A multi-tissue analysis identifies HLA complex group 9 gene methylation differences in bipolar disorder.

Epigenetic studies of DNA and histone modifications represent a new and important activity in molecular investigations of human disease. Our previous epigenome-wide scan identified numerous DNA methylation differences in post-mortem brain samples from individuals affected with major psychosis. In this article, we present the results of fine mapping DNA methylation differences at the human leukocyte antigen (HLA) complex group 9 gene (HCG9) in bipolar disorder (BPD). Sodium bisulfite conversion coupled with pyrosequencing was used to interrogate 28 CpGs spanning ∼700 bp region of HCG9 in 1402 DNA samples from post-mortem brains, peripheral blood cells and germline (sperm) of bipolar disease patients and controls. The analysis of nearly 40 000 CpGs revealed complex relationships between DNA methylation and age, medication as well as DNA sequence variation (rs1128306). Two brain tissue cohorts exhibited lower DNA methylation in bipolar disease patients compared with controls at an extended HCG9 region (P=0.026). Logistic regression modeling of BPD as a function of rs1128306 genotype, age and DNA methylation uncovered an independent effect of DNA methylation in white blood cells (odds ratio (OR)=1.08, P=0.0077) and the overall sample (OR=1.24, P=0.0011). Receiver operating characteristic curve A prime statistics estimated a 69-72% probability of correct BPD prediction from a case vs control pool. Finally, sperm DNA demonstrated a significant association (P=0.018) with BPD at one of the regions demonstrating epigenetic changes in the post-mortem brain and peripheral blood samples. The consistent multi-tissue epigenetic differences at HCG9 argue for a causal association with BPD.

Molecular studies of major depressive disorder: the epigenetic perspective.

Major depressive disorder (MDD) is a common and highly heterogeneous psychiatric disorder encompassing a spectrum of symptoms involving deficits to a range of cognitive, psychomotor and emotional processes. As is the norm for aetiological studies into the majority of psychiatric phenotypes, particular focus has fallen on the interplay between genetic and environmental factors. There are, however, several epidemiological, clinical and molecular peculiarities associated with MDD that are hard to explain using traditional gene- and environment-based approaches. Our goal in this study is to demonstrate the benefits of looking beyond conventional 'DNA+environment' and 'DNA x environment' aetiological paradigms. Epigenetic factors - inherited and acquired modifications of DNA and histones that regulate various genomic functions occurring without a change in nuclear DNA sequence - offer new insights about many of the non-Mendelian features of major depression, and provide a direct mechanistic route via which the environment can interact with the genome. The study of epigenetics, especially in complex diseases, is a relatively new field of research, and optimal laboratory techniques and analysis methods are still being developed. Incorporating epigenetic research into aetiological studies of MDD thus presents a number of methodological and interpretive challenges that need to be addressed. Despite these difficulties, the study of DNA methylation and histone modifications has the potential to transform our understanding about the molecular aetiology of complex diseases.

Epigenetics of complex diseases: from general theory to laboratory experiments.

Despite significant effort, understanding the causes and mechanisms of complex non-Mendelian diseases remains a key challenge. Although numerous molecular genetic linkage and association studies have been conducted in order to explain the heritable predisposition to complex diseases, the resulting data are quite often inconsistent and even controversial. In a similar way, identification of environmental factors causal to a disease is difficult. In this article, a new interpretation of the paradigm of "genes plus environment" is presented in which the emphasis is shifted to epigenetic misregulation as a major etiopathogenic factor. Epigenetic mechanisms are consistent with various non-Mendelian irregularities of complex diseases, such as the existence of clinically indistinguishable sporadic and familial cases, sexual dimorphism, relatively late age of onset and peaks of susceptibility to some diseases, discordance of monozygotic twins and major fluctuations on the course of disease severity. It is also suggested that a substantial portion of phenotypic variance that traditionally has been attributed to environmental effects may result from stochastic epigenetic events in the cell. It is argued that epigenetic strategies, when applied in parallel with the traditional genetic ones, may significantly advance the discovery of etiopathogenic mechanisms of complex diseases. The second part of this chapter is dedicated to a review of laboratory methods for DNA methylation analysis, which may be useful in the study of complex diseases. In this context, epigenetic microarray technologies are emphasized, as it is evident that such technologies will significantly advance epigenetic analyses in complex diseases.

Age related changes in 5-methylcytosine content in human peripheral leukocytes and placentas: an HPLC-based study.

The goal of the present study was to investigate inter-individual and age-dependent variation of global DNA methylation in human tissues. In this work, we examined 5-methyldeoxycytidine ((met)C) content by HPLC in human peripheral blood leukocytes obtained from 76 healthy individuals of ages varying from 4 to 94 years (yr), and 39 human placentas from various gestational stages. The HPLC analysis revealed a significant variation of (met)C across individuals and is consistent with the previous findings of age-dependent decrease of global methylation levels in human tissues. The age-dependent decrease of (met)C was relatively small, but statistically highly significant (p= 0.0002) in the aged group (65.9 +/- 8.9 [mean age +/- SD] yr; n = 22) in comparison to the young adult group (19.3 +/- 1.4 yr; n = 21). Males showed a subtle but statistically significant higher mean (met)C content than females. In contrast to the peripheral blood samples, DNA extracted from placentas exhibited gestational stage-dependent increase of methylation levels that appeared to inversely correlate with the expression levels of human endogenous retroviruses. These data may be helpful in further studies of DNA methylation, such as inheritance of epigenetic patterns, environment-induced changes, and involvement of epigenetic changes in disease.

Dopamine D4 receptor and tyrosine hydroxylase genes in bipolar disorder: evidence for a role of DRD4.

The involvement of the mesocorticolimbic dopamine system in behaviors that are compromised in patients with mood disorder has led to the investigation of dopamine system genes as candidates for bipolar disorder. In particular, the functional VNTRs in the exon III of the dopamine D4 (DRD4) and in intron I of the tyrosine hydroxylase (TH) genes have been investigated in numerous association studies that have produced contrasting results. Likewise, linkage studies in multiplex bipolar families have shown both positive and negative results for markers in close proximity to DRD4 and TH on 11p15.5. We performed a linkage disequilibrium analysis of the DRD4 and TH VNTRs in a sample of 145 nuclear families comprised of DSM-IV bipolar probands and their biological parents. An excess of transmissions and non transmissions was observed for the DRD4 4- and 2-repeat alleles respectively. The biased transmission showed a parent of origin effect (POE) since it was derived almost exclusively from the maternal meiosis (4-repeat allele maternally transmitted 40 times vs 20 times non-transmitted; chi(2) = 6.667; df = 1; P = 0.009; while paternally transmitted 26 times vs 21 times non-transmitted; chi(2) = 0.531; df = 1; P = 0.46). The analysis of TH did not reveal biased transmission of intron I VNTR alleles. Although replication of our study is necessary, the fact that DRD4 exhibit POE and is located on 11p15.5, in close proximity to a cluster of imprinted genes, suggests that genomic imprinting may be operating in bipolar disorder.

Lack of association between serotonin-2A receptor gene (HTR2A) polymorphisms and tardive dyskinesia in schizophrenia.

Tardive dyskinesia (TD) is a disabling neurological side effect associated with long-term treatment with typical antipsychotics. Family studies and animal models lend evidence for hereditary predisposition to TD. The newer atypical antipsychotics pose a minimal risk for TD which is in part attributed to their ability to block the serotonin-2A (5-HT(2A)) receptor. 5-HT(2A) receptors were also identified in the basal ganglia; a brain region that plays a critical role in antipsychotic-induced movement disorders. We tested the significance of variation in the 5-HT(2A) receptor gene (HTR2A) in relation to the TD phenotype. Three polymorphisms in HTR2A, one silent (C102T), one that alters the amino acid sequence (his452tyr) and one in the promoter region (A-1437G) were investigated in 136 patients refractory or intolerant to treatment with typical antipsychotics and with a DSM-IIIR diagnosis of schizophrenia. We did not find any significant difference in allele, genotype or haplotype frequencies of polymorphisms in HTR2A among patients with or without TD (P > 0.05). Further analysis using the ANCOVA statistic with a continuous measure of the TD phenotype (Abnormal Involuntary Movement Scale (AIMS) score) found that the AIMS scores were not significantly influenced by HTR2A polymorphisms, despite controlling for potential confounders such as age, gender and ethnicity (P > 0.05). Theoretically, central serotonergic function can be subject to genetic control at various other mechanistic levels including the rate of serotonin synthesis (tryptophane hydroxylase gene), release, reuptake (serotonin transporter gene) and degradation (monoamine oxidase gene). Analyses of these other serotonergic genes are indicated. In summary, polymorphisms in HTR2A do not appear to influence the risk for TD. Further studies evaluating in tandem multiple candidate genes relevant for the serotonergic system are warranted to dissect the genetic basis of the complex TD phenotype.

Human morbid genetics revisited: relevance of epigenetics.

Identification of genes predisposing their carrier to complex diseases is a much more complicated task than finding genes involved in simple mendelian diseases. The slow progress in the genetic research of complex diseases could be due to limitations in the basic research strategy, which is almost exclusively orientated to the detection of disease-related DNA mutations or polymorphisms. I argue in this article that epigenetic misregulation of genes is more consistent with the features of complex diseases than is DNA sequence variation, and therefore that epigenetic factors could be important in understanding the origins of complex diseases.

Long repeat tracts at SCA8 in major psychosis.

Expansion at a recently identified unstable trinucleotide repeat on chromosome 13q21 has been reported as the molecular cause for spinocerebellar ataxia type 8 (SCA8). The trinucleotide repeat, which consists of a [CTA]n repeat and adjacent [CTG]n repeat, was reported to have a pathogenic range of 107-127 CTG repeats (or 110-130 combined CTA and CTG repeats) in a large ataxia kindred. This repeat region was also cloned by our group from a bipolar affective disorder (BPAD) patient, who has approximately 600 combined repeats, and large alleles (>100 repeats) were reported to be present in 0.7% of controls and 1.5% of major psychosis patients (n = 710 and n = 1,120, respectively). We have followed up these findings by screening three new samples of BPAD and schizophrenia (SCZ) patients and controls, including 272 individuals from 14 BPAD families from Sweden, 130 individuals from 32 SCZ and BPAD families/trios from the Azores Islands, and 206 SCZ individuals from the United Kingdom and Ireland, and 219 matched controls. We found large repeat alleles above the SCA8 pathogenic range in individuals from 3 of 32 Azorean pedigrees and in 1 of 206 SCZ individuals from the United Kingdom, and repeat alleles within the SCA8 pathogenic range in 1 of 14 Swedish families. Although the rarity of major psychosis patients carrying the SCA8 expansion mutation would require a much larger sample size to reach statistical significance, these results support the previously reported observation of increased occurrence of large repeats at SCA8 in major psychosis. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:873-876, 2000.

The genes for major psychosis: aberrant sequence or regulation?

A number of recent clinical and molecular observations in major psychosis indicate that epigenetic factors may be operational in the origin of major mental illness. This article further develops the idea that epigenetic factors may play an etiopathogenic role in schizophrenia and bipolar affective disorder. The putative role of epigenetic factors is shown by the epigenetic interpretation of genetic association studies of the genes for serotonin 2A (HTR2A) and the dopamine D3 (DRD3) receptors in schizophrenia. The idea of epigenetic polymorphism of genetic alleles is introduced, and it is argued that epigenetic variation may explain a number of controversial and unclear findings in allelic and genotypic association studies of HTR2A and DRD3. In linkage analyses of multiplex families with bipolar affective disorder (BPAD), different loci on chromosome 18 indicated co-segregation of alleles of one parental sex with the disease phenotype, and this finding implies that the epigenetic mechanism of genomic imprinting may be involved. Evidence for genomic imprinting provides the background for epigenetic cloning of BPAD risk factors by searching for differentially modified genes on chromosome 18. Finally, epigenetic studies could be relevant to the better understanding of the molecular action of antipsychotic medications. In addition to this, if epimutations are detected in major psychosis, epigenetic treatment directed at correction of epigenetic status of a specific brain gene may eventually be developed.

The unstable trinucleotide repeat story of major psychosis.

New hopes for cloning susceptibility genes for schizophrenia and bipolar affective disorder followed the discovery of a novel type of DNA mutation, namely unstable DNA. One class of unstable DNA, trinucleotide repeat expansion, is the causal mutation in myotonic dystrophy, fragile X mental retardation, Huntington disease and a number of other rare Mendelian neurological disorders. This finding has led researchers in psychiatric genetics to search for unstable DNA sites as susceptibility factors for schizophrenia and bipolar affective disorder. Increased severity and decreased age at onset of disease in successive generations, known as genetic anticipation, was reported for undifferentiated psychiatric diseases and for myotonic dystrophy early in the twentieth century, but was initially dismissed as the consequence of ascertainment bias. Because unstable DNA was demonstrated to be a molecular substrate for genetic anticipation in the majority of trinucleotide repeat diseases including myotonic dystrophy, many recent studies looking for genetic anticipation have been performed for schizophrenia and bipolar affective disorder with surprisingly consistent positive results. These studies are reviewed, with particular emphasis placed on relevant sampling and statistical considerations, and concerns are raised regarding the interpretation of such studies. In parallel, molecular genetic investigations looking for evidence of trinucleotide repeat expansion in both schizophrenia and bipolar disorder are reviewed. Initial studies of genome-wide trinucleotide repeats using the repeat expansion detection technique suggested possible association of large CAG/CTG repeat tracts with schizophrenia and bipolar affective disorder. More recently, three loci have been identified that contain large, unstable CAG/CTG repeats that occur frequently in the population and seem to account for the majority of large products identified using the repeat expansion detection method. These repeats localize to an intron in transcription factor gene SEF2-1B at 18q21, a site named ERDA1 on 17q21 with no associated coding region, and the 3' end of a gene on 13q21, SCA8, that is believed to be responsible for a form of spinocerebellar ataxia. At present no strong evidence exists that large repeat alleles at either SEF2-1B or ERDA1 are involved in the etiology of schizophrenia or bipolar disorder. Preliminary evidence suggests that large repeat alleles at SCA8 that are non-penetrant for ataxia may be a susceptibility factor for major psychosis. A fourth, but much more infrequently unstable CAG/CTG repeat has been identified within the 5' untranslated region of the gene, MAB21L1, on 13q13. A fifth CAG/CTG repeat locus has been identified within the coding region of an ion transporter, KCNN3 (hSKCa3), on 1q21. Although neither large alleles nor instability have been observed at KCNN3, this repeat locus has been extensively analyzed in association and family studies of major psychosis, with conflicting findings. Studies of polyglutamine containing genes in major psychosis have also shown some intriguing results. These findings, reviewed here, suggest that, although a major role for unstable trinucleotides in psychosis is unlikely, involvement at a more modest level in a minority of cases cannot be excluded, and warrants further investigation.

Age of diagnosis-based linkage analysis in type 1 diabetes.

Genetic linkage studies of type 1 diabetes have produced a number of conflicting results, suggesting a high degree of locus heterogeneity in this disease. Approaches which model such heterogeneity will increase the power to fine map susceptibility loci. Here, using data from a genome scan of 356 affected sib pairs with type 1 diabetes, we performed heterogeneity analysis based on similarity of age at diagnosis of the sib pairs. We observed linkage to the region on chromosome 4p16.3 in sib pairs both diagnosed over the age of 10 years, whilst there was no evidence for linkage in sib pairs diagnosed before age 10 years. In contrast the sib pairs diagnosed before the age of 10 years demonstrated linkage to IDDM10, on chromosome 10p. Age of diagnosis-based heterogeneity analyses in complex diseases may be particularly helpful in mapping some susceptibility loci.

An unstable trinucleotide-repeat region on chromosome 13 implicated in spinocerebellar ataxia: a common expansion locus.

Larger CAG/CTG trinucleotide-repeat tracts in individuals affected with schizophrenia (SCZ) and bipolar affective disorder (BPAD) in comparison with control individuals have previously been reported, implying a possible etiological role for trinucleotide repeats in these diseases. Two unstable CAG/CTG repeats, SEF2-1B and ERDA1, have recently been cloned, and studies indicate that the majority of individuals with large repeats as detected by repeat-expansion detection (RED) have large repeat alleles at these loci. These repeats do not show association of large alleles with either BPAD or SCZ. Using RED, we have identified a BPAD individual with a very large CAG/CTG repeat that is not due to expansion at SEF2-1B or ERDA1. From this individual's DNA, we have cloned a highly polymorphic trinucleotide repeat consisting of (CTA)n (CTG)n, which is very long ( approximately 1,800 bp) in this patient. The repeat region localizes to chromosome 13q21, within 1.2 cM of fragile site FRA13C. Repeat alleles in our sample were unstable in 13 (5.6%) of 231 meioses. Large alleles (>100 repeats) were observed in 14 (1. 25%) of 1,120 patients with psychosis, borderline personality disorder, or juvenile-onset depression and in 5 (.7%) of 710 healthy controls. Very large alleles were also detected for Centre d'Etude Polymorphisme Humaine (CEPH) reference family 1334. This triplet expansion has recently been reported to be the cause of spinocerebellar ataxia type 8 (SCA8); however, none of our large alleles above the disease threshold occurred in individuals either affected by SCA or with known family history of SCA. The high frequency of large alleles at this locus is inconsistent with the much rarer occurrence of SCA8. Thus, it seems unlikely that expansion alone causes SCA8; other genetic mechanisms may be necessary to explain SCA8 etiology.

Age and sex based genetic locus heterogeneity in type 1 diabetes.

BACKGROUND: Two genome scans for susceptibility loci for type 1 diabetes using large collections of families have recently been reported. Apart from strong linkage in both studies of the HLA region on chromosome 6p, clear consistent evidence for linkage was not observed at any other loci. One possible explanation for this is a high degree of locus heterogeneity in type 1 diabetes, and we hypothesised that the sex of affected offspring, age of diagnosis, and parental origin of shared alleles may be the bases of heterogeneity at some loci. METHODS: Using data from a genome wide linkage study of 356 affected sib pairs with type 1 diabetes, we performed linkage analyses using parental origin of shared alleles in subgroups based on (1) sex of affected sibs and (2) age of diagnosis. RESULTS: Among the results obtained, we observed that evidence for linkage to IDDM4 on chromosome 11q13 occurred predominantly from opposite sex, rather than same sex sib pairs. At a locus on chromosome 4q, evidence for linkage was observed in sibs where one was diagnosed above the age of 10 years and the other diagnosed below 10 years of age. CONCLUSIONS: We show that heterogeneity tests based on age of diagnosis, sex of affected subject, and parental origin of shared alleles may be helpful in reducing locus heterogeneity in type 1 diabetes. If repeated in other samples, these findings may assist in the mapping of susceptibility loci for type 1 diabetes. Similar analyses can be recommended in other complex diseases.

Transmission ratio distortion in females on chromosome 10p11-p15.

A number of recent reports of linkage of markers on chromosome 10p to schizophrenia, and evidence for linkage in one study to bipolar affective disorder, provide encouragement for psychiatric genetics, after nonreplication of linkage findings at other chromosomal regions. The same region on chromosome 10 also demonstrates evidence for linkage to obesity, female alcoholism, and female type 1 diabetes. However, evidence for linkage can be confounded by the biological phenomenon of transmission ratio distortion. Transmission ratio distortion (also termed segregation distortion or meiotic drive) results in non-Mendelian segregation of alleles to live born offspring, and has not been investigated at the majority of loci for complex traits. We examined evidence for transmission ratio distortion using 40 Centre d'Etude du Polymorphisme Humain (CEPH) pedigrees across chromosome 10 using CEPH genotype data. Evidence for linkage of females to D10S211 was found (multipoint non-parametric linkage Z score [NPL] = 1.84, P = 0.040), while there was no linkage of this marker to male sex. The observation of possible transmission ratio distortion in females on chromosome 10p requires additional study, and may impact on the interpretation of positive linkage findings in this region. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 88:657-661, 1999.

Sex-based linkage analysis of alcoholism.

A high degree of locus heterogeneity is likely in alcoholism, and linkage heterogeneity analysis may be helpful in mapping susceptibility loci. The genetic contribution to alcoholism in females may be higher than in males, and therefore sex of affected individuals was used in linkage analysis. Families with female alcoholics demonstrated evidence for linkage to chromosomes 10p11-p15 and 21q22.1-q22.2 while those with male alcoholics did not provide evidence for linkage to these regions. Sharing of maternal and paternal alleles was also investigated separately, and evidence for linkage of maternal alleles on chromosomes 1 and 8, and paternal alleles on chromosome 2 was observed, suggesting parental origin effects. Mapping of complex traits may benefit from tests of linkage heterogeneity based on sex, and parental origin.

Haplotype analysis in the Collaborative Study on the Genetics of Alcoholism data: double recombinants.

The presence of close double recombinants in genotyping data may help identify genotyping errors. Alternatively, putative double recombinants may be associated with genetic mechanisms that may be related to disease. Phase-known apparent double recombination events were identified in the Collaborative Study on the Genetics of Alcoholism data, and compared to the sex-specific genetic map at each region. A number of double recombinants occurred within a short genetic distance. Also, in some families multiple double recombinants were observed flanking the same genetic marker, both suggesting possible genotyping error. An excess of paternal double recombinants was identified, which is consistent with reports of sex-specific differential meiotic interference.

IDDM9 and a locus for rheumatoid arthritis on chromosome 3q appear to be distinct.

Markers near a locus for type 1 diabetes on chromosome 3q22-q25 (IDDM9) demonstrate linkage to rheumatoid arthritis, however it is not clear whether these two loci overlap. Sex-specific linkage analysis may be of interest for rheumatoid arthritis on chromosome 3q since linkage of type 1 diabetes to IDDM9 derives predominantly from affected female sibpairs, and rheumatoid arthritis is more common in females than males. Using data from a recent genome scan for rheumatoid arthritis and sex-specific linkage analysis we show that linkage of rheumatoid arthritis to chromosome 3q peaks approximately 30 cM centromeric to IDDM9. Furthermore, there is no evidence for linkage to IDDM9 in females with rheumatoid arthritis.