Pilot GWAS of caries in African-Americans shows genetic heterogeneity.

BACKGROUND: Dental caries is the most common chronic disease in the US and disproportionately affects racial/ethnic minorities. Caries is heritable, and though genetic heterogeneity exists between ancestries for a substantial portion of loci associated with complex disease, a genome-wide association study (GWAS) of caries specifically in African Americans has not been performed previously. METHODS: We performed exploratory GWAS of dental caries in 109 African American adults (age > 18) and 96 children (age 3-12) from the Center for Oral Health Research in Appalachia (COHRA1 cohort). Caries phenotypes (DMFS, DMFT, dft, and dfs indices) assessed by dental exams were tested for association with 5 million genotyped or imputed single nucleotide polymorphisms (SNPs), separately in the two age groups. The GWAS was performed using linear regression with adjustment for age, sex, and two principal components of ancestry. A maximum of 1 million adaptive permutations were run to determine empirical significance. RESULTS: No loci met the threshold for genome-wide significance, though some of the strongest signals were near genes previously implicated in caries such as antimicrobial peptide DEFB1 (rs2515501; p = 4.54 × 10- 6) and TUFT1 (rs11805632; p = 5.15 × 10- 6). Effect estimates of lead SNPs at suggestive loci were compared between African Americans and Caucasians (adults N = 918; children N = 983). Significant (p < 5 × 10- 8) genetic heterogeneity for caries risk was found between racial groups for 50% of the suggestive loci in children, and 12-18% of the suggestive loci in adults. CONCLUSIONS: The genetic heterogeneity results suggest that there may be differences in the contributions of genetic variants to caries across racial groups, and highlight the critical need for the inclusion of minorities in subsequent and larger genetic studies of caries in order to meet the goals of precision medicine and to reduce oral health disparities.

Genetic Association of MMP10, MMP14, and MMP16 with Dental Caries.

Matrix metalloproteinases (MMPs), which degrade extracellular proteins as part of a variety of physiological processes, and their inhibitors have been implicated in the dental caries process. Here we investigated 28 genetic variants spanning the MMP10, MMP14, and MMP16 genes to detect association with dental caries experience in 13 age- and race-stratified (n = 3,587) samples from 6 parent studies. Analyses were performed separately for each sample, and results were combined across samples by meta-analysis. Two SNPs (rs2046315 and rs10429371) upstream of MMP16 were significantly associated with caries in an individual sample of white adults and via meta-analysis across 8 adult samples after gene-wise adjustment for multiple comparisons. Noteworthy is SNP rs2046315 (p = 8.14 × 10-8) association with caries in white adults. This SNP was originally nominated in a genome-wide-association study (GWAS) of dental caries in a sample of white adults and yielded associations in a subsequent GWAS of surface level caries in white adults as well. Therefore, in our study, we were able to recapture the association between rs2046315 and dental caries in white adults. Although we did not strengthen evidence that MMPs 10, 14, and 16 influence caries risk, MMP16 is still a likely candidate gene to pursue.

Genome-wide copy-number variation study of psychosis in Alzheimer's disease.

About 40-60% of patients with late-onset Alzheimer's disease (AD) develop psychosis, which represents a distinct phenotype of more severe cognitive and functional deficits. The estimated heritability of AD+P is ~61%, which makes it a good target for genetic mapping. We performed a genome-wide copy-number variation (CNV) study on 496 AD cases with psychosis (AD+P), 639 AD subjects with intermediate psychosis (AD intermediate P) and 156 AD subjects without psychosis (AD-P) who were recruited at the University of Pittsburgh Alzheimer's Disease Research Center using over 1 million single-nucleotide polymorphisms (SNPs) and CNV markers. CNV load analysis found no significant difference in total and average CNV length and CNV number in the AD+P or AD intermediate P groups compared with the AD-P group. Our analysis revealed a marginally significant lower number of duplication events in AD+P cases compared with AD-P controls (P=0.059) using multivariable regression model. The most interesting finding was the presence of a genome-wide significant duplication in the APC2 gene on chromosome 19, which was protective against developing AD+P (odds ratio=0.42; P=7.2E-10). We also observed suggestive associations of duplications with AD+P in the SET (P=1.95E-06), JAG2 (P=5.01E-07) and ZFPM1 (P=2.13E-07) genes and marginal association of a deletion in CNTLN (P=8.87E-04). We have identified potential novel loci for psychosis in Alzheimer's disease that warrant follow-up in large-scale independent studies.

Genetic Association of MPPED2 and ACTN2 with Dental Caries.

The first genome-wide association study of dental caries focused on primary teeth in children aged 3 to 12 yr and nominated several novel genes: ACTN2, EDARADD, EPHA7, LPO, MPPED2, MTR, and ZMPSTE24. Here we interrogated 156 single-nucleotide polymorphisms (SNPs) within these candidate genes for evidence of association with dental caries experience in 13 race- and age-stratified samples from 6 independent studies (n = 3600). Analysis was performed separately for each sample, and results were combined across samples via meta-analysis. MPPED2 was significantly associated with caries via meta-analysis across the 5 childhood samples, with 4 SNPs showing significant associations after gene-wise adjustment for multiple comparisons (p < .0026). These results corroborate the previous genome-wide association study, although the functional role of MPPED2 in caries etiology remains unknown. ACTN2 also showed significant association via meta-analysis across childhood samples (p = .0014). Moreover, in adults, genetic association was observed for ACTN2 SNPs in individual samples (p < .0025), but no single SNP was significant via meta-analysis across all 8 adult samples. Given its compelling biological role in organizing ameloblasts during amelogenesis, this study strengthens the hypothesis that ACTN2 influences caries risk. Results for the other candidate genes neither proved nor precluded their associations with dental caries.

Genome-wide association study of primary dentition pit-and-fissure and smooth surface caries.

Dental caries continues to be the most common chronic disease in children today. Despite the substantial involvement of genetics in the process of caries development, the specific genes contributing to dental caries remain largely unknown. We performed separate genome-wide association studies of smooth and pit-and-fissure tooth surface caries experience in the primary dentitions of self-reported white children in two samples from Iowa and rural Appalachia. In total, 1,006 children (ages 3-12 years) were included for smooth surface analysis, and 979 children (ages 4-14 years) for pit-and-fissure surface analysis. Associations were tested for more than 1.2 million single nucleotide polymorphisms, either genotyped or imputed. We detected genome-wide significant signals in KPNA4 (p value = 2.0E-9), and suggestive signals in ITGAL (p value = 2.1E-7) and PLUNC family genes (p value = 2.0E-6), thus nominating these novel loci as putative caries susceptibility genes. We also replicated associations observed in previous studies for MPPED2 (p value = 6.9E-6), AJAP1 (p value = 1.6E-6) and RPS6KA2 (p value = 7.3E-6). Replication of these associations in additional samples, as well as experimental studies to determine the biological functions of associated genetic variants, are warranted. Ultimately, efforts such as this may lead to a better understanding of caries etiology, and could eventually facilitate the development of new interventions and preventive measures.

Genome-wide association studies of pit-and-fissure- and smooth-surface caries in permanent dentition.

While genetics clearly influences dental caries risk, few caries genes have been discovered and validated. Recent studies have suggested differential genetic factors for primary dentition caries and permanent dentition caries, as well as for pit-and-fissure- (PF) and smooth- (SM) surface caries. We performed separate GWAS for caries in permanent-dentition PF surfaces (1,017 participants, adjusted for age, sex, and the presence of Streptococcus mutans) and SM surfaces (1,004 participants, adjusted for age, education group, and the presence of Streptococcus mutans) in self-reported whites (ages 14 to 56 yrs). Caries scores were derived based on visual assessment of each surface of each tooth; more than 1.2 million SNPs were either successfully genotyped or imputed and were tested for association. Two homologous genes were suggestively associated: BCOR (Xp11.4) in PF-surface caries (p value = 1.8E-7), and BCORL1 (Xq26.1) in SM-surface caries (p value = 1.0E-5). BCOR mutations cause oculofaciocardiodental syndrome, a Mendelian disease involving multiple dental anomalies. Associations of other plausible cariogenesis genes were also observed for PF-surface caries (e.g., INHBA, p value = 6.5E-6) and for SM-surface caries (e.g., CXCR1 and CXCR2, p value = 1.9E-6). This study supports the notion that genes differentially affect cariogenesis across the surfaces of the permanent dentition, and nominates several novel genes for investigation.

Clustering tooth surfaces into biologically informative caries outcomes.

UNLABELLED: Dental caries affects most adults worldwide; however, the risk factors for dental caries do not necessarily exert their effects uniformly across all tooth surfaces. Instead, the actions of some risk factors may be limited to a subset of teeth/surfaces. Therefore, we used hierarchical clustering on tooth surface-level caries data for 1,068 Appalachian adults (ages 18-75 yrs) to group surfaces based on co-occurrence of caries. Our cluster analysis yielded evidence of 5 distinct groups of tooth surfaces that differ with respect to caries: (C1) pit and fissure molar surfaces, (C2) mandibular anterior surfaces, (C3) posterior non-pit and fissure surfaces, (C4) maxillary anterior surfaces, and (C5) mid-dentition surfaces. These clusters were replicated in a national dataset (NHANES 1999-2000, N = 3,123). We created new caries outcomes defined as the number of carious tooth surfaces within each cluster. We show that some cluster-based caries outcomes are heritable (i.e., under genetic regulation; p < 0.05), whereas others are not. Likewise, we demonstrate the association between some cluster-based caries outcomes and potential risk factors such as age, sex, educational attainment, and toothbrushing habits. Together, these results suggest that the permanent dentition can be subdivided into groups of tooth surfaces that are useful for understanding the factors influencing cariogenesis. ABBREVIATIONS: COHRA, Center for Oral Health in Appalachia, the principal study sample; C1-5, clusters 1-5, groups of similarly behaving tooth surfaces identified through hierarchical clustering; DMFS index, decayed, missing, or filled surfaces, a traditional caries measure representing the number of affected surfaces across the entire dentition; DMFS1-5, partial DMFS indices representing the number of affected surfaces within a hierarchical cluster; and NHANES, National Health and Nutrition Examination Survey, the secondary study sample.

GWAS of dental caries patterns in the permanent dentition.

The importance of susceptibility genes in the risk for dental caries has been clearly established. While many candidate caries genes have been proposed, to date, few of them have been rigorously validated through observational and experimental studies. Moreover, most genetic epidemiological studies have analyzed global caries phenotypes that ignore the possibility that genes may exert differential effects across tooth surfaces of the dentition. Therefore, we performed genome-wide association studies (GWAS) of 5 novel dental caries phenotypes (developed by clustering the permanent dentition into categories of tooth surfaces based on co-occurrence of caries) to nominate new candidate caries genes. GWAS was performed in 920 self-reported white participants, aged 18 to 75 years, with genotype data on 518,997 genetic variants. We identified a significant genetic association between dental caries of the anterior mandibular teeth and LYZL2 (p value = 9e-9), which codes a bacteriolytic agent thought to be involved in host defense. We also identified a significant genetic association between caries of the mid- dentition tooth surfaces and AJAP1 (p value = 2e-8), a gene possibly involved in tooth development. Suggestive genetic associations were also observed for ABCG2, PKD2, the dentin/bone SCPP sub-family, EDNRA, TJFBR1, NKX2-3, IFT88, TWSG1, IL17D, and SMAD7 (p values < 7e-6). We nominate these novel genes for future study.

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 studies: prospects and challenges for oral health.

The genomic era of biomedical research has given rise to the genome-wide association study (GWAS) approach, which attempts to discover novel genes affecting an outcome by testing a large number (i.e., hundreds of thousands to millions) of genetic variants for association. This article discusses the issues surrounding the GWAS approach with emphasis on the prospects and challenges relevant to the oral health research community.

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.

Genome-wide association scan for childhood caries implicates novel genes.

Dental caries is the most common chronic disease in children and a major public health concern due to its increasing incidence, serious health and social co-morbidities, and socio-demographic disparities in disease burden. We performed the first genome-wide association scan for dental caries to identify associated genetic loci and nominate candidate genes affecting tooth decay in 1305 US children ages 3-12 yrs. Affection status was defined as 1 or more primary teeth with evidence of decay based on intra-oral examination. No associations met strict criteria for genome-wide significance (p < 10E-7); however, several loci (ACTN2, MTR, and EDARADD, MPPED2, and LPO) with plausible biological roles in dental caries exhibited suggestive evidence for association. Analyses stratified by home fluoride level yielded additional suggestive loci, including TFIP11 in the low-fluoride group, and EPHA7 and ZMPSTE24 in the sufficient-fluoride group. Suggestive loci were tested but not significantly replicated in an independent sample (N = 1695, ages 2-7 yrs) after adjustment for multiple comparisons. This study reinforces the complexity of dental caries, suggesting that numerous loci, mostly having small effects, are involved in cariogenesis. Verification/replication of suggestive loci may highlight biological mechanisms and/or pathways leading to a fuller understanding of the genetic risks for dental caries.

Inverted duplication pattern in anaphase bridges confirms the breakage-fusion-bridge (BFB) cycle model for 11q13 amplification.

The homogeneously staining region (hsr) involving chromosome band 11q13 includes amplified genes from this chromosome segment and carries a relatively poor prognosis in oral squamous cell carcinomas (OSCC), with shorter time to recurrence and reduced overall survival. We previously identified an inverted duplication pattern of genes within the 11q13 hsr in OSCC cells, supporting a breakage-fusion-bridge (BFB) cycle model for gene amplification. To validate our hypothesis that 11q13 gene amplification in OSCC occurs via BFB cycles, we carried out fluorescence in situ hybridization (FISH) using probes for band 11q13 on 29 OSCC cell lines. We demonstrate that all OSCC cell lines with 11q13 amplification express a significantly higher frequency of anaphase bridges containing 11q13 sequences compared to cell lines without amplification, providing further experimental evidence that 11q13 gene amplification in OSCC cells occurs via BFB cycles. Elucidation of mechanisms responsible for initiating and promoting gene amplification provides opportunities to identify new biomarkers to aid in the diagnosis and prognosis of oral cancer, and may be useful for developing novel therapeutic strategies for patients with OSCC.

Relationship of recombination patterns and maternal age among non-disjoined chromosomes 21.

Advancing maternal age has long been identified as the primary risk factor for human chromosome trisomy. More recently, altered patterns of meiotic recombination have been found to be associated with non-disjunction. We have used trisomy 21 as a model for human non-disjunction that occurs during the formation of oocytes to understand the role of maternal age and recombination. Patterns of recombination that increase the risk for non-disjunction of chromosome 21 include absence of any exchange, an exchange near the centromere or a single, telomeric exchange. Our recent work has shown that different susceptibility patterns are associated with the origin of the meiotic error and maternal age. For MI (meiosis I) errors, the proportion of oocytes with susceptible recombination patterns is highest among young mothers and decreases significantly in the oldest age group. In fact, the pattern of exchanges among the oldest age group mimics the pattern observed among normally disjoining chromosomes 21. These results suggest that oocytes of younger women, with functional meiotic apparatus and/or robust ovarian environment, are able to properly resolve all but the most susceptible exchange patterns. As women age, however, meiotic mechanisms erode, making it difficult to resolve even stable exchange events. Interestingly, our preliminary recombination results on MII errors reveal the opposite relationship with maternal age: susceptible pericentromeric exchanges occur most often in the older age group compared with the younger age group. If confirmed, we will have further evidence for multiple risk factors for non-disjunction that act at different times in the meiotic process.

Genetic association of ubiquilin with Alzheimer's disease and related quantitative measures.

The gene coding for ubiquilin 1 (UBQLN1) is located near a linkage peak on chromosome 9q22.2 and it also impacts the function of presenilin proteins involved in early-onset Alzheimer's disease (AD). Recently, genetic variation in UBQLN1 has been shown to affect the risk of AD in two independent family-based samples. The purpose of this study was to confirm the reported association in a large case-control sample and to also examine the association of UBQLN1 SNPs with quantitative measures of AD progression, namely age-at-onset (AAO), disease duration and Mini-Mental State Examination (MMSE) score. We examined the associations of three SNPs in the UBQLN1 gene (intron 6/A>C, intron 8/T>C and intron 9/A>G) in up to 978 LOAD cases and 808 controls. All SNPs were in significant linkage disequilibrium (P<0.0001). While modest significant associations were observed in the single-site regression analysis, 3-site haplotype analysis revealed significant associations (P<0.0001 for overall haplotype analysis). One common haplotype (H4) defined by intron 6/A-intron 8/C-intron 9/G alleles was associated with AD risk and one less common haplotype (H5) defined by intron 6/C-intron 8/C-intron 9/A alleles was associated with protection. The adjusted odds ratios with potentially one and two copies of risk haplotype H4 were 1.5 (95% CI: 0.99-2.26; P=0.054) and 3.66 (95% CI: 1.43-9.39; P=0.007), respectively, and odds ratio for haplotype H5 carriers was 0.31 (95% CI: 0.10-0.95; P=0.0398). In addition to disease risk, the homozygosity of the risk haplotype was also associated with older AAO, longer disease duration and lower MMSE score. In summary, our data from a large case-control cohort indicate that genetic variation in the UBQLN1 gene has a modest effect on risk, AAO and disease duration of AD. Our haplotype data suggest the presence of additional putative functional variants either in the UBQLN1 gene or nearby genes and provide strong justification for additional work in this region on chromosome 9.

Evaluation of human leukocyte N-formylpeptide receptor (FPR1) SNPs in aggressive periodontitis patients.

Polymorphonuclear neutrophils (PMNs) are attracted to sites of infection by N-formylpeptide (fMLP) chemoattractants. The high-affinity fMLP receptor (FPR1) of phagocytic cells interacts with bacterial fMLP and mediates chemotaxis, degranulation, and superoxide production. These cellular functions are disrupted in PMN from aggressive periodontitis (AP) patients. Two FPR1 gene single nucleotide polymorphisms (SNPs), c.329T>C and c.378C>G, have been associated with a localized form of AP in African-American patients. To evaluate the generality of these SNPs in AP patients, we sequenced a 363 bp interval of the FPR1 gene in an ethnically diverse group of patients (n=111) and controls (n=115). Neither c.329T>C nor c.378C>G were detected in the 452 alleles sequenced. Six SNPs were identified including two located in the FPR1 second extracellular loop that were significantly associated with the AP phenotype in African-American patients (p.R190W, P=0.0033; and p.N192K, P=0.0018). These two SNPs show three predominant haplotypes, each associated with a different disease risk in African-Americans. These data do not support the hypothesis that the FPR1 SNPs c.329T>C and c.378C>G play an etiologic role in aggressive periodontitis, but do suggest that SNPs in the second extracellular loop may be etiologically important.

Local high-capacity adenovirus-mediated mCTLA4Ig and mCD40Ig expression prolongs recombinant gene expression in skeletal muscle.

Multiple forms of muscular dystrophy are due to the absence of cytoskeletal muscle proteins that normally protect the integrity of muscle cells. The lack of any adequate treatments for these devastating diseases propels research toward the development of strategies for gene delivery to skeletal muscle. High-capacity adenoviral vectors (HC-AdV) devoid of all viral coding sequences have been developed to avoid expression of viral proteins by the gene therapy vector. However, the capsid proteins that are an essential component of the input viral vector and any residual helper virus in the vector preparation could induce an immune response. Furthermore, the therapeutic protein provided by a gene transfer vector presents the potential to induce an immune response in a patient who does not express a normal cellular protein due to genetic mutation. Therefore, we hypothesize that some immune suppression will be required with therapeutic gene delivery designed for the treatment of patients with inherited muscle diseases. In this study, we constructed and rescued three HC-AdVs expressing murine CTLA4Ig, murine CD40Ig, or both. The backbone vector without a gene insert was rescued as a negative control vector. The production of relevant proteins from each vector was determined in vitro. In vivo function of each of the immunosuppressant vectors was assayed by co-injection with an enhanced green fluorescent protein (EGFP)-expressing first-generation adenoviral vector (AdEGFP) into the tibialis anterior muscle of C57BL/10 mice. Higher levels of muscle EGFP expression were observed in animals receiving an immunosuppressant vector. Furthermore, the production of total anti-AdV and anti-EGFP antibodies was reduced in mice treated with each of the three immunosuppressant vectors. A second intramuscular administration of AdEGFP alone 4 weeks after the initial co-injection was successful in all immunosuppressant vector-treated groups, but not in the negative control vector-treated group. All groups had a high antibody response to adenoviral proteins after the second injection of AdEGFP alone, indicating that the initial co-injection did not tolerize against vector capsid antigens.

A survey of affected-sibship statistics for nonparametric linkage analysis.

We have compared the power of a large number of allele-sharing statistics for "nonparametric" linkage analysis with affected sibships. Our rationale was that there is an extensive literature comparing statistics for sibling pairs but that there has not been much guidance on how to choose statistics for studies that include sibships of various sizes. We concentrated on statistics that can be described as assigning scores to each identity-by-descent-sharing configuration that a pedigree might take on (Whittemore and Halpern 1994). We considered sibships of sizes two through five, 27 different genetic models, and varying recombination fractions between the marker and the trait locus. We tried to identify statistics whose power was robust over a wide variety of models. We found that the statistic that is probably used most often in such studies-S(all)-performs quite well, although it is not necessarily the best. We also found several other statistics (such as the R criterion, S(robdom), and the Sobel-and-Lange statistic C) that perform well in most situations, a few (such as S(-#geno) and the Feingold-and-Siegmund version of S(pairs)) that have high power only in very special situations, and a few (such as S(-#geno), the N criterion, and the Sobel-and-Lange statistic B) that seem to have low power for the majority of the trait models. For the most part, the same statistics performed well for all sibship sizes. We also used our results to give some suggestions regarding how to weight sibships of different sizes, in forming an overall statistic.

Estimating the frequency distribution of crossovers during meiosis from recombination data.

Estimation of tetrad crossover frequency distributions from genetic recombination data is a classic problem dating back to Weinstein (1936, Genetics 21, 155-199). But a number of important issues, such as how to specify the maximum number of crossovers, how to construct confidence intervals for crossover probabilities, and how to obtain correct p-values for hypothesis tests, have never been adequately addressed. In this article, we obtain some properties of the maximum likelihood estimate (MLE) for crossover probabilities that imply guidelines for choosing the maximum number of crossovers. We give these results for both normal meiosis and meiosis with nondisjunction. We also develop an accelerated EM algorithm to find the MLE more efficiently. We propose bootstrap-based methods to find confidence intervals and p-values and conduct simulation studies to check the validity of the bootstrap approach.

Molecular anatomy of an intracranial aneurysm: coordinated expression of genes involved in wound healing and tissue remodeling.

BACKGROUND AND PURPOSE: Approximately 6% of human beings harbor an unruptured intracranial aneurysm. Each year in the United States, >30 000 people suffer a ruptured intracranial aneurysm, resulting in subarachnoid hemorrhage. Despite the high incidence and catastrophic consequences of a ruptured intracranial aneurysm and the fact that there is considerable evidence that predisposition to intracranial aneurysm has a strong genetic component, very little is understood with regard to the pathology and pathogenesis of this disease. METHODS: To begin characterizing the molecular pathology of intracranial aneurysm, we used a global gene expression analysis approach (SAGE-Lite) in combination with a novel data-mining approach to perform a high-resolution transcript analysis of a single intracranial aneurysm, obtained from a 3-year-old girl. RESULTS: SAGE-Lite provides a detailed molecular snapshot of a single intracranial aneurysm. These data suggest that, at least in this specific case, aneurysmal dilation results in a highly dynamic cellular environment in which extensive wound healing and tissue/extracellular matrix remodeling are taking place. Specifically, we observed significant overexpression of genes encoding extracellular matrix components (eg, COL3A1, COL1A1, COL1A2, COL6A1, COL6A2, elastin) and genes involved in extracellular matrix turnover (TIMP-3, OSF-2), cell adhesion and antiadhesion (SPARC, hevin), cytokinesis (PNUTL2), and cell migration (tetraspanin-5). CONCLUSIONS: Although these are preliminary data, representing analysis of only one individual, we present a unique first insight into the molecular basis of aneurysmal disease and define numerous candidate markers for future biochemical, physiological, and genetic studies of intracranial aneurysm. Products of these genes will be the focus of future studies in wider sample sets.