From single-SNP to wide-locus: genome-wide association studies identifying functionally related genes and intragenic regions in small sample studies.

BACKGROUND: Genome-wide association studies (GWAS) have had limited success when applied to complex diseases. Analyzing SNPs individually requires several large studies to integrate the often divergent results. In the presence of epistasis, multivariate approaches based on the linear model (including stepwise logistic regression) often have low sensitivity and generate an abundance of artifacts. METHODS: Recent advances in distributed and parallel processing spurred methodological advances in nonparametric statistics. U-statistics for structured multivariate data (µStat) are not confounded by unrealistic assumptions (e.g., linearity, independence). RESULTS: By incorporating knowledge about relationships between SNPs, µGWAS (GWAS based on µStat) can identify clusters of genes around biologically relevant pathways and pinpoint functionally relevant regions within these genes. CONCLUSION: With this computational biostatistics approach increasing power and guarding against artifacts, personalized medicine and comparative effectiveness will advance while subgroup analyses of Phase III trials can now suggest risk factors for adverse events and novel directions for drug development.

Non-synonymous variants in the AMACR gene are associated with schizophrenia.

BACKGROUND: The AMACR gene is located in the schizophrenia susceptibility locus on chromosome 5p13, previously identified in a large Puerto Rican pedigree of Spanish origin. The AMACR-encoded protein is an enzyme involved in the metabolism of branched-chain fatty and bile acids. The enzyme deficiency causes structural and functional brain changes, and disturbances in fatty acid and oxidative phosphorylation pathways observed in individuals with schizophrenia. Therefore, AMACR is both a positional and functional candidate gene for susceptibility to schizophrenia. METHODS: The study had a two-step design: we performed mutation analysis of the coding and flanking regions of AMACR in affected members of the pedigree, and tested the detected sequence variants for association with schizophrenia in a Puerto Rican case-control sample (n=383) of Spanish descent. RESULTS AND CONCLUSION: We identified three missense variants segregating with the disorder in the family, rs2278008, rs2287939 and rs10941112. Two of them, rs2278008 and rs2287939, demonstrated significant differences in genotype (P = 4 × 10-4, P = 4 × 10-4) and allele (P = 1 × 10-4, P = 9.5 × 10-5) frequencies in unrelated male patients compare to controls, with the odds ratios (OR) 2.24 (95% CI: 1.48-3.40) and 2.25 (95% CI: 1.49-3.38), respectively. The G-C-G haplotype of rs2278008-rs2287939-rs10941112 revealed the most significant association with schizophrenia (P = 4.25 × 10-6, OR = 2.96; 95% CI: 1.85-4.76) in male subjects. There were no statistically significant differences in genotype, allele, and haplotype frequencies between female schizophrenia subjects and controls. Our results suggest that AMACR may play a significant role in susceptibility to schizophrenia in male patients.

Mutation analysis of the C1QTNF3 gene in patients with schizophrenia.

We previously identified a small region on chromosome 5p13 related to schizophrenia in a Puerto Rican pedigree. We screened one of the positional candidate genes, C1QTNF3, for mutations in affected family members. The direct sequencing identified 10 sequence variants, including five shared by all affected family members. Genotyping of the shared variants in a Puerto Rican sample of 118 cases and 136 controls did not reveal either allelic or genotype association with schizophrenia.

Polyspike and waves do not predict generalized tonic-clonic seizures in childhood absence epilepsy.

About 40% of children with childhood absence epilepsy develop generalized tonic-clonic seizures. It is commonly held that polyspike-wave pattern on the electroencephalogram (EEG) can predict this development of generalized tonic-clonic seizures. However, there is no firm evidence in support of this proposition. To test this assumption, we used survival analysis and compared the incidence of generalized tonic-clonic seizures in 115 patients with childhood absence epilepsy having either isolated 3-Hz spike-wave or coexisting 3 Hz and polyspike-waves and other variables. There was no evidence that polyspike-waves predicted development of generalized tonic-clonic seizures in patients with childhood absence epilepsy. Later age of onset (> or =8 years) and family histories of generalized tonic-clonic seizures were the only independent predictors. These results have implications for counseling and in the choice of first-line antiepileptic drugs used for childhood absence epilepsy, especially if valproate is chosen based on the observation of polyspike-waves.

Genome wide high density SNP-based linkage analysis of childhood absence epilepsy identifies a susceptibility locus on chromosome 3p23-p14.

Childhood absence epilepsy (CAE) is an idiopathic generalised epilepsy (IGE) characterised by typical absence seizures manifested by transitory loss of awareness with 2.5-4 Hz spike-wave complexes on ictal EEG. A genetic component to the aetiology is well recognised but the mechanism of inheritance and the genes involved are yet to be fully established. A genome wide single nucleotide polymorphism (SNP)-based high density linkage scan was carried out using 41 nuclear pedigrees with at least two affected members. Multipoint parametric and non-parametric linkage analyses were performed using MERLIN 1.1.1 and a susceptibility locus was identified on chromosome 3p23-p14 (Z(mean)=3.9, p<0.0001; HLOD=3.3, alpha=0.7). The linked region harbours the functional candidate genes TRAK1 and CACNA2D2. Fine-mapping using a tagSNP approach demonstrated disease association with variants in TRAK1.

A linkage search for joint panic disorder/bipolar genes.

There is comorbidity and a possible genetic connection between Bipolar disease (BP) and panic disorder (PD). Genes may exist that increase risk to both PD and BP. We explored this possibility using data from a linkage study of PD (120 multiplex families; 37 had > or =1 BP member). We calculated 2-point lodscores maximized over male and female recombination fractions by classifying individuals with PD and/or BP as affected (PD + BP). Additionally, to shed light on possible heterogeneity, we examine the pedigrees containing a bipolar member (BP+) separately from those that do not (BP-), using a Predivided-Sample Test (PST). Linkage evidence for common genes for PD + BP was obtained on chromosomes 2 (lodscore = 4.6) and chromosome 12 (lodscore = 3.6). These locations had already been implicated using a PD-only diagnosis, although at both locations this was larger when a joint PD + BP diagnosis was used. Examining the BP+ families and BP- families separately indicates that both BP+ and BP- pedigrees are contributing to the peaks on chromosomes 2 and 12. However, the PST indicates different evidence of linkage is obtained from BP+ and BP- pedigrees on chromosome 13. Our findings are consistent with risk loci for the combined PD + BP phenotype on chromosomes 2 and 12. We also obtained evidence of heterogeneity on chromosome 13. The regions on chromosomes 12 and 13 identified here have previously been implicated as regions of interest for multiple psychiatric disorders, including BP.

Non-replication of association studies: "pseudo-failures" to replicate?

Recently, serious doubts have been cast on the usefulness of association studies as a means to genetically dissect complex diseases because most initial findings fail to replicate in subsequent studies. The reasons usually invoked are population stratification, genetic heterogeneity, and inflated Type I errors. In this article, we argue that, even when these problems are addressed, the scientific community usually has unreasonably high expectations on replication success, based on initial low P values, a phenomenon known as the replication fallacy. We present a modified formula that gives the replication power of a second association study based on the P value of an initial study. When both studies have similar sample sizes, this formula shows that: (1) a P value only slightly lower than the nominal alpha results in only approximately 50% replication power; (2) very low P values are required to achieve a replication power of at least 80% (e.g., at alpha = 0.05, a P value of <0.005 is required). Because many initially significant findings result in low replication power, replication failure should not be surprising or be interpreted as necessarily refuting the initial findings. We refer to replication failures for which the replication power is low as "pseudo-failures."

Linkage and mutational analysis of CLCN2 in childhood absence epilepsy.

In order to assess the chloride channel gene CLCN2 as a candidate susceptibility gene for childhood absence epilepsy, parametric and non-parametric linkage analysis was performed in 65 nuclear pedigrees. This provided suggestive evidence for linkage with heterogeneity: NPL score=2.3, p<0.009; HLOD=1.5, alpha=0.44. Mutational analysis of the entire genomic sequence of CLCN2 was performed in 24 unrelated patients from pedigrees consistent with linkage, identifying 45 sequence variants including the known non-synonymous polymorphism rs2228292 (G2154C, Glu718Asp) and a novel variant IVS4+12G>A. Intra-familial association analysis using the pedigrees and a further 308 parent-child trios showed suggestive evidence for transmission disequilibrium of the G2154C minor allele: AVE-PDT chi(1)2 = 5.17, p<0.03. Case-control analysis provided evidence for a protective effect of the IVS4+12G>A minor allele: chi(1)2 = 7.27, p<0.008. The 65 nuclear pedigrees were screened for three previously identified mutations shown to segregate with a variety of idiopathic generalised epilepsy phenotypes (597insG, IVS2-14del11 and G2144A) but none were found. We conclude that CLCN2 may be a susceptibility locus in a subset of cases of childhood absence epilepsy.

A third-pass genome scan in panic disorder: evidence for multiple susceptibility loci.

BACKGROUND: Panic disorder (PD) is a common illness with a definite but "complex" genetic contribution and estimated heritability of 30-46%. METHODS: We report a genome scan in 120 multiplex PD pedigrees consisting of 1591 individuals of whom 992 were genotyped with 371 markers at an average spacing of 9cM. Parametric two-point, multipoint, and nonparametric analyses were performed using three PD models (Broad, Intermediate, Narrow) and allowing for homogeneity or heterogeneity. The two-point analyses were also performed allowing for independent male and female recombination fractions (theta). Genome-wide significance was empirically evaluated using simulations of this dataset. RESULTS: Evidence for linkage reached genome-wide significance in one region on chromosome 15q (near GABA-A receptor subunit genes) and was suggestive at loci on 2p, 2q and 9p using an averaged theta. Analyses allowing for sex-specific theta's were consistent except that support at one locus on 2q increased to genome-wide significance and an additional region of suggestive linkage on 12q was identified. However, differences in male and female recombination fractions predicted by the sex-specific approach were not consistent with current physical maps. CONCLUSIONS: These data provide evidence for chromosomal regions on 15q and 2q that may be important in genetic susceptibility to panic disorder. Although we are encouraged by the findings of analyses using sex-specific recombination fractions, we also note that further understanding of this analytic strategy will be important.

Evaluation of CACNA1H in European patients with childhood absence epilepsy.

CACNA1H was evaluated in a resource of Caucasian European patients with childhood absence epilepsy by linkage analysis and typing of sequence variants previously identified in Chinese patients. Linkage analysis of 44 pedigrees provided no evidence for a locus in the CACNA1H region and none of the Chinese variants were found in 220 unrelated patients.

Complex inheritance and parent-of-origin effect in juvenile myoclonic epilepsy.

BACKGROUND: Juvenile myoclonic epilepsy (JME) is an idiopathic generalized epilepsy (IGE) with complex inheritance. Previous studies have suggested maternal inheritance and female excess in IGEs but have not been specific for JME. We investigated evidence for maternal inheritance, female excess and patterns of familial seizure risk in a well-characterized sample of JME families. METHODS: We ascertained 89 families through a JME proband and 50 families through a non-JME IGE proband. JME families were divided into those with and without evidence of linkage to the EJM1 susceptibility locus on chromosome 6. We analyzed transmission in 43 multigenerational families, calculated the adjusted sex ratio for JME, and looked for evidence of seizure specific risk in 806 family members. RESULTS: We found evidence for preferential maternal transmission in both EJM1-linked and unlinked families (2.7:1), evidence even more marked when potential selection factors were excluded. The adjusted female: male risk ratio was very high in JME (RR=12.5; 95% CI: 1.9-83.7). Absence seizures in JME probands increased the overall risk of seizures in first degree relatives (15.8% vs. 7.0%, P=0.011), as well as first-degree relatives' specific risk of absence seizures (6% vs. 1.6%, P=0.01), but not myoclonic seizures. CONCLUSIONS: We have confirmed the finding of maternal inheritance in JME, which is not restricted to JME families linked to the EJM1 locus. The striking female excess in JME may relate to anatomical and/or endocrine sexual dimorphism in the brain. Evidence for independent inheritance of absence and myoclonic seizures in JME families reinforces a model in which combinations of loci confer susceptibility to the component seizure types of IGE.

Fine mapping of the 5p13 locus linked to schizophrenia and schizotypal personality disorder in a Puerto Rican family.

A locus involved in schizophrenia and related disorders in a Puerto Rican family has previously been mapped to chromosome 5p. The maximum two-point log of the odds (LOD) score of 3.72 was obtained for marker D5S111, and increased to 4.37 by multipoint analysis, assuming autosomal dominant inheritance with 90% penetrance. Additional genotyping and haplotype analysis placed the novel locus on 5p13.2-p13.3 within the interval between markers D5S1993 and D5S631. In the current study, we saturated the interval between markers D5S1993 and D5S631 with densely spaced polymorphic markers, genotyped these markers in the most informative branch of the family, and narrowed the critical region to 2.8 Mb. G-protein-coupled receptor gene [somatostatin and angiotensin-like peptide receptor (SALPR)] is one of the candidate genes within the critical interval. Sequence analysis of the coding region and the putative promoter of somatostatin and angiotensin-like peptide receptor did not reveal functionally significant variants in affected family members, although several polymorphisms were detected.

Malic enzyme 2 may underlie susceptibility to adolescent-onset idiopathic generalized epilepsy.

Idiopathic generalized epilepsy (IGE) is a class of genetically determined, phenotypically related epilepsy syndromes. Linkage analysis identified a chromosome 18 locus predisposing to a number of adolescent-onset IGEs. We report a single-nucleotide polymorphism (SNP) association analysis of the region around the marker locus with the high LOD score. This analysis, which used both case-control and family-based association methods, yielded strong evidence that malic enzyme 2 (ME2) is the gene predisposing to IGE. We also observed association among subgroups of IGE syndromes. An ME2-centered nine-SNP haplotype, when present homozygously, increases the risk for IGE (odds ratio 6.1; 95% confidence interval 2.9-12.7) compared with any other genotype. Both the linkage analysis and the association analysis support recessive inheritance for the locus, which is compatible with the fact that ME2 is an enzyme. ME2 is a genome-coded mitochondrial enzyme that converts malate to pyruvate and is involved in neuronal synthesis of the neurotransmitter gamma-aminobutyric acid (GABA). The results suggest that GABA synthesis disruption predisposes to common IGE and that clinical seizures are triggered when mutations at other genes, or perhaps other insults, are present.

BRD2 (RING3) is a probable major susceptibility gene for common juvenile myoclonic epilepsy.

Juvenile myoclonic epilepsy (JME) is a common form of generalized epilepsy that starts in adolescence. A major JME susceptibility locus (EJM1) was mapped to chromosomal region 6p21 in three independent linkage studies, and association was reported between JME and a microsatellite marker in the 6p21 region. The critical region for EJM1 is delimited by obligate recombinants at HLA-DQ and HLA-DP. In the present study, we found highly significant linkage disequilibrium (LD) between JME and a core haplotype of five single-nucleotide-polymorphism (SNP) and microsatellite markers in this critical region, with LD peaking in the BRD2 (RING3) gene (odds ratio 6.45; 95% confidence interval 2.36-17.58). DNA sequencing revealed two JME-associated SNP variants in the BRD2 (RING3) promoter region but no other potentially causative coding mutations in 20 probands from families with positive LOD scores. BRD2 (RING3) is a putative nuclear transcriptional regulator from a family of genes that are expressed during development. Our findings strongly suggest that BRD2 (RING3) is EJM1, the first gene identified for a common idiopathic epilepsy. These findings also suggest that abnormalities of neural development may be a cause of common idiopathic epilepsy, and the findings have implications for the generalizability of proposed pathogenetic mechanisms, derived from diseases that show Mendelian transmission, to their complex counterparts.

Further genetic evidence for a panic disorder syndrome mapping to chromosome 13q.

Substantial evidence supports that there is a genetic component to panic disorder (PD). Until recently, attempts at localizing genes for PD by using standard phenotypic data have not proven successful. Previous work suggests that a potential subtype of PD called the panic syndrome exists, and it is characterized by a number of medical conditions, most notably bladder/renal disorders. In the current study, a genome scan with 384 microsatellite markers was performed on 587 individuals in 60 multiplex pedigrees segregating PD and bladder/kidney conditions. Using both single-locus and multipoint analytic methods, we found significant linkage on chromosome 22 (maximum heterogeneity logarithm of odds score = 4.11 at D22S445) and on chromosome 13q (heterogeneity logarithm of odds score = 3.57 at D13S793) under a dominant-genetic model and a broad phenotypic definition. Multipoint analyses did not support the observation on chromosome 22. The chromosome 13 findings were corroborated by multipoint findings, and extend our previous findings from 19 of the 60 families. Several other regions showed elevated scores by using when one analytic method was used, but not the other. These results suggest that there are genes on chromosome 13q, and possibly on chromosome 22 as well, that influence the susceptibility toward a pleiotropic syndrome that includes PD, bladder problems, severe headaches, mitral valve prolapse, and thyroid conditions.

Phenotypic features of familial febrile seizures: case-control study.

OBJECTIVE: To identify phenotypic features of febrile seizures that can be used to reduce heterogeneity and thereby increase power in linkage analysis. BACKGROUND: Despite exciting discoveries in several rare pedigrees, the genetic basis of common febrile seizures remains a mystery. The major drawback of studying common febrile seizure disorder is etiologic and genetic heterogeneity. A linkage sample must therefore be classified a priori on phenotypic criteria likely to reflect genetically homogeneous subgroups. METHODS: Eighty-three cases (children with one or more febrile seizure plus first-degree family history of febrile seizures) and 101 controls (children with one or more febrile seizure but no first-degree family history of febrile seizures) were compared for association of phenotypic features in an unmatched case-control design. Odds ratios were calculated using univariate and multivariate methods. RESULTS: Recurrent febrile seizures was the only phenotypic feature significantly associated with first-degree family history of febrile seizures (OR 2.1, 95% CI 1.15 to 3.88). First-degree family history and later occurrence of afebrile seizures (OR 3.47, 95% CI 0.94 to 12.78) were independently associated with recurrent febrile seizures. Complex features did not show familial aggregation. CONCLUSIONS: The authors suggest recurrent and afebrile seizures as criteria on which to subgroup a linkage sample. These subgroups will not be evident at the time of the initial febrile seizure. Meticulous and prospective collection of phenotypic and family data are recommended.