screens suggest the presence of an autism susceptibility locus on chromosome 19p13.2-q13.4. The apolipoprotein E (APOE) gene on chromosome 19 encodes for a protein, apoE, whose different isoforms (E2, E3, E4) influence neuronal growth. APOE participates in lipid transport and metabolism, repair, growth, and maintenance of axons and myelin during neuronal development. The APOE protein competes with the Reelin protein for VLDL/APOER2 receptor binding. Several studies have reported evidence for an association between autism and the Reelin gene. Based on these data we tested for association between APOE and autism using family-based association methods in a data set of 322 autism families. Three promoter, one intronic, and one 3' UTR single nucleotide polymorphisms (SNPs) in the APOE gene (-491a/t, -427c/t, -219g/t, 113c/g, and 5361c/t) as well as the APOE functional polymorphism (E2, E3, E4) were examined and failed to reveal significant evidence that autism is associated with APOE.

Genetic polymorphisms of the N-acetyltransferase genes and risk of Parkinson's disease.

Recently, the authors demonstrated linkage in idiopathic PD to a region on chromosome 8p that contains the N-acetyltransferase genes, NAT1 and NAT2. The authors examined NAT1 and NAT2 for association with PD using family-based association methods and single nucleotide polymorphisms (SNPs). The authors did not find evidence for association with increased risk for PD between any individual NAT1 or NAT2 SNP or acetylation haplotype (N = 397 families, 1,580 individuals).

Complete genomic screen in Parkinson disease: evidence for multiple genes.

CONTEXT: The relative contribution of genes vs environment in idiopathic Parkinson disease (PD) is controversial. Although genetic studies have identified 2 genes in which mutations cause rare single-gene variants of PD and observational studies have suggested a genetic component, twin studies have suggested that little genetic contribution exists in the common forms of PD. OBJECTIVE: To identify genetic risk factors for idiopathic PD. DESIGN, SETTING, AND PARTICIPANTS: Genetic linkage study conducted 1995-2000 in which a complete genomic screen (n = 344 markers) was performed in 174 families with multiple individuals diagnosed as having idiopathic PD, identified through probands in 13 clinic populations in the continental United States and Australia. A total of 870 family members were studied: 378 diagnosed as having PD, 379 unaffected by PD, and 113 with unclear status. MAIN OUTCOME MEASURES: Logarithm of odds (lod) scores generated from parametric and nonparametric genetic linkage analysis. RESULTS: Two-point parametric maximum parametric lod score (MLOD) and multipoint nonparametric lod score (LOD) linkage analysis detected significant evidence for linkage to 5 distinct chromosomal regions: chromosome 6 in the parkin gene (MLOD = 5.07; LOD = 5.47) in families with at least 1 individual with PD onset at younger than 40 years, chromosomes 17q (MLOD = 2.28; LOD = 2.62), 8p (MLOD = 2.01; LOD = 2.22), and 5q (MLOD = 2.39; LOD = 1.50) overall and in families with late-onset PD, and chromosome 9q (MLOD = 1.52; LOD = 2.59) in families with both levodopa-responsive and levodopa-nonresponsive patients. CONCLUSIONS: Our data suggest that the parkin gene is important in early-onset PD and that multiple genetic factors may be important in the development of idiopathic late-onset PD.

Association of single-nucleotide polymorphisms of the tau gene with late-onset Parkinson disease.

CONTEXT: The human tau gene, which promotes assembly of neuronal microtubules, has been associated with several rare neurologic diseases that clinically include parkinsonian features. We recently observed linkage in idiopathic Parkinson disease (PD) to a region on chromosome 17q21 that contains the tau gene. These factors make tau a good candidate for investigation as a susceptibility gene for idiopathic PD, the most common form of the disease. OBJECTIVE: To investigate whether the tau gene is involved in idiopathic PD. DESIGN, SETTING, AND PARTICIPANTS: Among a sample of 1056 individuals from 235 families selected from 13 clinical centers in the United States and Australia and from a family ascertainment core center, we tested 5 single-nucleotide polymorphisms (SNPs) within the tau gene for association with PD, using family-based tests of association. Both affected (n = 426) and unaffected (n = 579) family members were included; 51 individuals had unclear PD status. Analyses were conducted to test individual SNPs and SNP haplotypes within the tau gene. MAIN OUTCOME MEASURE: Family-based tests of association, calculated using asymptotic distributions. RESULTS: Analysis of association between the SNPs and PD yielded significant evidence of association for 3 of the 5 SNPs tested: SNP 3, P =.03; SNP 9i, P =.04; and SNP 11, P =.04. The 2 other SNPs did not show evidence of significant association (SNP 9ii, P =.11, and SNP 9iii, P =.87). Strong evidence of association was found with haplotype analysis, with a positive association with one haplotype (P =.009) and a negative association with another haplotype (P =.007). Substantial linkage disequilibrium (P<.001) was detected between 4 of the 5 SNPs (SNPs 3, 9i, 9ii, and 11). CONCLUSIONS: This integrated approach of genetic linkage and positional association analyses implicates tau as a susceptibility gene for idiopathic PD.

Fine mapping and genetic heterogeneity in the pure form of autosomal dominant familial spastic paraplegia.

We evaluated seven families segregating pure, autosomal dominant familial spastic paraplegia (SPG) for linkage to four recently identified SPG loci on chromosomes 2q (1), 8q (2), 12q (3), and 19q (4). These families were previously shown to be unlinked to SPG loci on chromosomes 2p, 14q, and 15q. Two families demonstrated linkage to the new loci. One family (family 3) showed significant evidence for linkage to chromosome 12q, peaking at D12S1691 (maximum lod = 3.22). Haplotype analysis of family 3 did not identify any recombinants among affected individuals in the 12q candidate region. Family 5 yielded a peak lod score of 2.02 at marker D19S868 and excluded linkage to other known SPG loci. Haplotype analysis of family 5 revealed several cross-overs in affected individuals, thereby potentially narrowing the SPG12 candidate region to a 5-cM region between markers D19S868 and D19S220. Three of the families definitively excluded all four loci examined, providing evidence for further genetic heterogeneity of pure, autosomal dominant SPG. In conclusion, these data confirm the presence of SPG10 (chromosome 12), potentially reduce the minimum candidate region for SPG12 (chromosome 19q), and suggest there is at least one additional autosomal dominant SPG locus.

Identification and expression analysis of spastin gene mutations in hereditary spastic paraplegia.

Pure hereditary spastic paraplegia (SPG) type 4 is the most common form of autosomal dominant hereditary SPG, a neurodegenerative disease characterized primarily by hyperreflexia and progressive spasticity of the lower limbs. It is caused by mutations in the gene encoding spastin, a member of the AAA family of ATPases. We have screened the spastin gene for mutations in 15 families consistent with linkage to the spastin gene locus, SPG4, and have identified 11 mutations, 10 of which are novel. Five of the mutations identified are in noninvariant splice-junction sequences. Reverse transcription-PCR analysis of mRNA from patients shows that each of these five mutations results in aberrant splicing. One mutation was found to be "leaky," or partially penetrant; that is, the mutant allele produced both mutant (skipped exon) and wild-type (full-length) transcripts. This phenomenon was reproduced in in vitro splicing experiments, with a minigene splicing-vector construct only in the context of the endogenous splice junctions flanking the splice junctions of the skipped exon. In the absence of endogenous splice junctions, only mutant transcript was detected. The existence of at least one leaky mutation suggests that relatively small differences in the level of wild-type spastin expression can have significant functional consequences. This may account, at least in part, for the wide ranges in age at onset, symptom severity, and rate of symptom progression that have been reported to occur both among and within families with SPG linked to SPG4. In addition, these results suggest caution in the interpretation of data solely obtained with minigene constructs to study the effects of sequence variation on splicing. The lack of full genomic sequence context in these constructs can mask important functional consequences of the mutation.

A duplication in chromosome 4q35 is associated with hereditary benign intraepithelial dyskeratosis.

Hereditary benign intraepithelial dyskeratosis (HBID) is an autosomal dominant disorder characterized by elevated epithelial plaques on the ocular and oral mucous membranes. It has been reported primarily, but not exclusively, in individuals of American Indian heritage in North Carolina. We have examined and obtained DNA on two large families affected by HBID. Using genetic linkage analysis we have localized the HBID gene to chromosome 4 (4q35) with a peak LOD score of 8.97. Molecular analysis of these data reveals that all individuals affected with HBID in both families demonstrate the presence of three alleles for two tightly linked markers, D4S1652 and D4S2390, which map to the telomeric region of 4q35. This suggests the presence of a duplication segregating with the disease phenotype that is most likely involved in its causation.

Isolation of genomic DNA from mammalian cells.

This unit describes simple, cost-effective preparation of DNA from whole blood or cultured cells that yields high-molecular-weight DNA suitable for both Southern blotting and the polymerase chain reaction. Preparation time may be shortened by substituting a high-salt precipitation procedure for the dialysis step; however, this results in a smaller average fragment size. The isolation of DNA from buccal swabs, collected from the inside of the cheek, is also described. The DNA is suitable for PCR analysis. Preparation of buffered phenol for DNA extraction is described in a support protocol. This unit describes simple, cost-effective preparation of DNA from whole blood or cultured cells that yields high-molecular-we.