Electroencephalogram findings in patients with posterior cortical atrophy.

AIM OF THE STUDY: The aim of this study is to evaluate standard scalp EEG findings in patients with posterior cortical atrophy (PCA), an atypical variant of Alzheimer's disease (AD). CLINICAL RATIONALE: PCA is a topographically selective variant of AD. Patients with typical AD have an increased likelihood of seizures, which may negatively impact overall functional performance and cognition. It is currently unknown what the typical EEG findings are for patients with PCA. MATERIALS AND METHODS: A retrospective chart review was performed on patients identified either with autopsy confirmed (n=13) or clinically (n=126) as PCA. RESULTS: 139 patients were included though only 23 (16.5%) had undergone EEG recording. The EEG was normal in 6 (26%), while an abnormal EEG was present in 17 (74%). Interictal epileptic discharges (IEDs) were found in 2 of the 23 patients (9%). CONCLUSIONS: This study of limited sample size suggests that there may be an increased predilection to find IEDs within PCA when compared to typical AD. Larger cohorts are required to determine frequency of abnormal EEGs in PCA, roles of AEDs in therapy, and in the selection of preferred AED. CLINICAL IMPLICATIONS: Patients with PCA would potentially benefit from an EEG for assessment of IEDs which may provide the clinician with a therapeutic opportunity.

Genome-wide association study of Alzheimer's disease.

In addition to apolipoprotein E (APOE), recent large genome-wide association studies (GWASs) have identified nine other genes/loci (CR1, BIN1, CLU, PICALM, MS4A4/MS4A6E, CD2AP, CD33, EPHA1 and ABCA7) for late-onset Alzheimer's disease (LOAD). However, the genetic effect attributable to known loci is about 50%, indicating that additional risk genes for LOAD remain to be identified. In this study, we have used a new GWAS data set from the University of Pittsburgh (1291 cases and 938 controls) to examine in detail the recently implicated nine new regions with Alzheimer's disease (AD) risk, and also performed a meta-analysis utilizing the top 1% GWAS single-nucleotide polymorphisms (SNPs) with P<0.01 along with four independent data sets (2727 cases and 3336 controls) for these SNPs in an effort to identify new AD loci. The new GWAS data were generated on the Illumina Omni1-Quad chip and imputed at ~2.5 million markers. As expected, several markers in the APOE regions showed genome-wide significant associations in the Pittsburg sample. While we observed nominal significant associations (P<0.05) either within or adjacent to five genes (PICALM, BIN1, ABCA7, MS4A4/MS4A6E and EPHA1), significant signals were observed 69-180 kb outside of the remaining four genes (CD33, CLU, CD2AP and CR1). Meta-analysis on the top 1% SNPs revealed a suggestive novel association in the PPP1R3B gene (top SNP rs3848140 with P = 3.05E-07). The association of this SNP with AD risk was consistent in all five samples with a meta-analysis odds ratio of 2.43. This is a potential candidate gene for AD as this is expressed in the brain and is involved in lipid metabolism. These findings need to be confirmed in additional samples.

Genome-wide association analysis of age-at-onset in Alzheimer's disease.

The risk of Alzheimer's disease (AD) is strongly determined by genetic factors and recent genome-wide association studies (GWAS) have identified several genes for the disease risk. In addition to the disease risk, age-at-onset (AAO) of AD has also strong genetic component with an estimated heritability of 42%. Identification of AAO genes may help to understand the biological mechanisms that regulate the onset of the disease. Here we report the first GWAS focused on identifying genes for the AAO of AD. We performed a genome-wide meta-analysis on three samples comprising a total of 2222 AD cases. A total of ~2.5 million directly genotyped or imputed single-nucleotide polymorphisms (SNPs) were analyzed in relation to AAO of AD. As expected, the most significant associations were observed in the apolipoprotein E (APOE) region on chromosome 19 where several SNPs surpassed the conservative genome-wide significant threshold (P<5E-08). The most significant SNP outside the APOE region was located in the DCHS2 gene on chromosome 4q31.3 (rs1466662; P=4.95E-07). There were 19 additional significant SNPs in this region at P<1E-04 and the DCHS2 gene is expressed in the cerebral cortex and thus is a potential candidate for affecting AAO in AD. These findings need to be confirmed in additional well-powered samples.

TMEM106B regulates progranulin levels and the penetrance of FTLD in GRN mutation carriers.

OBJECTIVES: To determine whether TMEM106B single nucleotide polymorphisms (SNPs) are associated with frontotemporal lobar degeneration (FTLD) in patients with and without mutations in progranulin (GRN) and to determine whether TMEM106B modulates GRN expression. METHODS: We performed a case-control study of 3 SNPs in TMEM106B in 482 patients with clinical and 80 patients with pathologic FTLD-TAR DNA-binding protein 43 without GRN mutations, 78 patients with FTLD with GRN mutations, and 822 controls. Association analysis of TMEM106B with GRN plasma levels was performed in 1,013 controls and TMEM106B and GRN mRNA expression levels were correlated in peripheral blood samples from 33 patients with FTLD and 150 controls. RESULTS: In our complete FTLD patient cohort, nominal significance was identified for 2 TMEM106B SNPs (top SNP rs1990622, p(allelic) = 0.036). However, the most significant association with risk of FTLD was observed in the subgroup of GRN mutation carriers compared to controls (corrected p(allelic) = 0.0009), where there was a highly significant decrease in the frequency of homozygote carriers of the minor alleles of all TMEM106B SNPs (top SNP rs1990622, CC genotype frequency 2.6% vs 19.1%, corrected p(recessive) = 0.009). We further identified a significant association of TMEM106B SNPs with plasma GRN levels in controls (top SNP rs1990622, corrected p = 0.002) and in peripheral blood samples a highly significant correlation was observed between TMEM106B and GRN mRNA expression in patients with FTLD (r = -0.63, p = 7.7 × 10(-5)) and controls (r = -0.49, p = 2.2 × 10(-10)). CONCLUSIONS: In our study, TMEM106B SNPs significantly reduced the disease penetrance in patients with GRN mutations, potentially by modulating GRN levels. These findings hold promise for the development of future protective therapies for FTLD.

Gene expression levels as endophenotypes in genome-wide association studies of Alzheimer disease.

BACKGROUND: Late-onset Alzheimer disease (LOAD) is a common disorder with a substantial genetic component. We postulate that many disease susceptibility variants act by altering gene expression levels. METHODS: We measured messenger RNA (mRNA) expression levels of 12 LOAD candidate genes in the cerebella of 200 subjects with LOAD. Using the genotypes from our LOAD genome-wide association study for the cis-single nucleotide polymorphisms (SNPs) (n = 619) of these 12 LOAD candidate genes, we tested for associations with expression levels as endophenotypes. The strongest expression cis-SNP was tested for AD association in 7 independent case-control series (2,280 AD and 2,396 controls). RESULTS: We identified 3 SNPs that associated significantly with IDE (insulin degrading enzyme) expression levels. A single copy of the minor allele for each significant SNP was associated with approximately twofold higher IDE expression levels. The most significant SNP, rs7910977, is 4.2 kb beyond the 3' end of IDE. The association observed with this SNP was significant even at the genome-wide level (p = 2.7 x 10(-8)). Furthermore, the minor allele of rs7910977 associated significantly (p = 0.0046) with reduced LOAD risk (OR = 0.81 with a 95% CI of 0.70-0.94), as expected biologically from its association with elevated IDE expression. CONCLUSIONS: These results provide strong evidence that IDE is a late-onset Alzheimer disease (LOAD) gene with variants that modify risk of LOAD by influencing IDE expression. They also suggest that the use of expression levels as endophenotypes in genome-wide association studies may provide a powerful approach for the identification of disease susceptibility alleles.

Postnatal downregulation of inhibitory neuromuscular transmission to the longitudinal muscle of the guinea pig ileum.

Neuromuscular transmission is crucial for normal gut motility but little is known about its postnatal maturation. This study investigated excitatory/inhibitory neuromuscular transmission in vitro using ileal nerve-muscle preparations made from neonatal (< or =48 h postnatal) and adult ( approximately 4 months postnatal) guinea pigs. In tissues from neonates and adults, nicotine (0.3-30 micromol L(-1)) contracted longitudinal muscle preparations in a tetrodotoxin (TTX) (0.3 micromol L(-1))-sensitive manner. The muscarinic receptor antagonist, scopolamine (1 micromol L(-1)), reduced substantially nicotine-induced contractions in neonatal tissues but not adult tissues. In the presence of N(omega)-nitro-l-arginine (NLA, 100 micromol L(-1)) to block nitric oxide (NO) mediated inhibitory neuromuscular transmission, scopolamine-resistant nicotine-induced contractions were revealed in neonatal tissues. NLA enhanced the nicotine-induced contractions in neonatal but not in adult tissues. Electrical field stimulation (20 V; 0.3 ms; 5-25 Hz, scopolamine 1 micromol L(-1) present) caused NLA and TTX-sensitive longitudinal muscle relaxations. Frequency-response curves in neonatal tissues were left-shifted compared with those obtained in adult tissues. Immunohistochemical studies revealed that NO synthase (NOS)-immunoreactivity (ir) was present in nerve fibres supplying the longitudinal muscle in neonatal and adult tissues. However, quantitative studies demonstrated that fluorescence intensity of NOS-ir nerve fibres was higher in neonatal than adult tissues. Nerve fibres containing substance P were abundant in longitudinal muscle in adult but not in neonatal tissues. Inhibitory neuromuscular transmission is relatively more effective in the neonatal guinea pig small intestine. Delayed maturation of excitatory motor pathways might contribute to paediatric motility disturbances.

Otoacoustic emissions and brainstem evoked potentials in compound carriers of connexin 26 mutations.

This study compares the effects of mutations in the gap junction protein connexin 26 (Cx26), on outer hair cells (OHCs), inner hair cells (IHCs) and auditory nerve/brainstem among carriers of these mutations. One hundred and twenty eight individuals, from a village with widespread consanguinity and congenital deafness, due to three Cx26 mutations, were selected among relatives of deaf persons, and divided into non-carriers, carriers of one mutation, homozygous to one mutation, or compound heterozygous carriers of two different mutations. Distortion product otoacoustic emissions (DPOAEs), auditory brainstem responses (ABRs) and audiometric evaluation were compared in these genetic groups. Hearing loss among homozygotes and compound heterozygotes was comparable and ranged from mild to profound. Most ABRs from these groups showed no responses or partial responses (peaks III, V) with prolonged latencies, but some individuals had all peaks at normal latencies. DPOAEs were absent, except sporadic responses. Carriers of one mutation had significantly smaller DPOAEs compared to non-carriers, although normal pure tone audiograms and ABRs were found in these groups. In conclusion, based on DPOAEs, Cx26 mutations may impact OHC function among carriers of one or two Cx26 mutations. IHC/nerve impairment among homozygotes and compound heterozygotes is variable. OHCs may be more susceptible to Cx26 mutations compared to IHCs and the auditory nerve and brainstem pathway activated by them.

The effects of a connexin 26 mutation--35delG--on oto-acoustic emissions and brainstem evoked potentials: homozygotes and carriers.

The purpose of this study was to examine whether outer hair cells (OHCs), inner hair cells and the brainstem auditory pathway are impaired due to a mutation in a gap junction protein, connexin 26 (Cx26), 35delG. Fifty-six individuals, from a village with widespread consanguinity and profound, non-syndromic congenital deafness, due to 35delG mutation, were selected among relatives of deaf people. The individuals were either non-carriers (n=20), heterozygous (n=20) or homozygous (n=16) for the mutation. Distortion product oto-acoustic emissions (DPOAEs) and auditory brainstem evoked potentials (ABEPs) in mutation non-carriers, in heterozygotes (carriers) and in subjects homozygous for the mutation were compared in addition to audiometric evaluation. Most deaf homozygotes had no DPOAEs, except some sporadic responses at 1000, 8000 and 10000 Hz. This was also observed in audiometry which showed profound hearing loss in most cases. Two cases were unique: one had moderate to severe hearing loss and the other had severe to profound hearing loss. A significant difference was found between non-carriers and carriers of 35delG: non-carriers had larger DPOAE responses than heterozygotes at all frequencies. The prevalence of responses got lower with higher frequencies in both groups, but between 6000 and 10000 Hz 50-70% of the carriers had no DPOAE responses, compared to 30-60% of non-carriers. In both groups responses diminished with age, but no significant interaction was found between age and the genetic group. ABEPs among homozygotes were variable: in most homozygotes ABEPs were absent or partial (waves III, V) with prolonged latencies, but two subjects had ABEPs within normal limits, in one ear. ABEPs were normal with no differences between carriers and non-carriers. We suggest that OHC function is affected by the 35delG mutation in Cx26. In addition, the hearing of carriers of this mutation may be impaired at very high frequencies (8000-10000 Hz), which are not assessed in routine audiometry or ABEP testing.

High-throughput variation detection and genotyping using microarrays.

The genetic dissection of complex traits may ultimately require a large number of SNPs to be genotyped in multiple individuals who exhibit phenotypic variation in a trait of interest. Microarray technology can enable rapid genotyping of variation specific to study samples. To facilitate their use, we have developed an automated statistical method (ABACUS) to analyze microarray hybridization data and applied this method to Affymetrix Variation Detection Arrays (VDAs). ABACUS provides a quality score to individual genotypes, allowing investigators to focus their attention on sites that give accurate information. We have applied ABACUS to an experiment encompassing 32 autosomal and eight X-linked genomic regions, each consisting of approximately 50 kb of unique sequence spanning a 100-kb region, in 40 humans. At sufficiently high-quality scores, we are able to read approximately 80% of all sites. To assess the accuracy of SNP detection, 108 of 108 SNPs have been experimentally confirmed; an additional 371 SNPs have been confirmed electronically. To access the accuracy of diploid genotypes at segregating autosomal sites, we confirmed 1515 of 1515 homozygous calls, and 420 of 423 (99.29%) heterozygotes. In replicate experiments, consisting of independent amplification of identical samples followed by hybridization to distinct microarrays of the same design, genotyping is highly repeatable. In an autosomal replicate experiment, 813,295 of 813,295 genotypes are called identically (including 351 heterozygotes); at an X-linked locus in males (haploid), 841,236 of 841,236 sites are called identically.

Assignment of a novel bifurcated SET domain gene, SETDB1, to human chromosome band 1q21 by in situ hybridization and radiation hybrids.

We have identified a human gene encoding an unusual bifurcated SET domain protein containing a large "insertion" between the most highly conserved parts of the SET domain. The existence of an evolutionarily related C. elegans gene encoding a similarly bifurcated SET domain suggests that SET domains may generally be composed of two functionally distinct subdomains. We mapped this gene, called SETDB1, to human chromosome 1q21. This region is targeted by a large number of recurrent translocations, suggesting that like the SET domain protein MLL, mutant forms of SETDB1 may be associated with human neoplasias.

Allele frequency distributions in pooled DNA samples: applications to mapping complex disease genes.

Genetic studies of complex hereditary disorders require for their mapping the determination of genotypes at several hundred polymorphic loci in several hundred families. Because only a minority of markers are expected to show linkage and association in family data, a simple screen of genetic markers to identify those showing linkage in pooled DNA samples can greatly facilitate gene identification. All studies involving pooled DNA samples require the comparison of allele frequencies in appropriate family samples and subsamples. We have tested the accuracy of allele frequency estimates, in various DNA samples, by pooling DNA from multiple individuals prior to PCR amplification. We have used the ABI 377 automated DNA sequencer and GENESCAN software for quantifying total amplification using a 5' fluorescently labeled forward PCR primer and relative peak heights to estimate allele frequencies in pooled DNA samples. In these studies, we have genotyped 11 microsatellite markers in two separate DNA pools, and an additional four markers in a third DNA pool, and compared the estimated allele frequencies with those determined by direct genotyping. In addition, we have evaluated whether pooled DNA samples can be used to accurately assess allele frequencies on transmitted and untransmitted chromosomes, in a collection of families for fine-structure gene mapping using allelic association. Our studies show that accurate, quantitative data on allele frequencies, suitable for identifying markers for complex disorders, can be identified from pooled DNA samples. This approach, being independent of the number of samples comprising a pool, promises to drastically reduce the labor and cost of genotyping in the initial identification of disease loci. Additional applications of DNA pooling are discussed. These developments suggest that new statistical methods for analyzing pooled DNA data are required.

Two different connexin 26 mutations in an inbred kindred segregating non-syndromic recessive deafness: implications for genetic studies in isolated populations.

Non-syndromic recessive deafness (NSRD) is the most common form of prelingual hereditary hearing loss. To date, 10 autosomal NSRD loci (DFNBs) have been identified by genetic mapping; at least three times as many additional loci are expected to be identified. We have performed linkage analyses in two inter-related inbred kindreds, comprised of >50 affecteds, from a single Israeli-Arab village segregating NSRD. Genetic mapping by two-point and multi-point linkage analysis in 10 candidate regions identified the segregating gene to be on human chromosome 13q11 (DFNB1). Haplotype analysis, using eight microsatellite markers spanning 15 cM in 13q11, suggested the segregation of two different mutations in this kindred: affected individuals were homozygotes for either haplotype or compound heterozygotes. The gene for the connexin 26 gap junction protein, recently shown to be mutant in both dominant and recessive deafness, maps to this locus. We identified two distinct mutations, W77R and Gdel35, both of which likely inactivate connexin 26. The Gdel35 change likely occurs at a mutational hotspot within the connexin 26 gene. The recombination of marker alleles at the polymorphisms studied in 13q11, at known map distances from the mutations, allowed us to estimate the age of the mutations to be 3-5 generations (75-125 years). This study independently confirms the identity of connexin 26 as an NSRD gene. Importantly, we demonstrate that in small populations with high rates of consanguinity, as compared with large outbred populations, recessive mutations may have very recent origin and show allelic diversity.

Genetic studies of the mouse mutations mahogany and mahoganoid.

The mouse mutations mahogany (mg) and mahoganoid (md) are negative modifiers of the Agouti coat color gene, which encodes a paracrine signaling molecule that induces a swithc in melanin synthesis from eumelanin to pheomelanin. Animals mutant for md or mg synthesize very little or no pheomelanin depending on Agouti gene background. The Agouti protein is normally expressed in the skin and acts as an antagonist of the melanocyte receptor for alpha-MSH (Mc1r); however, ectopic expression of Agouti causes obesity, possibly by antagonizing melanocortin receptors expressed in the brain. To investigate where md and mg lie in a genetic pathway with regard to Agouti and Mc1r signaling, we determined the effects of these mutations in animals that carried either a loss-of-function Mc1r mutation (recessive yellow, Mc1re) or a gain-of-function Agouti mutation (lethal yellow, Ay). We found that the Mc1re mutation suppressed the effects of md and mg, but that md and mg suppressed the effects of Ay on both coat color and obesity. Plasma levels of alpha-MSH and of ACTH were unaffected by md or mg. These results suggest that md and mg interfere directly with Agouti signaling, possibly at the level of protein production or receptor regulation.