Association of dopamine‐ and serotonin‐related genes with canine aggression

Human‐directed canine aggression was studied using 50 aggressive and 81 non‐aggressive dogs. We examined 62 single nucleotide polymorphisms (SNPs) occurring in or in the close vicinity of 16 neurotransmitter‐related genes. Allelic associations with aggression were identified for DRD1, HTR1D, HTR2C and SLC6A1. Risk or protective haplotypes for aggressive behaviour based on 2–5 SNPs were identified. The frequency of aggressive dogs varied significantly between the haplotypes within loci and the odds ratios of aggression in dogs with risk haplotypes compared with protective haplotypes varied from 4.4 (HTR2C) to 9.0 (SLC6A1). A risk haplotype across the neurotransmitter receptor gene HTR1D harboured a non‐synonymous SNP with a potential effect on protein function. We identified no haplotypes in complete association with the recorded phenotypes, supporting a complex inheritance of aggression.     

Linkage disequilibrium and sequence diversity in a 500-kbp region around the adh1 locus in elite maize germplasm

Linkage disequilibrium (LD) at the adh1 locus was examined in two sets of maize inbreds. A set of 32 was chosen to represent most of the genetic diversity in the cultivated North American elite maize breeding pool. A second set of 192 inbreds was chosen to sample more deeply the two major heterotic groups in elite maize germplasm. Analysis of several loci in the vicinity of the adh1 gene shows that LD as measured by D and r² extends greater than 500 kbp in this germplasm. The presence of this exceptionally long segment of high LD may be suggestive of selection acting on one of the genes in the vicinity of adh1 or of a locally reduced rate of recombination.Electronic Supplementary Material Supplementary material is available for this article at .     

Patterns of linkage disequilibrium and haplotype distribution in disease candidate genes

Background

The adequacy of association studies for complex diseases depends critically on the existence of linkage disequilibrium (LD) between functional alleles and surrounding SNP markers.

Results

We examined the patterns of LD and haplotype distribution in eight candidate genes for osteoporosis and/or obesity using 31 SNPs in 1,873 subjects. These eight genes are apolipoprotein E (APOE), type I collagen α1 (COL1A1), estrogen receptor-α (ER-α), leptin receptor (LEPR), parathyroid hormone (PTH)/PTH-related peptide receptor type 1 (PTHR1), transforming growth factor-β1 (TGF-β1), uncoupling protein 3 (UCP3), and vitamin D (1,25-dihydroxyvitamin D₃) receptor (VDR). Yin yang haplotypes, two high-frequency haplotypes composed of completely mismatching SNP alleles, were examined. To quantify LD patterns, two common measures of LD, D' and r², were calculated for the SNPs within the genes. The haplotype distribution varied in the different genes. Yin yang haplotypes were observed only in PTHR1 and UCP3. D' ranged from 0.020 to 1.000 with the average of 0.475, whereas the average r² was 0.158 (ranging from 0.000 to 0.883). A decay of LD was observed as the intermarker distance increased, however, there was a great difference in LD characteristics of different genes or even in different regions within gene.

Conclusion

The differences in haplotype distributions and LD patterns among the genes underscore the importance of characterizing genomic regions of interest prior to association studies.     

Multilocus OCA2 genotypes specify human iris colors

Human iris color is a quantitative, multifactorial phenotype that exhibits quasi-Mendelian inheritance. Recent studies have shown that OCA2 polymorphism underlies most of the natural variability in human iris pigmentation but to date, only a few associated polymorphisms in this gene have been described. Herein, we describe an iris color score (C) for quantifying iris melanin content in-silico and undertake a more detailed survey of the OCA2 locus (n = 271 SNPs). In 1,317 subjects, we confirmed six previously described associations and identified another 27 strongly associated with C that were not explained by continental population stratification (OR 1.5–17.9, P = 0.03 to <0.001). Haplotype analysis with respect to these 33 SNPs revealed six haplotype blocks and 11 hap-tags within these blocks. To identify genetic features for best-predicting iris color, we selected sets of SNPs by parsing P values among possible combinations and identified four discontinuous and non-overlapping sets across the LD blocks (p-Selected SNP sets). In a second, partially overlapping sample of 1,072, samples with matching diplotypes comprised of these p-Selected OCA2 SNPs exhibited a rate of C concordance of 96.3% (n = 82), which was significantly greater than that obtained from randomly selected samples (62.6%, n = 246, P<0.0001). In contrast, the rate of C concordance using diplotypes comprised of the 11 identified hap-tags was only 83.7%, and that obtained using diplotypes comprised of all 33 SNPs organized as contiguous sets along the locus (defined by the LD block structure) was only 93.3%. These results confirm that OCA2 is the major human iris color gene and suggest that using an empirical database-driven system, genotypes from a modest number of SNPs within this gene can be used to accurately predict iris melanin content from DNA. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic analysis of adiponectin and obesity in Hispanic families: the IRAS Family Study

Adiponectin, coded for by the APM1 gene, is a novel adipocyte-derived hormone implicated in energy homeostasis and obesity. Several genetic studies have observed evidence of association between APM1 gene polymorphisms and features of the metabolic syndrome, such as insulin resistance and obesity. As part of a comprehensive genetic analysis of the APM1 gene, we have screened 96 unrelated individuals for polymorphisms in the promoter, coding regions, and 3untranslated region (UTR). Three promoter single-nucleotide polymorphisms (SNPs), two rare coding SNPs (G113A and T1233C), and 13 SNPs in the 3UTR were identified. Eighteen SNPs were genotyped in 811 Hispanic individuals from 45 families in the IRAS Family Study (IRASFS). SNPs were tested for association with six obesity quantitative traits (body mass index, waist, waist:hip ratio, subcutaneous adipose tissue, visceral adipose tissue, and visceral:subcutaneous ratio). Significant evidence of association to at least one of the obesity traits was identified in seven of the 18 SNPs (<0.001–0.05). The promoter SNP INS CA-11156 was the most consistently associated SNP and was associated significantly with all measures of obesity, except the visceral:subcutaneous ratio (P-values 0.009–0.03). Haplotype analysis supported this evidence of association, with haplotypes containing an insertion of one CA repeat at position –11156 consistently being associated with lower obesity values (P-value <0.001–0.05). The adiponectin polymorphisms, in particular those in the promoter region, thus show significant association with obesity measures in the Hispanic population. Additional studies are needed to confirm our findings and determine which polymorphism causes the functional effect.     

Evaluation of LD decay and various LD-decay estimators in simulated and SNP-array data of tetraploid potato

Key message

The number of SNPs required for QTL discovery is justified by the distance at which linkage disequilibrium has decayed. Simulations and real potato SNP data showed how to estimate and interpret LD decay.

Abstract

The magnitude of linkage disequilibrium (LD) and its decay with genetic distance determine the resolution of association mapping, and are useful for assessing the desired numbers of SNPs on arrays. To study LD and LD decay in tetraploid potato, we simulated autotetraploid genotypes and used it to explore the dependence on: (1) the number of haplotypes in the population (the amount of genetic variation) and (2) the percentage of haplotype specific SNPs (hs-SNPs). Several estimators for short-range LD were explored, such as the average r ², median r ², and other percentiles of r ² (80, 90, and 95 %). For LD decay, we looked at LD_½,90, the distance at which the short-range LD is halved when using the 90 % percentile of r ² at short range, as estimator for LD. Simulations showed that the performance of various estimators for LD decay strongly depended on the number of haplotypes, although the real value of LD decay was not influenced very much by this number. The estimator LD_½,90 was chosen to evaluate LD decay in 537 tetraploid varieties. LD_½,90 values were 1.5 Mb for varieties released before 1945 and 0.6 Mb in varieties released after 2005. LD_½,90 values within three different subpopulations ranged from 0.7 to 0.9 Mb. LD_½,90 was 2.5 Mb for introgressed regions, indicating large haplotype blocks. In pericentromeric heterochromatin, LD decay was negligible. This study demonstrates that several related factors influencing LD decay could be disentangled, that no universal approach can be suggested, and that the estimation of LD decay has to be performed with great care and knowledge of the sampled material.

     

Polymorphism ofTcrb andTcrg genes in Biozzi mice: Segregation analysis of a newTcrg haplotype with antibody responsiveness

Tcrb andTcrg gene polymorphism was investigated in high (H) and low (L) responder Biozzi mice from selection I, II, and GS by Southern blot analysis with appropriateV andC probes. No polymorphism of theTcrb haplotype was detected between H and L mice in all selections which were all found to be of the BALB/c type. The H-I and H-II g genotype was of BALB/c and DBA/2 type, respectively. In contrast, a newTcrg haplotype shared by L-I and L-II mice was identified and characterized by C1, 2, 3, C4, V1, 2, 3, V5, and V6 restriction fragment length polymorphisms (RFLPs).Tcrg genotypes were not fixed in the GS selection and two additional new haplotypes were identified in two L-GS mice. An attempt was made to correlate the L-Ig genotype with the low responder status by analyzingg haplotypes among highest and lowest responder (H-1 x L-I)F₂ hybrids immunized with sheep red blood cells (SRBC). No correlation was found in this segregation study, whereas a highly significant one was established with theH-2 haplotype, a locus already known to participate in the genetic control of H-I/L-I difference. The lack of correlation between SRBC response and theTcrg genotype was consistent with the heterogenousg haplotypes found in mice of the GS selection. Together, the present results suggest that H and L mice have the sameTcrab potential repertoire and that T-cell receptor (Tcr) genes cannot be considered as immune response genes in this model. Our results also indicate that the F₂ segregation analysis, given a polymorphic gene, is suitable for an investigation of its immune response functions.     

Evidence for epistasis between <Emphasis Type="Italic">SLC6A4</Emphasis> and <Emphasis Type="Italic">ITGB3</Emphasis> in autism etiology and in the determination of platelet serotonin levels

Autism is a neurodevelopmental disorder of unclear etiology. The consistent finding of platelet hyperserotonemia in a proportion of patients and its heritability within affected families suggest that genes involved in the serotonin system play a role in this disorder. The role in autism etiology of seven candidate genes in the serotonin metabolic and neurotransmission pathways and mapping to autism linkage regions (SLC6A4, HTR1A, HTR1D, HTR2A, HTR5A, TPH1 and ITGB3) was analyzed in a sample of 186 nuclear families. The impact of interactions among these genes in autism was assessed using the multifactor-dimensionality reduction (MDR) method in 186 patients and 181 controls. We further evaluated whether the effect of specific gene variants or gene interactions associated with autism etiology might be mediated by their influence on serotonin levels, using the quantitative transmission disequilibrium test (QTDT) and the restricted partition method (RPM), in a sample of 109 autistic children. We report a significant main effect of the HTR5A gene in autism (P = 0.0088), and a significant three-locus model comprising a synergistic interaction between the ITGB3 and SLC6A4 genes with an additive effect of HTR5A (P < 0.0010). In addition to the previously reported contribution of SLC6A4, we found significant associations of ITGB3 haplotypes with serotonin level distribution (P = 0.0163). The most significant models contributing to serotonin distribution were found for interactions between TPH1 rs4537731 and SLC6A4 haplotypes (P = 0.002) and between HTR1D rs6300 and SLC6A4 haplotypes (P = 0.013). In addition to the significant independent effects, evidence for interaction between SLC6A4 and ITGB3 markers was also found. The overall results implicate SLC6A4 and ITGB3 gene interactions in autism etiology and in serotonin level determination, providing evidence for a common underlying genetic mechanism and a molecular explanation for the association of platelet hyperserotonemia with autism. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Association of several polymorphic loci of serotoninergic genes with unipolar depression

Serotoninergic system is one of the major brain neurotransmitter systems that is involved in the development of depressive spectrum disorders. Regulatory genes of this system are the principle candidate genes predisposing to unipolar depression. Using PCR-RFLP analysis, we have conducted a study of polymorphic loci of several genes of this system: C1019G of serotonin receptor 1A gene, (HTR1A); A-1438G of serotonin receptor 2A gene, (HTR2A); G861C of serotonin receptor 1B gene, (HTR1B); Stin2VNTR and 5-HTTLPR of serotonin transporter gene (SLC6A4) in patients with unipolar depression from Tatar and Russian population. The results of the study suggest that genotype 10/10 of the SLC6A4 gene as well as genotype G/G and allele G of the HTR2A gene can predispose to increased risk of unipolar depression development in ethnic Russians. In contrast, genotype 12/10 of the SLC6A4 gene is a marker of low risk of the disease in both groups.     

Analysis of serotonin receptor 2A gene (HTR2A): Association study with autism spectrum disorder in the Indian population and investigation of the gene expression in peripheral blood leukocytes

Several studies suggest involvement of serotoninergic system in the pathophysiology of Autism Spectrum Disorder (ASD). The 5-HT receptor binding studies using ³H-lysergic acid diethylamide (³H-LSD) and linkage analysis provided evidences to consider HTR2A as a potential candidate gene for ASD. The three SNPs, −1438A/G (rs6311), 102T/C (rs6313) and 1354C/T (rs6314) of HTR2A have been well studied in the etiology of various neuropsychiatric disorders. But studies on association of this gene with ASD are limited to two reports from American and Korean populations. Additionally there are reports, which demonstrated paternal imprinting of HTR2A with expression from only one allele. So far no reports are available on HTR2A and its association with any neuropsychiatric disorders from Indian population. Therefore, the present study investigates association of the above mentioned three markers of HTR2A with ASD in Indian population using population and family-based approaches. The study also deals with allelic expression pattern of HTR2A in Peripheral Blood Leukocytes (PBLs) to understand the parental imprinting status. The genotyping analyses were carried out for probands, parents and controls. The subsequent association analyses did not show association of these markers with ASD. So, HTR2A is unlikely to be a genetic marker for ASD in Indian population. The expression analyses showed absence of monoallelic expression, suggesting lack of parental imprinting of HTR2A gene. However, we noticed methylation of the CpG sites at −1438A/G and 102T/C loci of HTR2A gene. Further bioinformatics analysis revealed absence of CpG islands in the promoter of the gene supporting biallelic expression pattern of HTR2A in PBLs.     

Restriction fragment length polymorphisms associated with immunoglobulin heavy chain gamma genes in Tunisians

Summary DNA polymorphisms in the human immunoglobulin gamma () region have been studied in random Arabo-Berber Tunisians and in a large Tunisian Berber kindred. Haplotypes have then been designated, based on variation in the BamHI restriction fragments containing the C1, C2, C4, and C genes. Two new haplotypes, in addition to the four previously described, have been observed. These new haplotypes, designated H5 and H6, were confirmed by family studies. The H5 haplotype was associated with black African Gm haplotypes · (Gm^{1,17;..;5,6,11} and Gm^1,17;..;5,11) (Gm^{a,z;..;blc3bo} and Gm^a,z;..;blbo) and probably represents a common haplotype in the black population. The haplotype H6 may be derived from H5. One of 39 random Tunisians was homozygous for a multigene deletion. DNA polymorphisms of the C genes, in conjuction with Gm markers, provide highly variable genetic markers important for the characterization of human populations.     

A Hidden Markov Model Combining Linkage and Linkage Disequilibrium Information for Haplotype Reconstruction and Quantitative Trait Locus Fine Mapping

Faithful reconstruction of haplotypes from diploid marker data (phasing) is important for many kinds of genetic analyses, including mapping of trait loci, prediction of genomic breeding values, and identification of signatures of selection. In human genetics, phasing most often exploits population information (linkage disequilibrium), while in animal genetics the primary source of information is familial (Mendelian segregation and linkage). We herein develop and evaluate a method that simultaneously exploits both sources of information. It builds on hidden Markov models that were initially developed to exploit population information only. We demonstrate that the approach improves the accuracy of allele phasing as well as imputation of missing genotypes. Reconstructed haplotypes are assigned to hidden states that are shown to correspond to clusters of genealogically related chromosomes. We show that these cluster states can directly be used to fine map QTL. The method is computationally effective at handling large data sets based on high-density SNP panels.ARRAY technology now allows genotyping of large cohorts for thousands to millions of single nucleotide polymorphisms (SNPs), which are becoming available for a growing list of organisms including human and domestic animals. Among other applications, these advances permit systematic scanning of the genome to map trait loci by association (e.g., Wellcome Trust Case Control Consortium 2007; Charlier et al. 2008), to predict genomic breeding values for complex traits (Meuwissen et al. 2001; Goddard and Hayes 2009), or to identify signatures of selection (e.g., Voight et al. 2006).Present-day genotyping platforms do not directly provide information about linkage phase; i.e., co-inherited alleles at adjacent heterozygous markers (haplotypes) are not identified as such. As haplotype information may considerably empower genetic analyses, indirect phasing strategies have been devised: haplotypes can be reconstructed from unphased genotypes using either familial information (Mendelian segregation and linkage) and/or population information (linkage disequilibrium, LD, and surrogate parents) (e.g., Windig and Meuwissen 2004; Scheet and Stephens 2006; Kong et al. 2008).Haplotype-based approaches are routinely applied in animal genetics for combined linkage and LD mapping of QTL (e.g., Meuwissen and Goddard 2000; Blott et al. 2003). In these studies, phasing has so far relied on familial information provided by the extended pedigrees typical of livestock (e.g., Windig and Meuwissen 2004). This approach, however, leaves a nonnegligible proportion of genotypes unphased, especially for the less connected individuals. After phasing, identity-by-descent (IBD) probabilities conditional on haplotype data—needed for QTL mapping—are computed for all chromosome pairs, using familial as well as population information (hence combined linkage and LD mapping – L + LD) (e.g., Meuwissen and Goddard 2001). However, the use of high-density SNP chips and the analysis of ever larger cohorts render the computation of pairwise IBD probabilities a bottleneck.We herein propose a more efficient, heuristic approach based on hidden Markov models (HMM). It simultaneously phases and sorts haplotypes in clusters that can be used directly for mapping or other purposes. The proposed method exploits familial as well as population information, and imputes missing genotypes. We herein describe the accuracy of the proposed method and its use for L + LD mapping of QTL.     

Structure and evolution of the promoter regions of the DQA genes

HLA-DQ antigens are unique among the class II antigens in that their chains are highly polymorphic. In the present study, we characterized the general structure of the promoter regions of the DQA genes derived from different DR haplotypes and defined their nucleotide sequence polymorphisms. The promoter of each DQA1 allele contains three sequence motifs which are not present in non-DQA related class II genes: one identical to a tumor necrosis factor (TNF) response element, one similar to an NFB binding element, and one similar to a W motif. All DQA alleles lack TATA and CCAAT boxes in the proximal promoter region but carry other sequence elements characteristic of MHC class II genes, including S, X, X2, and Y boxes, and a pyrimidine-rich tract upstream of the X box. Nucleotide sequence polymorphisms among the various DQA1 alleles were noted within the promoter region, with some of the differences mapping within, or close to, regulatory elements that are important for the expression of MHC class II genes. All DQA1 alleles carry an unrearranged, full length, Alu-Sx related repeat immediately upstream of the proximal promoter region. This repeat was not present in the DQA2 (DXA) genes analyzed, confirming that DQ locus duplication probably occurred before integration of the Alu repeat into the primordial DQA1 locus, some 31–43 million years (myr) ago. The DQA2 promoter region is highly conserved between DR4 and DR3 haplotypes, with the degree of conservation exceeding that expected from the neutral mutation rate.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence data base and have been assigned the accession numbers M97 454-M97 464. Correspondence to: E. Morzycka-Wroblewska.     

A mitochondrial mutator plasmid that causes senescence under dietary restricted conditions

Background

The definition of human MHC class I haplotypes through association of HLA-A, HLA-Cw and HLA-B has been used to analyze ethnicity, population migrations and disease association.

Results

Here, we present HLA-E allele haplotype association and population linkage disequilibrium (LD) analysis within the ~1.3 Mb bounded by HLA-B/Cw and HLA-A to increase the resolution of identified class I haplotypes. Through local breakdown of LD, we inferred ancestral recombination points both upstream and downstream of HLA-E contributing to alternative block structures within previously identified haplotypes. Through single nucleotide polymorphism (SNP) analysis of the MHC region, we also confirmed the essential genetic fixity, previously inferred by MHC allele analysis, of three conserved extended haplotypes (CEHs), and we demonstrated that commercially-available SNP analysis can be used in the MHC to help define CEHs and CEH fragments.

Conclusion

We conclude that to generate high-resolution maps for relating MHC haplotypes to disease susceptibility, both SNP and MHC allele analysis must be conducted as complementary techniques.     

A Recombination Hotspot in a Schizophrenia-Associated Region of GABRB2

Background

Schizophrenia is a major disorder with complex genetic mechanisms. Earlier, population genetic studies revealed the occurrence of strong positive selection in the GABRB2 gene encoding the β₂ subunit of GABA_A receptors, within a segment of 3,551 bp harboring twenty-nine single nucleotide polymorphisms (SNPs) and containing schizophrenia-associated SNPs and haplotypes.

Methodology/Principal Findings

In the present study, the possible occurrence of recombination in this ‘S1–S29’ segment was assessed. The occurrence of hotspot recombination was indicated by high resolution recombination rate estimation, haplotype diversity, abundance of rare haplotypes, recurrent mutations and torsos in haplotype networks, and experimental haplotyping of somatic and sperm DNA. The sub-segment distribution of relative recombination strength, measured by the ratio of haplotype diversity (H_d) over mutation rate (θ), was indicative of a human specific Alu-Yi6 insertion serving as a central recombining sequence facilitating homologous recombination. Local anomalous DNA conformation attributable to the Alu-Yi6 element, as suggested by enhanced DNase I sensitivity and obstruction to DNA sequencing, could be a contributing factor of the increased sequence diversity. Linkage disequilibrium (LD) analysis yielded prominent low LD points that supported ongoing recombination. LD contrast revealed significant dissimilarity between control and schizophrenic cohorts. Among the large array of inferred haplotypes, H26 and H73 were identified to be protective, and H19 and H81 risk-conferring, toward the development of schizophrenia.

Conclusions/Significance

The co-occurrence of hotspot recombination and positive selection in the S1–S29 segment of GABRB2 has provided a plausible contribution to the molecular genetics mechanisms for schizophrenia. The present findings therefore suggest that genome regions characterized by the co-occurrence of positive selection and hotspot recombination, two interacting factors both affecting genetic diversity, merit close scrutiny with respect to the etiology of common complex disorders.     

ENU mutagenesis identifies mice with morbid obesity and severe hyperinsulinemia caused by a novel mutation in leptin

Background

Obesity is a multifactorial disease that arises from complex interactions between genetic predisposition and environmental factors. Leptin is central to the regulation of energy metabolism and control of body weight in mammals.

Methodology/Principal Findings

To better recapitulate the complexity of human obesity syndrome, we applied N-ethyl-N-nitrosourea (ENU) mutagenesis in combination with a set of metabolic assays in screening mice for obesity. Mapping revealed linkage to the chromosome 6 within a region containing mouse Leptin gene. Sequencing on the candidate genes identified a novel T-to-A mutation in the third exon of Leptin gene, which translates to a V145E amino acid exchange in the leptin propeptide. Homozygous Leptin^145E/145E mutant mice exhibited morbid obesity, accompanied by adipose hypertrophy, energy imbalance, and liver steatosis. This was further associated with severe insulin resistance, hyperinsulinemia, dyslipidemia, and hyperleptinemia, characteristics of human obesity syndrome. Hypothalamic leptin actions in inhibition of orexigenic peptides NPY and AgRP and induction of SOCS1 and SOCS3 were attenuated in Leptin^145E/145E mice. Administration of exogenous wild-type leptin attenuated hyperphagia and body weight increase in Leptin^145E/145E mice. However, mutant V145E leptin coimmunoprecipitated with leptin receptor, suggesting that the V145E mutation does not affect the binding of leptin to its receptor. Molecular modeling predicted that the mutated residue would form hydrogen bond with the adjacent residues, potentially affecting the structure and formation of an active complex with leptin receptor within that region.

Conclusions/Significance

Thus, our evolutionary, structural, and in vivo metabolic information suggests the residue 145 as of special function significance. The mouse model harboring leptin V145E mutation will provide new information on the current understanding of leptin biology and novel mouse model for the study of human obesity syndrome.     

DNA polymorphism of the C2 locus

The extent of the C2 locus in the HLA class III region has been determined by Southern blotting techniques and by DNA sequence analysis. The gene is 18 kb in length and therefore provides a marked contrast to the adjacent factor B gene of 6 kb. A novel restriction fragment length polymorphism (RFLP) has been identified using the endonuclease Sst I and a genomic probe derived from the 5 region of the C2 gene. Four variants have been detected in a sample of unrelated individuals with haplotypes carrying the C2C allele. Further analysis using C2 and factor B cDNA probes has determined the relationship between this and the other RFLPs previously identified in this region of the genome. Together, the three polymorphisms identified so far make the subdivision of previously indistinguishable haplotypes possible. They therefore constitute a series of markers which increase the resolution of genetic variation in the C2 locus and they may be important in studies of diseases associated with this region of the major histocompatibility complex.