Genomic View of Bipolar Disorder Revealed by Whole Genome Sequencing in a Genetic Isolate
[['Bipolar affective disorder is a life-long mental illness characterized by recurrent episodes of depression and mania or hypomania , with a typical age of onset in young adulthood .', 'Twin and family studies have shown that bipolar disorder has a strong genetic component with heritability estimated to be in the range of 80% [1] .', 'As such , families with bipolar disorder have been extensively studied by linkage analysis [2] and a considerable number of possible susceptibility loci have been reported .', 'The largest linkage study to date on 972 unrelated families identified loci on chromosomes 6q21 and 9q21 [3] .', 'In addition , genome-wide association studies ( GWAS ) using large cohorts of patients and control subjects have identified single nucleotide polymorphisms ( SNPs ) in CACNA1C ( alpha 1C subunit of the L-type voltage-gated calcium channel ) and ANK3 ( ankyrin 3 ) implicating these as potential susceptibility genes for bipolar disorder [4]–[6] .', 'A recent meta-analysis of GWA data from over 13 , 600 individuals identified a risk locus for major mood disorders at 3p21 . 1 [7] .', 'A large-scale international GWAS effort replicated the association for CACNA1C and identified new susceptibility alleles in ODZ4 [8] .', 'Although genome-wide association studies remain an important approach for identifying common variants conferring risk for common psychiatric disorders , each individual locus or allele identified to date explains only a very small proportion of familial clustering and relatively small changes in risk [9]–[11] .', 'Genetic studies of isolated populations have led to the identification of genetic variants underlying a number of Mendelian disorders , particularly those caused by autosomal recessive mutations [12] , [13] .', 'More recently , homozygosity mapping of rare recessive mutations in consanguineous families has identified mutations and copy number variants ( CNVs ) in neurodevelopmental disorders , such as autism and hereditary sensory and autonomic neuropathy type IV [14] , [15] .', 'Genetically isolated populations , characterized by reduced genetic , phenotypic and environmental heterogeneity , serve as a powerful model for studies of allelic diversity associated with common neurodevelopmental and psychiatric disorders .', 'The Old Order Amish are a genetic isolate of European ancestry currently residing in several states in North America , with a concentration in Pennsylvania and Ohio [16] .', 'Bipolar disorder type I ( BPI ) and bipolar disorder type II ( BPII ) in the Amish occur with similar prevalence , pattern of symptoms , clinical course and response to mood-stabilizing medicines as observed in the general North American population [17]–[19] .', 'Alcohol and drug abuse , which often complicate psychiatric diagnoses , are rare among the Amish .', 'Their lifestyle provides a remarkably uniform environment in which behavioral changes can be readily and longitudinally ascertained .', 'The original genetic linkage study of a large extended Old Order Amish pedigree with bipolar affective disorder reported positive findings with DNA markers around the HRAS1 and INS loci on chromosome 11 [20] .', 'When the original Amish pedigree was further extended and updated clinical data were incorporated , linkage at the chromosome 11 locus was excluded [21] .', 'An early genome-wide linkage scan using 551 microsatellite markers revealed possible bipolar susceptibility loci on chromosomes 6 , 13 , and 15 , suggesting that bipolar disorder , even in a genetic isolate , is likely inherited as a complex trait [22] .', 'To identify the genetic basis of bipolar disorder in the Amish , our research returned to the expanded multigenerational Old Order Amish pedigree available as the Amish Study of Major Affective Disorders at the Coriell Institute for Medical Research and applied contemporary genomic and statistical methodology by integrating genotype and whole-genome sequence data .', 'This investigation combines linkage analyses on multiallelic microsatellite genotypes for the large pedigree ( n\u200a=\u200a497 ) with the analysis of Illumina Omni 2 . 5M SNP genotypes ( n\u200a=\u200a388 ) and whole-genome sequences of 50 family members .', 'Our findings reveal multiple linkage regions that each harbor a considerable number of sequence variants , supporting initially reported locus heterogeneity .', 'Dissection of exonic and intronic variants that reside in these linkage peaks has identified credible candidate genes that will be further examined in large-scale population-based studies .', 'Our results underscore the complexity of the genetic etiology of bipolar disorder in this genetic isolate and have important implications for the study of bipolar disorder in the general population .'], ['The Amish Study of Major Affective Disorders includes biomaterials and clinical data for a large extended family ( 497 family members ) ( Figures 1A and 1B ) .', 'The Amish Study Psychiatric Board ( see Methods ) established clinical diagnoses for each family member using Research Diagnostic Criteria ( RDC ) [23] and DSM-III/IV criteria [24] .', 'We obtained:', 'a ) genotypes from a panel of 1991 multi-allelic microsatellite markers ( deCODE panel ) for the entire pedigree ( 497 individuals ) and', 'b ) high-density SNP genotypes ( Illumina Omni 2 . 5 M SNP arrays ) for a subset of 388 individuals .', 'Copy number variants ( CNVs ) were called using the PennCNV software [25] .', 'To establish an initial catalogue of potential sequence variants underlying bipolar disorder , we also obtained whole genome sequences ( WGS ) for 50 individuals comprising 18 parent-child trios and 8 additional parent-child pairs from the extended pedigree ( Figure S1 ) .', 'We selected individuals for whole genome sequencing that included', 'a ) the most distantly related subfamilies;', 'b ) affected individuals ( n\u200a=\u200a23 ) diagnosed with BPI or BPII and', 'c ) a small subset of healthy siblings ( ages between 48 and 78 , well past the age of disease onset , which is usually prior to age 35 ) .', 'Whole genome sequencing was performed by Complete Genomics Inc . ( CGI; Mountain View , CA ) using a sequence-by-ligation method [26] .', 'Median sequencing depth was ∼50× across all 50 samples ( Figure S2 ) .', 'By combining genotype data for the entire extended pedigree with whole genome sequences for a selected set of parent-child trios across different sub-pedigrees , we were able to infer the genetic background for the entire pedigree and address the impact of both common and rare variants within this large multigenerational family .', 'In order to address genetic and phenotypic heterogeneity , analysis was restricted to 49 nuclear families with at least one BPI subject .', 'Among the 364 selected subjects were 92 individuals with Major Affective Disorder including bipolar disorder I ( BPI ) ( n\u200a=\u200a62 ) , bipolar disorder II ( BPII ) ( n\u200a=\u200a15 ) and Major Depressive Disorder , recurrent form ( MDD-R ) ( n\u200a=\u200a15 ) ; 52 individuals with minor psychiatric diagnoses ( classified as “unknown” phenotype ) and 220 “well” subjects .', 'Three diagnostic hierarchies were defined for the analysis: the first and most stringent classification included only BPI individuals as affected ( BPI phenotype ) , the second included BPI and BPII individuals as affected ( narrow phenotype ) and the third included BPI , BPII and MDD-R as affected ( bipolar spectrum disorder or BPS ) .', 'To quantify the fraction of phenotypic variance accounted for by genetic causes , we estimated the narrow sense heritability ( h2 ) of bipolar susceptibility in the Amish pedigree using the Sequential Oligogenic Linkage Analysis Routines ( SOLAR ) [27] software .', 'We observed significant heritability for all three phenotype definitions ( BPI , narrow , BPS ) .', 'The most heritable phenotype was BPS with a heritability of 0 . 626 ( P\u200a=\u200a0 . 0000015 ) ( Table 1 ) .', 'To quantify the overall degree of relatedness of individuals within the extended pedigree and to provide quality control of our genotype data , pair-wise kinship coefficients were calculated based on the SNP array genotypes using the KING software [28] ( Figure S3 ) .', 'Observed ranges of kinship coefficients for known family relationships matched well with expectations and coefficients obtained from an independent dataset of 569 unrelated parent pairs ( Figure 1C ) .', 'To estimate the overall level of consanguinity and identify the most consanguineous nuclear families , we investigated relatedness between 32 pairs of parents .', 'While the overall relatedness of parent pairs in the pedigree is higher than for the control parent pairs , the bulk of the distribution falls into the range of kinship expected for unrelated individuals ( kinship coefficients <0 . 0442 ) .', 'A single , four-member family showed a kinship coefficient of 0 . 0453 between the parents , which falls into the expected range for third degree relatives .', 'The overall low kinship coefficient estimates between pairs of parents suggests that inbreeding among close relatives is uncommon in this pedigree , and it is unlikely to bias subsequent analyses .', 'The sequencing of 50 Amish genomes identified 11 . 1 million sequence variants within the 97% called genome fraction .', 'From the ∼3 . 79 million SNPs and indels detected in each individual , 20 , 000 fell within exons , with an average of 10 , 000 synonymous SNPs and roughly the same number of non-synonymous , frame shift or gain/loss of a stop-codon variants .', 'The overall rate of novel SNP variants per sample is ∼1 . 3% with respect to dbSNP 138 .', 'We initially searched for exonic missense variants common in the 23 individuals with BPI/II and rare ( MAF<2% ) in the 1000 Genomes Project data , the EVS database and a set of 54 HapMap WGS made publicly available by CGI .', 'Of the resulting list of 514 variants predicted to be “damaging/deleterious” by both Polyphen2 [29] and SIFT [30] none were both present in all BP I/II samples and absent or rare in 1000 Genomes data .', 'The same was true when we prioritized exonic variants based on the degree of nucleotide-level evolutionary conservation using Genomic Evolutionary Rate Profiling ( GERP ) conservation scores ( [31]; see Table S1 for the 50 most constrained variants ) .', 'This gives further support to previously reported genetic analysis [22] , which revealed a complex mode of inheritance and genetic heterogeneity of bipolar disorder in this large multigenerational family .', 'Table 2 shows the top 30 putatively damaging missense SNPs enriched among this Amish pedigree and rare in the general population ( see Table S2 for the full list of 514 variants ) .', 'To explore the role of common variants in bipolar susceptibility and to prioritize variants under linkage peaks ( see below ) , we performed family based genome-wide association analysis on 388 samples genotyped with Illumina Omni 2 . 5 Million SNP arrays .', 'We applied two methods for association analysis:', '( a ) EMMAX [32] , a mixed model association method accounting for population structure , including pairwise relatedness and', '( b ) FBAT [33] , an extension of the classical transmission distortion test ( TDT ) [34] to larger families .', 'These two methods are complementary , as EMMAX quantifies differences in allele frequency in affected and unaffected subjects while FBAT considers deviations from random segregation of alleles in affected and unaffected children .', 'After conducting quality control of the SNP genotype data ( see Methods ) , 1 . 3M SNPs were retained for association analysis ( see Figure S4 for quantile-quantile plots ) .', 'We did not observe genome-wide significant association ( p<5e-8 ) with either method ( Figure S5 ) .', 'We hypothesized that meaningful associations may be found among SNPs that showed the lowest P values ( p<\u200a=\u200a5e-4 ) using both methods ( Table S3 ) .', 'The two SNPs with the strongest association ( p<5e-5 for both algorithms ) were rs13317247 ( FBAT p\u200a=\u200a1 . 8e-5 , EMMAX p\u200a=\u200a6 . 5e-6 ) in the intron of the metabotropic glutamate receptor 7 ( GRM7 ) gene on chromosome 3 ( Figure S6 top ) and rs13207753 ( FBAT p\u200a=\u200a3 . 4e-5 , EMMAX p\u200a=\u200a4 . 23e-5 ) in the intergenic region between Tubulin-tyrosine ligase-like protein 2 ( TTLL2 ) and t-complex 10 homolog ( TCP10 ) on chromosome 6 ( Figure S6 bottom ) .', 'The association signal for either SNP did not replicate in a previously published case-control study of 2 , 836 BP and 2 , 744 controls ( GAIN [35] ) .', 'However , it is noteworthy that there was a suggestive association signal located at rs58674863 ( p\u200a=\u200a3 . 3e-4 ) within the same intron of GRM7 , 22 . 3 Kb distal from rs13207753 ( Figure S7 ) .', 'Recent large-scale genome-wide association studies have implicated a number of loci with susceptibility to bipolar disorder and schizophrenia [8] , [36] and we interrogated each of these regions in the Amish pedigree .', 'Thus , we retrieved 38 SNP association hits from a recent large-scale GWAS on BP [8] ( Table S2 therein ) and 24 candidate regions implicated in schizophrenia [36] ( Table 3 therein ) .', 'To convert the single SNP associations into candidate regions we applied an offset of 50 kb up- and downstream of each SNP .', 'We identified 7 , 571 SNPs within 62 distinct candidate regions .', 'To address the question of whether variation in these candidate regions contributes to BP susceptibility , we performed family-based burden tests using the rareFBAT test [37] .', 'The rareFBAT test allows combined testing of the additive effects of defined sets of variants , in this case the variants within the 62 candidate regions .', 'No significant nor suggestive ( P<5×10−4 ) association was observed ( Table S4 ) , implying that these previously reported regions do not account for a major portion of the genetic risk in this pedigree .', 'Nevertheless , the whole-genome sequencing identified a number of rare and putative damaging variants in ANK3 , ODZ2 , ODZ4 and ITIH3 genes , located within or surrounding significant genome-wide association hits for bipolar disorder ( Table S5 ) .', 'To understand the role of rare variants in susceptibility to affective disorders in this pedigree , we combined linkage , association and whole genome sequence analysis ( Figure 2 ) .', 'As expected from the results of previous studies , our results thus far suggested a polygenic mode of inheritance of BP within the Amish pedigree .', 'In contrast to association , linkage analysis aggregates the effects of multiple , rare causal variants if they are in genomic proximity .', 'Thus we performed linkage analysis to detect chromosomal regions that are likely to harbor susceptibility variants .', 'The linkage analysis identified candidate genomic regions and also the subset of nuclear families supporting each linkage signal ( from this point referred to as “linked families” ) .', 'Further analysis of these families identified the haplotypes with maximum allele sharing in affected siblings [38] .', 'Based on phasing and imputation of variants identified in the WGS , we then characterized each haplotype in terms of sequence variants present .', 'As a final step , we examined association signals ( from GWAS data generated using FBAT and EMMAX , see above ) and carried-out family-based burden association analysis ( using the rareFBAT test ) on genes under the linkage peaks .', 'We performed linkage analysis of the 1991 microsatellite markers for 364 individuals in the extended pedigree and 7 sub-pedigrees with nuclear families with high quality imputation ( see Methods ) .', 'Parametric and non-parametric linkage analyses of the microsatellite genotypes were performed using the MERLIN software [39] .', 'The parametric linkage analysis , using the multipoint LOD and heterogeneity LOD scores ( HLOD ) [40] , [41] was performed for dominant and recessive models .', 'Standard linear and exponential LOD scores were used for non-parametric linkage ( NPL ) analysis .', 'NPL is based on allele sharing , i . e . identity-by-descent between affected siblings at a given chromosome position .', 'Highly comparable linkage results were obtained with microsatellite genotypes and 10K SNP genotypes , with slightly higher LOD score values in microsatellite data due to the larger sample size ( 484 versus 388 genotyped samples ) .', 'Linkage analysis of all 49 nuclear families and the sub-pedigrees detected suggestive linkage ( LOD>2 . 2 ) on six and eleven chromosomes respectively ( Tables S6A and S6B ) .', 'Within the full set of 49 nuclear families the highest linkage we observed was on 7q21 ( LOD 2 . 99 at D7S518 , BPS phenotype ) and 18p11 ( expLOD 2 . 76 at D18S453 , BPI phenotype ) .', 'The analysis of subpedigrees yielded nominally significant linkage peaks on 2p25 ( expLOD 3 . 01 at D2S2211 , Narrow phenotype ) and 4p16 . 3 ( HLOD 3 . 95 at D4S3360 , Narrow phenotype ) observed within subpedigree 310/410 as well as 16p13 ( expLOD 3 . 15 at D16S3127 ) within sub-pedigree 310 ( Figures 3A and 4 ) .', 'Several of the detected peaks were reported in previous linkage studies of bipolar disorder .', 'The 7q21 region showed significant evidence for linkage ( P\u200a=\u200a0 . 0003 ) in a joint meta-analysis of data from 11 BP and 18 schizophrenia studies [42] .', 'Interestingly , the authors note that the signal at the locus “appears to be due to the inclusion of results for the broad susceptibility models such as those including unipolar depression” .', 'This is consistent with the strongest signal being observed under the BPS phenotype in our data .', 'Moreover , the 7q21 . 3-q22 . 3 region has been reported to yield suggestive linkage ( NPL LOD\u200a=\u200a2 . 28 ) for a data set of Ashkenazi Jews and Belgian Europeans [43] .', 'The 18p11 . 22 region is one among the most frequently reported bipolar disorder linkage peaks .', 'Linkage in the 18p11 region was first reported based on affected sibling analysis of 22 BP families [44] and subsequently confirmed by a study of 28 families with apparent unilineal transmission of BP [45] .', 'Also , a study of the effect of age-of-onset by ordered subset analysis found significant linkage at the 18p11 . 2 locus [46] .', 'In addition to these findings , suggestive linkage was reported in a non-parametric linkage analysis of a data set of 22 Caucasian families [47] and in an association study of 363 parent-child trios of Caucasian descent [48] .', 'Finally , the microsatellite marker with the highest linkage signal on chromosome 18 detected in our study ( D18S453 ) maps less than 1 Mb distal to the translocation breakpoint [t ( 18;21 ) ( p11 . 1;p11 . 1 ) ] segregating with schizophrenia in a seven member family [49] .', 'A number of groups reported evidence for linkage for bipolar and other psychiatric disorders in the 4p15 . 2–4p16 . 3 region ( see references in [47] , [50]–[53] ) , including a genome-wide linkage study of mental health wellness in the Old Order Amish which found significant evidence for linkage on chromosome 4p , about 5 Mb distal of the peak we detected [53] .', 'Importantly , the analysis of identity-by-state sharing under each linkage peak identified considerable haplotype heterogeneity , with multiple contributing haplotypes across the linked nuclear families at each peak ( Table S7 ) .', 'The notable exception to this was the three-marker haplotype on 4p16 . 3 ( D4S3360-D4S2936-D4S412 ) covering the highest observed linkage peak ( HLOD 3 . 96 ) .', 'Here among the four contributing haplotypes ( 4-5-5 , 5-4-6 , 5-3-2 , 4-2-2 at above listed microsatellite loci ) , a single haplotype , 5-3-2 , was shared by 3 out of 4 linked nuclear families .', 'The 5-3-2 haplotype was present in 9/10 affected individuals within the contributing families .', 'There was significant haplotype association of the 5-3-2 haplotype ( FBAT p\u200a=\u200a0 . 0267 ) as well as for the global omnibus test ( FBAT global p\u200a=\u200a0 . 0291 ) in affected individuals within linked families ( Table S8 , Figure 3B ) .', 'Whole-genome sequencing identified multiple non-synonymous variants under each peak ( Table 3 ) .', 'Many of these exonic , putative damaging variants ( by consensus of Polyphen2 and SIFT ) are common ( MAF>5% ) in European populations based on 1000 Genomes and the EVS data .', 'Such variants are found at 2p25 ( in the SNTG2 and TTC15 genes ) and 4p16 . 3 ( in POLN and EVC genes ) .', 'However a significant number of variants under the linkage peaks are either novel or rare ( e . g . LAMB1 , MKL2 , ZNF732 , ZAN and ANKS4B ) or moderately frequent ( e . g ERCC4 and MLL5 ) .', 'Using the current level of resolution , it is difficult to evaluate whether these variants contribute to nominal association signals within the five regions detected by the analysis of SNP array data using EMMAX and FBAT ( Figure S8 ) .', 'We also analyzed sequence variation on linkage-driving haplotypes by focusing on exonic missense variants and SNPs associated with the expression level of genes under linkage peaks ( ‘eSNPs’ see Methods ) [54] .', 'To test for association in the presence of allelic heterogeneity we then performed rareFBAT burden tests for all genes under the five linkage peaks with either exonic missense or regulatory eSNP variants ( Figure S8 ) .', 'While the included variants were selected based on their presence on linkage-driving haplotypes , the tests were performed on data from the entire pedigree , i . e . both linked and unlinked families .', 'The standard Null hypothesis of the rareFBAT test is “no linkage and no association” .', 'Since there was a subset of linked families at each linkage peak , this Null might lead to slightly liberal p-values .', 'To account for this we also report p-values for testing the Null hypothesis of “linkage and no association” , which can be seen as a conservative correction as the majority of families are unlinked ( see Methods ) .', 'Forty-two ( 42 ) of the 543 genes under the five linkage peaks were nominally associated ( P<\u200a=\u200a0 . 05; Figure S8; Tables S9 , S10 , S11 , S12 , S13 , S14 ) .', 'However , significance was not retained after correction for multiple testing by False Discovery Rate ( FDR ) [55] .', 'We performed Ingenuity Pathway Analysis ( IPA ) of nominally significant genes but observed no strong enrichment for a specific pathway , biological function or tissue-specific gene expression ( see Figure S9 for an example IPA analysis of the top 100 genes on Table S2 ) .', 'Under the 7q21 peak , the most strongly associated genes were the tectonin beta-propeller repeat containing 1 ( TECPR1 , P\u200a=\u200a2 . 8×10−5 ) and the neighboring distal-less homeobox 5 and 6 genes ( DLX5 and DLX6 ) ( P\u200a=\u200a0 . 008 and P\u200a=\u200a0 . 003 respectively ) .', 'The most strongly associated gene under the 16p13 peak was the ATP-binding cassette , sub-family C , member 6 ( ABCC6 ) gene ( P\u200a=\u200a0 . 00055 ) .', 'The 2p25 region harbors the integrin beta 1 binding protein 1 ( ITGB1BP1 ) gene , which is involved in cell adhesion .', 'The class of cell adhesion molecules has been implicated to play a role in bipolar disorder in multiple gene set enrichment analyses studies [56] , [57] .', 'Thus , while our results do not allow the unequivocal identification of a causal gene under the identified linkage peaks , the gene-wise burden tests analysis yields a number of plausible candidate genes ( Table S9 ) .'], ['We employed several strategies to comprehensively delineate the role of both common and rare variants as susceptibility alleles for bipolar affective disorder in a large extended pedigree with a high incidence of the disorder .', 'The role of common variants was initially explored using family-based genome-wide association analysis of high-density SNP genotypes for the entire family .', 'To address the role of rare variants we employed linkage analysis to prioritize sequence variants from WGS on specific haplotypes enriched in affected subjects .', "A combination of WGS with dense SNP genotypes that allow long-range haplotype phasing has been successfully utilized to identify variants that influence risk for sick sinus syndrome , gout , gliomas , ovarian cancer and Alzheimer's disease [58]–[62] .", 'However , it appears this has not yet been attempted for a genetically and phenotypically complex mental disorder .', 'Our extensive analysis of a single exceptionally large family with affective disorder revealed an unanticipated level of genetic complexity with no convergence of evidence in support for a limited number of loci conferring disease risk in this family .', 'Our linkage analysis identified several nominally significant peaks in different sub-pedigrees under different disease models .', 'These results are consistent with a polygenic mode of inheritance contributing to the disease susceptibility and phenotypic presentation .', 'Polygenic inheritance usually implies that interacting risk factors in several genes across the genome lead to disease susceptibility .', 'Our fine mapping of imputed variants in regions with the strongest linkage and association signals did not reveal a single SNP ( or haplotype ) but rather several non-coding and exonic SNPs .', 'Due to the long haplotype blocks observed in founder populations , a linkage signal may reflect a combined effect of multiple causal alleles within the same chromosomal region as commonly described in the dissection of behavioral traits in model organisms [63]–[65] .', 'The combined high-density SNP genotyping , microsatellite genotyping , and whole-genome sequencing are expected to capture nearly all of the genetic variants co-segregating with the BPI phenotype .', 'The lack of any single variant or haplotype exhibiting a significant linkage signal under both parametric and non-parametric analysis suggests that the BP1 disorder within this pedigree is likely to be genetically heterogeneous and caused by aggregation of different moderately frequent alleles in different sub-pedigrees .', 'Using WGS we identified a number of non-synonymous , likely deleterious variants that are rare in the 1000 Genomes Project dataset ( 2% ) but present in 10–30% of the BP subjects and their first-degree relatives .', 'Such differences in allele frequency , resulting from a population bottleneck and expansion from a relatively small number of founders , have been previously described in several isolated populations ( see [66] , [67] for reviews ) .', 'Understanding the phenotypic consequences of these Amish-enriched variants will be important not only in the context of their potential contribution as risk factors ( or modifiers ) for mental illness but also for other , yet undiagnosed disorders segregating in the Amish [68] .', 'For example , the variant rs113270504 , which leads to a Val540Met substitution in the G Protein-Coupled Receptor 124 ( GPR124 ) , is rare in non-Amish populations ( <2% in 1000 Genomes and EVS ) .', 'The corresponding gene is essential for angiogenesis in the central nervous system and the development of the blood-brain barrier [69] , [70] .', 'Another variant , rs78247304 in KCNH7 ( Arg394His ) , potassium voltage-gated channel , subfamily H , member 7 , is of considerable interest .', 'Voltage-gated ion channels have previously been implicated in a number of psychiatric disorders , including bipolar disorder , due to their known involvement in neuronal excitability and synaptic transmission [71] .', 'Association at the KCNH7 locus with bipolar disorder was also reported at rs6736615 , an intronic variant , in a recent case-control study of 400 Taiwanese subjects [72] .', 'Moreover , an exome-sequencing study of a small number of Amish subjects with mental illness identified rs78247304 as a likely risk variant and showed that the Arg394His substitution leads to altered voltage dependence and activation kinetics of the channel in patch clamp experiments [73] .', 'In addition , we have identified three genes ( ADAM18 , INSL6 , IFT81 ) that play roles in spermatogenesis and fertilization .', 'To determine whether mutations in these genes may underlie bipolar disorder and/or other complex traits , it will be necessary to explore additional Amish families with mental illness and comorbid medical conditions .', 'Our analysis of previously published genome-wide significant GWAS loci did not show any convergence with the results from our family-based GWAS .', 'Recently , two publications employed whole exome sequencing to identify rare variants associated with bipolar disorder using a family-based design [74] , [75] .', 'The analysis of a single family with three affected daughters and an unaffected brother identified variants that segregate with bipolar disorder ( and are not present in 200 controls ) in eight brain expressed genes [74] .', 'We examined genetic variation in these genes in our data and identified several low frequency , potentially damaging variants in JMJD1C , also known as thyroid hormone receptor beta-biding protein8 ( TRIP8 ) , involved in hormone-dependant transcriptional regulation .', 'Among several genes identified in the second publication [75] , the most striking are variants in Odd oz 2 , ODZ2 ( also known as TEN-M2 ) .', 'A rare variant in this gene and in a paralogous gene ODZ4 , with an intronic SNP ( rs12576775 ) associated with bipolar disorder at a genome-wide level [8] , segregate in this large Amish pedigree , but again do not reside in regions with the nominal association or linkage signal ( Table S5 ) .', 'Isolated founder populations provide a unique opportunity to evaluate the genetic architecture of human disease [67] .', 'Family-based linkage scans provide initial insights into the number of disease conferring loci , while association studies can define the role of common alleles or alleles that are rare in other populations , but moderately frequent in the isolate .', 'The lack of statistical power observed in our study may be due to the modest number of family members and ( or ) a small effect size of risk-alleles .', 'Therefore , to establish a causal link between specific variants and disease risk will require more in-depth analysis in additional families and population-based cohorts in the same or related Amish communities .', 'Moreover , to address the high level of genetic heterogeneity , it will be necessary to phenotypically dissect the clinical diagnoses and explore subtle differences in manifestation or medical comorbidities between subpedigrees [76] .', 'The Iceland-based deCODE study highlights the value of combining genomic characterization of a genetic isolate with the comprehensive phenotypic data to enable identification of variants associated with several human diseases and other traits [58] , [77] , [78] .', 'Recently a systematic framework for studying the genetic architecture of complex traits has been proposed [79] .', 'By simulating data under a range of disease models , the method determines which genetic architectures are consistent with the empirical data .', 'For example , the study shows that while the number of known risk loci for type 2 diabetes is consistent with a range of disease architectures , disease models with extremely low or high values for the true number of risk loci can be ruled out .', 'Performing a similar analysis of bipolar disorder would require the integration of linkage , GWAS and , crucially , several of the upcoming next-generation sequencing studies .', 'Leveraging family-based design with the population-based studies of BP-associated quantitative traits in a founder population will be critical for effective gene and human-genetics driven drug discovery .', 'Although candidates detected by our comprehensive approach were not significant after the correction for multiple testing , we provide supportive evidence and a biological rational for further investigating these variants in large cohorts , especially when more genome and exome data become available .'], ['The genetic-epidemiologic study of bipolar disorder among the Old Order Amish , in settlements throughout Pennsylvania , was documented extensively , including ascertainment protocols and diagnostic methods [17] , [18] , [80] .', 'Ascertainment of patient cases used a “scribe network” with assigned codes to ensure patient confidentiality .', 'Structured interviews ( SADS-L ) [81] , [82] were conducted with the patient and close others .', 'Signed , informed consents were obtained to access medical records .', 'Two forms were used:
['Introduction', 'Results', 'Discussion', 'Methods']
['Bipolar disorder is a common , heritable mental illness characterized by recurrent episodes of mania and depression .', 'Despite considerable effort to elucidate the genetic underpinnings of bipolar disorder , causative genetic risk factors remain elusive .', 'We conducted a comprehensive genomic analysis of bipolar disorder in a large Old Order Amish pedigree .', 'Microsatellite genotypes and high-density SNP-array genotypes of 388 family members were combined with whole genome sequence data for 50 of these subjects , comprising 18 parent-child trios .', 'This study design permitted evaluation of candidate variants within the context of haplotype structure by resolving the phase in sequenced parent-child trios and by imputation of variants into multiple unsequenced siblings .', 'Non-parametric and parametric linkage analysis of the entire pedigree as well as on smaller clusters of families identified several nominally significant linkage peaks , each of which included dozens of predicted deleterious variants .', 'Close inspection of exonic and regulatory variants in genes under the linkage peaks using family-based association tests revealed additional credible candidate genes for functional studies and further replication in population-based cohorts .', 'However , despite the in-depth genomic characterization of this unique , large and multigenerational pedigree from a genetic isolate , there was no convergence of evidence implicating a particular set of risk loci or common pathways .', 'The striking haplotype and locus heterogeneity we observed has profound implications for the design of studies of bipolar and other related disorders .']
['Bipolar disorder is a common , heritable mental illness characterized by recurrent episodes of mania and depression .', 'Despite considerable efforts genetic studies have yet to reveal the precise genetic underpinnings of the disorder .', 'In this study we have analyzed a large extended pedigree of Old Order Amish that segregates bipolar disorder .', 'Our study design integrates both dense genotype and whole-genome sequence data .', 'In a combined linkage and association analysis we identify five chromosomal regions with nominally significant or suggestive evidence for linkage , several of which constitute replication of earlier linkage findings for bipolar disorder in non-Amish families .', 'Association analysis of genetic variants in each of the linkage regions yielded a number of plausible candidate genes for bipolar disorder .', 'The striking genetic heterogeneity we observed in this genetic isolate has profound implications for the study of bipolar disorder in the general population .']
['genome-wide association studies', 'genome sequencing', 'medicine', 'genome analysis tools', 'genomics', 'genetic mutation', 'mental health', 'linkage maps', 'genome scans', 'genetic association studies', 'genetics', 'psychiatry', 'biology', 'human genetics', 'genetics of disease', 'mood disorders', 'clinical genetics']