DISC1 exon 11 rare variants found more commonly in schizoaffective spectrum cases than controls

We previously performed a linkage study using families identified through probands meeting criteria for DSM‐IV schizoaffective disorder, bipolar type (SABP) and observed a genome‐wide significant signal (LOD = 3.54) at chromosome 1q42 close to DISC1. An initial sequencing study of DISC1 using 14 unrelated DSM‐IV SABP samples from the linkage study identified 2 non‐synonymous coding SNPs in exon 11 in 2 separate individuals. Here we provide evidence of additional rare coding SNPs within exon 11. In sequencing exon 11 in 506 cases and 1,211 controls for variants that occurred only once, 4 additional rare variants were found in cases (P‐value = 0.008, Fisher's exact trend test). © 2011 Wiley‐Liss, Inc.     

Association analysis of the DISC1 gene with schizophrenia in the Japanese population and DISC1 immunoreactivity in the postmortem brain

The Disrupted-in-Schizophrenia 1 (DISC1) gene plays a role in the regulation of neural development. Previous evidence from genetic association and biological studies implicates the DISC1 gene as having a role in the pathophysiology of schizophrenia. In the present study, we explored the association between DISC1 missense mutation rs821616 (Ser704Cys) single nucleotide polymorphism (SNP) and four other SNPs (rs1772702, rs1754603, rs821621, rs821624) in the related haplotype block and schizophrenia in the Japanese population. We could not find a significant association of selected SNPs with schizophrenia after correction for multiple testing. We performed a meta-analysis of the Ser704Cys variant in schizophrenia using data from the present study and five previous Japanese population studies, and found no association with schizophrenia. We also examined DISC1 immunoreactivity in postmortem prefrontal cortex specimens of schizophrenia patients compared to control samples. The immunoreactivity revealed a significant decrease of DISC1 protein expression in the schizophrenia samples after ruling out potential confounding factors. However, the Ser704Cys variant did not show effects on DISC1 immunoreactivity. These results provide evidence that this functional genetic variation of DISC1 do not underlie the pathophysiology of schizophrenia in the Japanese population.     

Investigating the genetic association of HCP5, SPATA2, TNIP1, TNFAIP3 and COG6 with psoriasis in Chinese population

Psoriasis is a chronic inflammatory skin disease occurred under the interaction of genetic and environmental factors. The genes HLA complex P5 (HCP5), spermatogenesis associated 2 (SPATA2), tumour necrosis factor alpha‐induced protein 3 (TNFAIP3), TNFAIP3‐interacting protein 1 (TNIP1) and the component of oligomeric Golgi complex 6 (COG6) were reported to be associated with psoriasis in western populations by genome‐wide association studies. The aim of this study was to investigate whether the HCP5, TNIP1, TNFAIP3, SPATA2 and COG6 genes were genetic risk factors for psoriasis in Chinese population. One single nucleotide polymorphism (SNP) from each gene was evaluated using Chinese patients with psoriasis (n = 201) and controls (n = 300). The results demonstrated that SNPs rs2395029, rs17728338 and rs610604 from the HCP5, TNIP1 and TNFAIP3 genes, respectively, were associated with psoriasis in the studied population at both genotype level and allelic level (P < 0.05). Thus, the data suggested that HCP5, TNIP1 and TNFAIP3 may play a role in common pathogenic of psoriasis in Chinese and confer risk factors for psoriasis in various ethnic populations. These results provide potential makers for diagnosing, treating and preventing the psoriasis.     

Association study of Nogo‐related genes with schizophrenia in a Japanese case–control sample

Many studies have suggested that myelin dysfunction may be causally involved in the pathogenesis of schizophrenia. Nogo (RTN4), myelin‐associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMG) all bind to the common receptor, Nogo‐66 receptor 1 (RTN4R). We examined 68 single nucleotide polymorphisms (SNPs) (51 with genotyping and 17 with imputation analysis) from these four genes for genetic association with schizophrenia, using a 2,120 case–control sample from the Japanese population. Allelic tests showed nominally significant association of two RTN4 SNPs (P = 0.047 and 0.037 for rs11894868 and rs2968804, respectively) and two MAG SNPs (P = 0.034 and 0.029 for rs7249617 and rs16970218, respectively) with schizophrenia. The MAG SNP rs7249617 also showed nominal significance in a genotypic test (P = 0.017). In haplotype analysis, the MAG haplotype block including rs7249617 and rs16970218 showed nominal significance (P = 0.008). These associations did not remain significant after correction for multiple testing, possibly due to their small genetic effect. In the imputation analysis of RTN4, the untyped SNP rs2972090 showed nominally significant association (P = 0.032) and several imputed SNPs showed marginal associations. Moreover, in silico analysis (PolyPhen) of a missense variant (rs11677099: Asp357Val), which is in strong linkage disequilibrium with rs11894868, predicted a deleterious effect on Nogo protein function. Despite a failure to detect robust associations in this Japanese cohort, our nominally positive signals, taken together with previously reported biological and genetic findings, add further support to the “disturbed myelin system theory of schizophrenia” across different populations. © 2011 Wiley‐Liss, Inc.     

The association of telomere length and genetic variation in telomere biology genesa

Telomeres cap chromosome ends and are critical for genomic stability. Many telomere‐associated proteins are important for telomere length maintenance. Recent genome‐wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) in genes encoding telomere‐associated proteins (RTEL1 and TERT‐CLPTM1) as markers of cancer risk. We conducted an association study of telomere length and 743 SNPs in 43 telomere biology genes. Telomere length in peripheral blood DNA was determined by Q‐PCR in 3,646 participants from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial and Nurses' Health Study. We investigated associations by SNP, gene, and pathway (functional group). We found no associations between telomere length and SNPs in TERT‐CLPTM1L or RTEL1. Telomere length was not significantly associated with specific functional groups. Thirteen SNPs from four genes (MEN1, MRE11A, RECQL5, and TNKS) were significantly associated with telomere length. The strongest findings were in MEN1 (gene‐based P=0.006), menin, which associates with the telomerase promoter and may negatively regulate telomerase. This large association study did not find strong associations with telomere length. The combination of limited diversity and evolutionary conservation suggest that these genes may be under selective pressure. More work is needed to explore the role of genetic variants in telomere length regulation. Hum Mutat 31:1050–1058, 2010. Published 2010 Wiley‐Liss, Inc.     

Evaluation of risk loci for schizophrenia derived from genome‐wide association studies in a German population

In the genome‐wide association study (GWAS) on schizophrenia [O'Donovan et al. (2008); Nat Genet 40:1053–1055] a UK‐sample of 479 cases with DSM‐IV schizophrenia was genotyped in comparison to control subjects with follow up of 12 putative loci in international replication sets of approximately 15,000 cases and controls. In these cohorts and a combined bipolar and schizophrenia UK‐sample, six single nucleotide polymorphisms (SNPs) supported association, with the strongest evidence for SNP‐marker rs1344706 at the zinc finger ZNF804A locus on chromosome 2q32.1 (P = 1.61 × 10^?7). We attempted replication of these findings in a German population of 2,154 individuals (632 with affective disorders, 937 with schizophrenia, and 585 controls), but found none of the GWAS risk alleles significantly associated with psychosis. Particularly rs1344706, initially surpassing the genome‐wide significance level in an extended phenotype of schizophrenia and affective disorder, produced consistently negative results. At the ZNF804A locus estimated Odds ratios reached 1.08 (0.93–1.26 95% CI) for the schizophrenia sample and 1.04 (0.90–1.20 95% CI) for the combined set of cases with schizophrenia and affective disorder. The main limitation of our study may be the reduced power of the sample size, but our data may be useful for future meta‐analysis of GWA data sets. Although GWAS have proven extraordinary successful in identifying susceptibility genes for complex genetic disorders, the hypothesis of common genetic variants in the complex group of the schizophrenic psychoses with small effect size but relatively high frequency is still put to further scrutiny. © 2011 Wiley‐Liss, Inc.     

Positive association of phencyclidine-responsive genes, PDE4A and PLAT, with schizophrenia

As schizophrenia‐like symptoms are produced by administration of phencyclidine (PCP), a noncompetitive antagonist of N‐methyl‐D ‐aspartate (NMDA) receptors, PCP‐responsive genes could be involved in the pathophysiology of schizophrenia. We injected PCP to Wistar rats and isolated five different parts of the brain in 1 and 4 hr after the injection. We analyzed the gene expression induced by the PCP treatment of these tissues using the AGILENT rat cDNA microarray system. We observed changes in expression level in 90 genes and 21 ESTs after the treatment. Out of the 10 genes showing >2‐fold expressional change evaluated by qRT‐PCR, we selected 7 genes as subjects for the locus‐wide association study to identify susceptibility genes for schizophrenia in the Japanese population. In haplotype analysis, significant associations were detected in combinations of two SNPs of BTG2 (P = 1.4 × 10^?6), PDE4A (P = 1.4 × 10^?6), and PLAT (P = 1 × 10^?3), after false discovery rate (FDR) correction. Additionally, we not only successfully replicated the haplotype associations in PDE4A (P = 6.8 × 10^?12) and PLAT (P = 0.015), but also detected single‐point associations of one SNP in PDE4A (P = 0.0068) and two SNPs in PLAT (P = 0.0260 and 0.0104) in another larger sample set consisting of 2,224 cases and 2,250 controls. These results indicate that PDE4A and PLAT may be susceptibility genes for schizophrenia in the Japanese population. © 2011 Wiley‐Liss, Inc.     

Addressing Population‐Specific Multiple Testing Burdens in Genetic Association Studies

The number of effectively independent tests performed in genome‐wide association studies (GWAS) varies by population, making a universal P‐value threshold inappropriate. We estimated the number of independent SNPs in Phase 3 HapMap samples by: (1) the LD‐pruning function in PLINK, and (2) an autocorrelation‐based approach. Autocorrelation was also used to estimate the number of independent SNPs in whole genome sequences from 1000 Genomes. Both approaches yielded consistent estimates of numbers of independent SNPs, which were used to calculate new population‐specific thresholds for genome‐wide significance. African populations had the most stringent thresholds (1.49 × 10^?7 for YRI at r² = 0.3), East Asian populations the least (3.75 × 10^?7 for JPT at r² = 0.3). We also assessed how using population‐specific significance thresholds compared to using a single multiple testing threshold at the conventional 5 × 10^?8 cutoff. Applied to a previously published GWAS of melanoma in Caucasians, our approach identified two additional genes, both previously associated with the phenotype. In a Chinese breast cancer GWAS, our approach identified 48 additional genes, 19 of which were in or near genes previously associated with the phenotype. We conclude that the conventional genome‐wide significance threshold generates an excess of Type 2 errors, particularly in GWAS performed on more recently founded populations.     

Inherited balanced translocation t(9;17)(q33.2;q25.3) concomitant with a 16p13.1 duplication in a patient with schizophrenia

We report two rare genetic aberrations in a schizophrenia patient that may act together to confer disease susceptibility. A previously unreported balanced t(9;17)(q33.2;q25.3) translocation was observed in two schizophrenia‐affected members of a small family with diverse psychiatric disorders. The proband also carried a 1.5 Mbp microduplication at 16p13.1 that could not be investigated in other family members. The duplication has been reported to predispose to schizophrenia, autism and mental retardation, with incomplete penetrance and variable expressivity. The t(9;17) (q33.2;q25.3) translocation breakpoint occurs within the open reading frames of KIAA1618 on 17q25.3, and TTLL11 (tyrosine tubulin ligase like 11) on 9q33.2, causing no change in the expression level of KIAA1618 but leading to loss of expression of one TTLL11 allele. TTLL11 belongs to a family of enzymes catalyzing polyglutamylation, an unusual neuron‐specific post‐translational modification of microtubule proteins, which modulates microtubule development and dynamics. The 16p13.1 duplication resulted in increased expression of NDE1, encoding a DISC1 protein partner mediating DISC1 functions in microtubule dynamics. We hypothesize that concomitant TTLL11‐NDE1 deregulation may increase mutation load, among others, also on the DISC1 pathway, which could contribute to disease pathogenesis through multiple effects on neuronal development, synaptic plasticity, and neurotransmission. Our data illustrate the difficulties in interpreting the contribution of multiple potentially pathogenic changes likely to emerge in future next‐generation sequencing studies, where access to extended families will be increasingly important. © 2011 Wiley‐Liss, Inc.     

A Genome‐Wide Assessment of Variability in Human Serum Metabolism

The study of the genetic regulation of metabolism in human serum samples can contribute to a better understanding of the intermediate biological steps that lead from polymorphism to disease. Here, we conducted a genome‐wide association study (GWAS) to discover metabolic quantitative trait loci (mQTLs) utilizing samples from a study of prostate cancer in Swedish men, consisting of 402 individuals (214 cases and 188 controls) in a discovery set and 489 case‐only samples in a replication set. A global nontargeted metabolite profiling approach was utilized resulting in the detection of 6,138 molecular features followed by targeted identification of associated metabolites. Seven replicating loci were identified (PYROXD2, FADS1, PON1, CYP4F2, UGT1A8, ACADL, and LIPC) with associated sequence variants contributing significantly to trait variance for one or more metabolites (P = 10^?13–10^?91). Regional mQTL enrichment analyses implicated two loci that included FADS1 and a novel locus near PDGFC. Biological pathway analysis implicated ACADM, ACADS, ACAD8, ACAD10, ACAD11, and ACOXL, reflecting significant enrichment of genes with acyl‐CoA dehydrogenase activity. mQTL SNPs and mQTL‐harboring genes were over‐represented across GWASs conducted to date, suggesting that these data may have utility in tracing the molecular basis of some complex disease associations.     

Genome‐Wide Association Study Confirms SNPs in SNCA and the MAPT Region as Common Risk Factors for Parkinson Disease

Parkinson disease (PD) is a chronic neurodegenerative disorder with a cumulative prevalence of greater than one per thousand. To date three independent genome‐wide association studies (GWAS) have investigated the genetic susceptibility to PD. These studies implicated several genes as PD risk loci with strong, but not genome‐wide significant, associations. In this study, we combined data from two previously published GWAS of Caucasian subjects with our GWAS of 604 cases and 619 controls for a joint analysis with a combined sample size of 1752 cases and 1745 controls. SNPs in SNCA (rs2736990, p‐value = 6.7 × 10^?8; genome‐wide adjusted p = 0.0109, odds ratio (OR) = 1.29 [95% CI: 1.17–1.42] G vs. A allele, population attributable risk percent (PAR%) = 12%) and the MAPT region (rs11012, p‐value = 5.6 × 10^?8; genome‐wide adjusted p = 0.0079, OR = 0.70 [95% CI: 0.62–0.79] T vs. C allele, PAR%= 8%) were genome‐wide significant. No other SNPs were genome‐wide significant in this analysis. This study confirms that SNCA and the MAPT region are major genes whose common variants are influencing risk of PD.     

Genetic Variation in the Peroxisome Proliferator‐Activated Receptor (PPAR) and Peroxisome Proliferator‐Activated Receptor Gamma Co‐activator 1 (PGC1) Gene Families and Type 2 Diabetes

We used a two‐stage study design to evaluate whether variations in the peroxisome proliferator‐activated receptors (PPAR) and the PPAR gamma co‐activator 1 (PGC1) gene families (PPARA, PPARG, PPARD, PPARGC1A, and PPARGC1B) are associated with type 2 diabetes (T2D) risk. Stage I used data from a genome‐wide association study (GWAS) from Shanghai, China (1019 T2D cases and 1709 controls) and from a meta‐analysis of data from the Asian Genetic Epidemiology Network for T2D (AGEN‐T2D). Criteria for selection of single nucleotide polymorphisms (SNPs) for stage II were: (1) P < 0.05 in single marker analysis in Shanghai GWAS and P < 0.05 in the meta‐analysis or (2) P < 10^?3 in the meta‐analysis alone and (3) minor allele frequency ≥ 0.10. Nine SNPs from the PGC1 family were assessed in stage II (an independent set of middle‐aged men and women from Shanghai with 1700 T2D cases and 1647 controls). One SNP in PPARGC1B, rs251464, was replicated in stage II (OR = 0.87; 95% CI: 0.77–0.99). Gene‐body mass index (BMI) and gene–exercise interactions and T2D risk were evaluated in a combined dataset (Shanghai GWAS and stage II data: 2719 cases and 3356 controls). One SNP in PPARGC1A, rs12640088, had a significant interaction with BMI. No interactions between the PPARGC1B gene and BMI or exercise were observed.     

DISC1 in adult ADHD patients: An association study in two European samples

The DISC1 gene was named after its discovery in a Scottish pedigree with schizophrenia (SCZ) patients. However, subsequent studies have shown association of DISC1 variants with a range of different neurocognitive phenotypes and psychiatric disorders, including bipolar disorder (BPD), and major depression. Attention‐deficit/hyperactivity disorder (ADHD) shares some symptoms with BPD and ADHD patients often suffer from comorbid affective disorders. We wanted to examine the role of DISC1 in ADHD, and with comorbid symptoms of mood disorders. Eleven single nucleotide polymorphisms (SNPs) previously implicated in SCZ and BPD, and a DISC1 duplication involving exon 1, were genotyped in 561 adult ADHD cases and 713 controls of Norwegian ancestry. The intronic SNP rs1538979 was associated with ADHD in the Norwegian sample [odds ratio (OR): 1.33, 95% confidence interval (CI) 1.03–1.73, P = 0.03] and replicated in a Spanish adult ADHD sample of 694 cases and 735 controls, using the tagging SNP rs11122330 (meta‐analysis: P = 0.008, OR 1.25, 95% CI 1.06–1.47). In the Norwegian ADHD sample we also observed an association between the Phe607‐variant of rs6675281 and a positive score on the Mood Disorder Questionnaire (MDQ; OR = 1.44, 95% CI 1.08–1.93, P = 0.01). To our knowledge, this is the first study to show an association between DISC1 variants and ADHD. Our study suggests that further studies are warranted to resolve if DISC1 variation is involved in several common neurodevelopmental disorders including ADHD. © 2013 Wiley Periodicals, Inc.     

A molecule‐based genetic association approach implicates a range of voltage‐gated calcium channels associated with schizophrenia

Traditional genome‐wide association studies (GWAS) have successfully detected genetic variants associated with schizophrenia. However, only a small fraction of heritability can be explained. Gene‐set/pathway‐based methods can overcome limitations arising from single nucleotide polymorphism (SNP)‐based analysis, but most of them place constraints on size which may exclude highly specific and functional sets, like macromolecules. Voltage‐gated calcium (Ca_v) channels, belonging to macromolecules, are composed of several subunits whose encoding genes are located far away or even on different chromosomes. We combined information about such molecules with GWAS data to investigate how functional channels associated with schizophrenia. We defined a biologically meaningful SNP‐set based on channel structure and performed an association study by using a validated method: SNP‐set (sequence) kernel association test. We identified eight subtypes of Ca_v channels significantly associated with schizophrenia from a subsample of published data (N = 56,605), including the L‐type channels (Ca_v1.1, Ca_v1.2, Ca_v1.3), P‐/Q‐type Ca_v2.1, N‐type Ca_v2.2, R‐type Ca_v2.3, T‐type Ca_v3.1, and Ca_v3.3. Only genes from Ca_v1.2 and Ca_v3.3 have been implicated by the largest GWAS (N = 82,315). Each subtype of Ca_v channels showed relatively high chip heritability, proportional to the size of its constituent gene regions. The results suggest that abnormalities of Ca_v channels may play an important role in the pathophysiology of schizophrenia and these channels may represent appropriate drug targets for therapeutics. Analyzing subunit‐encoding genes of a macromolecule in aggregate is a complementary way to identify more genetic variants of polygenic diseases. This study offers the potential of power for discovery the biological mechanisms of schizophrenia.     

Functional annotations improve the predictive score of human disease‐related mutations in proteins

Single nucleotide polymorphisms (SNPs) are the simplest and most frequent form of human DNA variation, also valuable as genetic markers of disease susceptibility. The most investigated SNPs are missense mutations resulting in residue substitutions in the protein. Here we propose SNPs&GO, an accurate method that, starting from a protein sequence, can predict whether a mutation is disease related or not by exploiting the protein functional annotation. The scoring efficiency of SNPs&GO is as high as 82%, with a Matthews correlation coefficient equal to 0.63 over a wide set of annotated nonsynonymous mutations in proteins, including 16,330 disease‐related and 17,432 neutral polymorphisms. SNPs&GO collects in unique framework information derived from protein sequence, evolutionary information, and function as encoded in the Gene Ontology terms, and outperforms other available predictive methods. Hum Mutat 30:1–8, 2009. © 2009 Wiley‐Liss, Inc.     

Expression of DISC1 binding partners is reduced in schizophrenia and associated with DISC1 SNPs

DISC1 has been identified as a schizophrenia susceptibility gene based on linkage and SNP association studies and clinical data suggesting that risk SNPs impact on hippocampal structure and function. In cell and animal models, C-terminus-truncated DISC1 disrupts intracellular transport, neural architecture and migration, perhaps because it fails to interact with binding partners involved in neuronal differentiation such as fasciculation and elongation protein zeta-1 (FEZ1), platelet-activating factor acetylhydrolase, isoform Ib, PAFAH1B1 or lissencephaly 1 protein (LIS1) and nuclear distribution element-like (NUDEL). We hypothesized that altered expression of DISC1 and/or its molecular partners may underlie its pathogenic role in schizophrenia and explain its genetic association. We examined the expression of DISC1 and these selected binding partners as well as reelin, a protein in a related signaling pathway, in the hippocampus and dorsolateral prefrontal cortex of postmortem human brain patients with schizophrenia and controls. We found no difference in the expression of DISC1 or reelin mRNA in schizophrenia and no association with previously identified risk DISC1 SNPs. However, the expression of NUDEL, FEZ1 and LIS1 was each significantly reduced in the brain tissue from patients with schizophrenia and expression of each showed association with high-risk DISC1 polymorphisms. Although, many other DISC1 binding partners still need to be investigated, these data implicate genetically linked abnormalities in the DISC1 molecular pathway in the pathophysiology of schizophrenia.     

A functional variant on 20q13.33 related to glioma risk alters enhancer activity and modulates expression of multiple genes

Genome‐wide association studies (GWAS) have identified single‐nucleotide polymorphisms (SNPs) associated with glioma risk on 20q13.33, but the biological mechanisms underlying this association are unknown. We tested the hypothesis that a functional SNP on 20q13.33 impacted the activity of an enhancer, leading to an altered expression of nearby genes. To identify candidate functional SNPs, we identified all SNPs in linkage disequilibrium with the risk‐associated SNP rs2297440 that mapped to putative enhancers. Putative enhancers containing candidate functional SNPs were tested for allele‐specific effects in luciferase enhancer activity assays against glioblastoma multiforme (GBM) cell lines. An enhancer containing SNP rs3761124 exhibited allele‐specific effects on activity. Deletion of this enhancer by CRISPR‐Cas9 editing in GBM cell lines correlated with an altered expression of multiple genes, including STMN3, RTEL1, RTEL1‐TNFRSF6B, GMEB2, and SRMS. Expression quantitative trait loci (eQTL) analyses using nondiseased brain samples, isocitrate dehydrogenase 1 (IDH1) wild‐type glioma, and neurodevelopmental tissues showed STMN3 to be a consistent significant eQTL with rs3761124. RTEL1 and GMEB2 were also significant eQTLs in the context of early CNS development and/or in IDH1 wild‐type glioma. We provide evidence that rs3761124 is a functional variant on 20q13.33 related to glioma/GBM risk that modulates the expression of STMN3 and potentially other genes across diverse cellular contexts.     

Population‐based identity‐by‐descent mapping combined with exome sequencing to detect rare risk variants for schizophrenia

Genome‐wide association studies (GWASs) are highly effective at identifying common risk variants for schizophrenia. Rare risk variants are also important contributors to schizophrenia etiology but, with the exception of large copy number variants, are difficult to detect with GWAS. Exome and genome sequencing, which have accelerated the study of rare variants, are expensive so alternative methods are needed to aid detection of rare variants. Here we re‐analyze an Irish schizophrenia GWAS dataset (n = 3,473) by performing identity‐by‐descent (IBD) mapping followed by exome sequencing of individuals identified as sharing risk haplotypes to search for rare risk variants in coding regions. We identified 45 rare haplotypes (>1 cM) that were significantly more common in cases than controls. By exome sequencing 105 haplotype carriers, we investigated these haplotypes for functional coding variants that could be tested for association in independent GWAS samples. We identified one rare missense variant in PCNT but did not find statistical support for an association with schizophrenia in a replication analysis. However, IBD mapping can prioritize both individual samples and genomic regions for follow‐up analysis but genome rather than exome sequencing may be more effective at detecting risk variants on rare haplotypes.