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PubMed1991

Mouse major histocompatibility complex and lung development: haplotype variation, H-2 immunolocalization, and progressive maturation.

The association of the mouse major histocompatibility complex (H-2), lung maturation, and corticosteroid responsiveness has recently been demonstrated in congenic B10 (H-2b) and B10.A (H-2a) mice (Hu et al.: American Journal of Medical Genetics 35:126-131, 1990). We have investigated additional haplotypes [B10.BR (H-2k) and B10.D2 (H-2d)] to confirm that there is a strong association between H-2 haplotype variation and the degree of pulmonary maturation. Lungs of B10.D2, B10.BR, B10, and B10.A congenic mice achieve haplotypic specific maturation: B10.D2 lungs greater than B10 lungs = B10.BR lung greater than B10.A lungs. It appears that the expression of these developmental potentials is under corticosteroid regulation. Further, to test the hypothesis that H-2 antigens would be expressed earlier in embryonic lungs with the H-2b (B10) or H-2k (B10.BR) haplotype than with the H-2a (B10.A) haplotype, we investigated the spatiotemporal patterns of H-2 antigen localization in B10, B10.BR, and B10.A congenic mouse strains with and without corticosteroid treatment. The spatial patterns of H-2 antigen localization was similar in the B10, B10.BR, and B10.A mouse lungs; however, these patterns appeared earlier in both untreated and treated B10 and B10.BR mice as compared with untreated B10.A mice, suggesting an H-2 haplotype associated rate of pulmonary maturation. Following corticosteroid treatment, all congenic strains had a temporally comparable spatial distribution of H-2 antigens. Our results provide preliminary evidence suggesting that both a lung "developmental gene(s)" and a "glucocorticoid responsiveness gene(s)" are most likely outside the K-D subregions of the H-2 complex. A model of the H-2 regulation of lung maturation and corticosteroid responsiveness is discussed.

Animals↗

PubMed2003

Haplotypes at the OPRM1 locus are associated with susceptibility to substance dependence in European-Americans.

Our objective was to investigate the relationship between the gene encoding the mu-opioid receptor (OPRM1) and susceptibility to substance dependence in European-American (EA) and African-American (AA) subjects. Eight single nucleotide polymorphisms (SNPs) at the OPRM1 locus, i.e., -2044C/A, -1793T/A, -1699insT, -1469T/C, -1320A/G, -111C/T, +17C/T (Ala6Val), and +118A/G (Asn40Asp) were genotyped in 676 subjects: 318 EA subjects and 124 AA subjects with substance dependence, and 179 EA normal controls, and 55 AA normal controls. Affection status was defined by each unique combination of alcohol, cocaine, and opioid dependence and analysis of association examined in relation to the possible combinations. We used a newly implemented permutation method to evaluate statistical significance. In EAs, a significant difference in haplotype frequency distributions was found between controls and "alcohol + opioid" dependent patients (P = 0.0036). This finding is also supported by logistic regression analysis and a simulation method. The frequencies of allele -2044A and haplotypes including -2044A are higher in these patients than in controls. In AAs, no allele, haplotype, or genotype frequencies were significantly different between cases and controls. There were highly significant differences in the allele, haplotype, and genotype frequencies between EA and AA controls. Four of the variants [-1793T/A, -1699insT, -1320A/G, and -111C/T] are in virtually complete linkage disequilibrium (LD) to compose a sequence pattern, which does not associate with any of the seven categories of substance dependence. In EAs, allele -2044A and haplotypes that include -2044A are the susceptibility allele and haplotypes for substance dependence. These findings suggest that OPRM1 may play a role in the pathophysiology of substance dependence and this role is population- and diagnosis-specific.

Alcoholism↗

PubMed2003

Tumor necrosis factor haplotype analysis amongst schizophrenia probands from four distinct populations in the Asia-Pacific region.

A single nucleotide polymorphism (TNF(-308A)) within the promoter region of the gene encoding tumor necrosis factor (TNF), has been significantly associated with schizophrenia in a study of Italian patients and control subjects Boin et al. [2001: Mol Psychiatry 6:79-82]. We have applied case-control analyses to examine TNF promoter haplotypes (containing TNF(-308) and two additional promoter variants: TNF(-376) and TNF(-238)) in four schizophrenia cohorts drawn from Australian, Indian Fijian, Indigenous Fijian, and Brahmin populations. In addition, we have applied the sibling transmission disequilibrium (STD) test to promoter haplotypes within 81 trios drawn from Australian Caucasian pedigrees with multiple schizophrenia cases, and 86 trios drawn from the Brahmin population of Tamil Nadu province in Southern India. Within each of these cohorts, we found no evidence of recombination between these tightly linked promoter variants, supporting previous studies which demonstrated that only a subset of the eight possible haplotypes exist. Of the four observed haplotypes, we and others have observed only one carries the TNF(-308A) variant allele. We report no significant differences in TNF promoter haplotype frequencies between the patient and control groups within each population, although the Indian Fijian cohort showed a trend towards reduced TNF(-308A) alleles amongst schizophrenia cases (P = 0.07). We found no evidence of bias in TNF promoter haplotype transmission to schizophrenia probands. Very similar results were obtained when only the TNF(-308) polymorphism was considered. Taken together, these data provide no support for the involvement of TNF promoter variants TNF(-308), TNF(-376), and TNF(-238) in schizophrenia susceptibility within four ethnically distinct cohorts.

Australia↗

PubMed2007

Haplotype association study between DRD1 gene and bipolar type I affective disorder in two samples from Canada and Sardinia.

Based on the dopaminergic hypothesis, the dopamine D(1) receptor gene (DRD1) is considered to be a good candidate gene involved in the susceptibility of bipolar disorder (BP). Genetic association between three DRD1 single nucleotide polymorphisms (SNPs) (-800T/C, -48A/G, and 1403T/C) and bipolar type I (BP I) disorder was performed in a case-control sample of Sardinian origin (170 BP I and 209 controls) and in an enlarged sample (229 families) of BP I trios from Toronto. The haplotype analyses generated significant global chi-square in both samples (P-value 0.024 in Toronto and 0.00042 in Sardinian). The main representative haplotypes in both samples were the -800T/-48A/1403C and the -800C/-48G/1403T. Considering each group individually, the -800C/-48G/1403T was transmitted more frequently from parents to BP I probands in Toronto sample (nominally P-value = 0.047) and was more frequent in cases than in control subjects in Sardinian sample although showing no significant evidence of association (nominally P-value = 0.16) When the estimated haplotype counts of both samples were combined, the global chi(2) was significant (P-value = 0.00085) and the nominal P-value for the haplotype -800C/-48G/1403T was 0.01. The fact that the same haplotype shows a similar trend for association in samples originating from different ethnic backgrounds seems to imply that the -800C/-48G/1403T haplotype may be considered as a risk factor for BP I disorder.

Adult↗

PubMed2005

Haplotype trees and modern human origins.

A haplotype is a multisite haploid genotype at two or more polymorphic sites on the same chromosome in a defined DNA region. An evolutionary tree of the haplotypes can be estimated if the DNA region had little to no recombination. Haplotype trees can be used to reconstruct past human gene-flow patterns and historical events, but any single tree captures only a small portion of evolutionary history, and is subject to error. A fuller view of human evolution requires multiple DNA regions, and errors can be minimized by cross-validating inferences across loci. An analysis of 25 DNA regions reveals an out-of-Africa expansion event at 1.9 million years ago. Gene flow with isolation by distance was established between African and Eurasian populations by about 1.5 million years ago, with no detectable interruptions since. A second out-of-Africa expansion occurred about 700,000 years ago, and involved interbreeding with at least some Eurasian populations. A third out-of-Africa event occurred around 100,000 years ago, and was also characterized by interbreeding, with the hypothesis of a total Eurasian replacement strongly rejected (P < 10(-17)). This does not preclude the possibility that some Eurasian populations could have been replaced, and the status of Neanderthals is indecisive. Demographic inferences from haplotype trees have been inconsistent, so few definitive conclusions can be made at this time. Haplotype trees from human parasites offer additional insights into human evolution and raise the possibility of an Asian isolate of humanity, but once again not in a definitive fashion. Haplotype trees can also indicate which genes were subject to positive selection in the lineage leading to modern humans. Genetics provides many insights into human evolution, but those insights need to be integrated with fossil and archaeological data to yield a fuller picture of the origin of modern humans.

Emigration and Immigration↗

PubMed2004

Novel haplotypes in 17q21 are associated with progressive supranuclear palsy.

Progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) are sporadic neurodegenerative diseases presenting as atypical parkinsonian disorders, characterized by the presence of tau-positive neurofibrillary tangles. Recently, an extended haplotype (H1E) of 787.6 kb that comprises several genes including MAPT showed increased association with PSP. The objective of this study was to determine the size of the H1E haplotype associated with PSP and CBD in different populations and to identify specific subhaplotypes in the background of H1E haplotype. Nineteen single nucleotide polymorphisms (SNPs) in the 17q21 region were genotyped in two case-control samples. The SNPs that were associated with higher risk for the disease in the homozygous state delimit a region of more that 1 Mb. Haplotype analyses in the Spanish sample showed that the most frequent haplotype found among the patients (H1E'), which extends 1.04 Mb and contains several genes such as MAPT, CRHR1, IMP5, Saitohin, WTN3, and NSF. A specific subhaplotype (H1E'A) was present in 16% of PSP patients but was not observed in the controls. Furthermore, the H2E'A haplotype, was rarely present in the disease group suggesting that it plays a protective role. The identification of these specific subhaplotypes that modify risk for PSP/CBD supports the hypothesis that a pathogenic allele exists in a subgroup of PSP patients.

Adult↗

PubMed2003

The effect of HLA-DR on susceptibility to rheumatoid arthritis is influenced by the associated lymphotoxin alpha-tumor necrosis factor haplotype.

OBJECTIVE: HLA-DRB1, a major genetic determinant of susceptibility to rheumatoid arthritis (RA), is located within 1,000 kb of the gene encoding tumor necrosis factor (TNF). Because certain HLA-DRB1*04 subtypes increase susceptibility to RA, investigation of the role of the TNF gene is complicated by linkage disequilibrium (LD) between TNF and DRB1 alleles. By adequately controlling for this LD, we aimed to investigate the presence of additional major histocompatibility complex (MHC) susceptibility genes. METHODS: We identified 274 HLA-DRB1*04-positive cases of RA and 271 HLA-DRB1*04-positive population controls. Each subject was typed for 6 single-nucleotide polymorphisms within a 4.5-kb region encompassing TNF and lymphotoxin alpha (LTA). LTA-TNF haplotypes in these unrelated individuals were determined using a combination of family data and the PHASE software program. RESULTS: Significant differences in LTA-TNF haplotype frequencies were observed between different subtypes of HLA-DRB1*04. The LTA-TNF haplotypes observed were very restricted, with only 4 haplotypes constituting 81% of all haplotypes present. Among individuals carrying DRB1*0401, the LTA-TNF 2 haplotype was significantly underrepresented in cases compared with controls (odds ratio 0.5 [95% confidence interval 0.3-0.8], P = 0.007), while in those with DRB1*0404, the opposite effect was observed (P = 0.007). CONCLUSION: These findings suggest that the MHC contains genetic elements outside the LTA-TNF region that modify the effect of HLA-DRB1 on susceptibility to RA.

Arthritis, Rheumatoid↗

PubMed2004

A functional haplotype of the PADI4 gene associated with rheumatoid arthritis in a Japanese population is not associated in a United Kingdom population.

OBJECTIVE: In the era of postgenomic research, linkage- and association-based strategies are beginning to reveal novel complex disease genes. Using such an approach, a functional haplotype of the peptidylarginine deiminase 4 gene (PADI4) has recently been identified as a gene conferring susceptibility to rheumatoid arthritis (RA) in a Japanese population. In the present study, we investigated the association of single-nucleotide polymorphisms (SNPs) in the PADI4 gene with RA in a UK population. METHODS: Association with 4 exonic SNPs (padi4_89*G/A, padi4_90*T/C, padi4_92*G/C, and padi4_104*T/C), mapping to the PADI4 gene and defining a haplotype previously reported to be associated with RA, was investigated. Genotyping was performed using 5' allelic discrimination assays. Estimated haplotypes were generated using the expectation-maximization algorithm, and frequencies of the SNPs and haplotypes were compared between unrelated Caucasian RA patients from the UK (n = 839) and population controls (n = 481). RESULTS: Allele frequencies for the 4 SNPs in the UK population were similar to those reported in the Japanese control population, but none of these was associated with RA. As in the Japanese population, the SNPs in the UK population defined 2 major haplotypes, but neither was associated with RA (P = 0.79). CONCLUSION: A PADI4 susceptibility haplotype associated with RA in a Japanese population is not associated with RA in a UK population. Other genes involved in the citrullinating pathway remain strong candidate RA-susceptibility genes and require further investigation.

Adult↗

PubMed2005

Association of SLC11A1 (NRAMP1) with persistent oligoarticular and polyarticular rheumatoid factor-negative juvenile idiopathic arthritis in Finnish patients: haplotype analysis in Finnish families.

OBJECTIVE: The SLC11A1 (formerly called NRAMP1) gene is important in natural resistance to a variety of intracellular infections mediated by macrophages and has been proposed as a candidate gene for autoimmune disease susceptibility. The aim of this study was to examine susceptibility in Finnish patients with persistent oligoarticular and polyarticular rheumatoid factor (RF)-negative juvenile idiopathic arthritis (JIA) due to the presence of the SLC11A1 locus on chromosome 2. METHODS: A total of 234 Finnish JIA nuclear families and 639 elderly Finnish controls without a history of JIA were evaluated for association with JIA at 3 intragenic single-nucleotide polymorphisms: an intragenic insertion/deletion, a promoter microsatellite (NRAMP1), and a 3' microsatellite (D2S1471). RESULTS: Analysis of marker haplotypes demonstrated a strong association of Finnish JIA with 6-marker, 4-marker, and 2-marker haplotypes. Most impressively, 1 of the 6-marker haplotypes showed an odds ratio (OR) of 4.0 (95% confidence interval [95% CI] 2.6-6.2) in all JIA patients, 3.5 (95% CI 1.9-6.5) in those with persistent oligoarticular JIA, and 4.1 (95% CI 2.5-6.7) in those with polyarticular RF-negative JIA. Stratification of the haplotype data suggested that susceptibility to JIA in the haplotype spanning the SLC11A1 locus is independent (P < 0.01) of an association with a DRB1 JIA shared epitope (DRB1*JIASE) that includes well-characterized strong susceptibility to DRB1*08 and *11 alleles. This SLC11A1 haplotype also had an additive effect with DRB1*JIASE in those with polyarticular, but not those with persistent oligoarticular, disease (P = 0.06, OR 2.9 [95% CI 0.9-9.2] versus P = 0.5, OR 1.6 [95% CI 0.4-6.0]). CONCLUSION: Taken together, these data provide support for the existence of a locus at or near SLC11A1 that is a strong susceptibility factor for JIA in Finnish patients.

Adolescent↗

PubMed2004

PTCH polymorphism is associated with the rate of increase in basal cell carcinoma numbers during follow-up: preliminary data on the influence of an exon 12-exon 23 haplotype.

After first presentation with a basal cell carcinoma (BCC), patients demonstrate interindividual diversity in the rate of development of further BCCs (number/year of follow-up). The mechanism for this variation is unknown. In this study, we evaluated whether PTCH variants mediate this phenomenon. We used negative binomial regression analysis to identify associations between BCC numbers/year and host characteristics, parameters of exposure to ultraviolet radiation (UVR), and PTCH exon 12(1686) C/T, intron 15(2560+9) G/C, and exon 23(3944) C/T genotypes and haplotypes in 279 BCC cases who presented with an initial tumor on the head/neck. PTCH genotypes were not significantly associated with BCCs/year, although cases with two copies of the C1686-C3944 haplotype developed significantly fewer BCCs/year than those without this haplotype (rate ratio = 0.44; 95% CI = 0.27-0.71). Cases with one copy of T1686-T3944 developed more BCCs/year (rate ratio = 2.46; 95% CI = 1.27-3.97) than those without the haplotype. We found no significant associations between BCCs/year and the other PTCH haplotypes studied. We reexamined the association of C1686-C3944 with BCCs/year in a model that included UVR exposure parameters (sunburning in childhood, sunbathing score, intermittency of exposure between 40 and 60 years of age, exposure in hours/year) and skin type, gender, and age at first presentation. The association between C1686-C3944 and BCCs/year remained significant (rate ratio = 0.44; 95% CI = 0.26-0.73 for two copies of the haplotype). The data show that allelic variation in PTCH contributes to the rate of development of BCC.

Adult↗

PubMed2002

Haplotype frequency estimation in patient populations: the effect of departures from Hardy-Weinberg proportions and collapsing over a locus in the HLA region.

Haplotype analyses are an important area in the study of the genetic components of human disease. Associations between markers and disease loci that are not evident with a single marker locus may be identified in multi-locus marker analyses using estimated haplotype frequencies (HFs). Procedures that make use of the expectation-maximization (EM) algorithm to estimate HFs from unphased genotype data are in common use in genetic studies. The EM algorithm uses these unphased genotype frequencies along with the assumption of Hardy-Weinberg proportions (HWP) to converge on HF estimates. In this paper, we assess the accuracy of EM estimates of HFs in patients with type I diabetes for whom the true haplotypes are known, but the data are analyzed ignoring family information to allow comparison between estimated and true frequencies. The data consist of six HLA loci with high levels of polymorphism and a range of departures from HWP and linkage equilibrium. While the overall accuracy of the EM estimates is good, there can be large over- and underestimates of particular HFs, even for common haplotypes, especially when the loci involved deviate significantly from HWP. Estimating HFs for three or more loci and then collapsing over loci so as to generate two locus haplotypes can improve the accuracy of the estimation. The collapsing procedure is most beneficial when one of the loci in the two-locus haplotype of interest deviates significantly from HWP and the locus collapsed over is in linkage disequilibrium with the other loci.

Algorithms↗

PubMed2004

Comparison of prospective and retrospective methods for haplotype inference in case-control studies.

We compare bias and power of three methods for haplotype inference on disease risk using unphased genotype data from a case-control study. We examine the prospective score test of Schaid et al., a novel modification of the prospective estimating equations of Zhao et al. and the retrospective likelihood of Epstein and Satten. We find that all three approaches are roughly comparable when the haplotype effect on disease odds follows a multiplicative model. However, for dominant and recessive models of haplotype effect, the retrospective-likelihood method has increased efficiency with respect to the prospective methods. As all three methods assume haplotype frequencies are in Hardy-Weinberg Equilibrium (HWE), we compare the robustness of each procedure to departures from HWE. We find the prospective methods are robust to departure from HWE, while the retrospective-likelihood method is biased for dominant and recessive models of haplotype effect. To remedy this limitation of the retrospective-likelihood method, we propose a modification that allows for a non-negative fixation index (common to all haplotype pairs) and show it dramatically reduces the bias of the retrospective likelihood when HWE is violated.

Algorithms↗

PubMed2004

SNPs, haplotypes, and model selection in a candidate gene region: the SIMPle analysis for multilocus data.

Modern molecular techniques make discovery of numerous single nucleotide polymorphims (SNPs) in candidate gene regions feasible. Conventional analysis relies on either independent tests with each variant or the use of haplotypes in association analysis. The first technique ignores the dependencies between SNPs. The second, though it may increase power, often introduces uncertainty by estimating haplotypes from population data. Additionally, as the number of loci expands for a haplotype, ambiguity in interpretation increases for determining the underlying genetic components driving a detected association. Here, we present a genotype-level analysis to jointly model the SNPs via a SNP interaction model with phase information (SIMPle) to capture the underlying haplotype structure. This analysis estimates both the risk associated with each variant and the importance of phase between pairwise combinations of SNPs. Thus, rather than selecting between genotype- or haplotype-level approaches, the SIMPle method frames the analysis of multilocus data in a model selection paradigm, the aim to determine which SNPs, phase terms, and linear combinations best describe the relation between genetic variation and a trait of interest. To avoid unstable estimation due to sparse data and to incorporate both the dependencies among terms and the uncertainty in model selection, we propose a Bayes model averaging procedure. This highlights key SNPs and phase terms and yields a set of best representative models. Using simulations, we demonstrate the utility of the SIMPle model to identify crucial SNPs and underlying haplotype structures across a variety of causal models and genetic architectures.

Bayes Theorem↗

PubMed2005

Haplotype sharing transmission/disequilibrium tests that allow for genotyping errors.

The present study introduces new Haplotype Sharing Transmission/Disequilibrium Tests (HS-TDTs) that allow for random genotyping errors. We evaluate the type I error rate and power of the new proposed tests under a variety of scenarios and perform a power comparison among the proposed tests, the HS-TDT and the single-marker TDT. The results indicate that the HS-TDT shows a significant increase in type I error when applied to data in which either Mendelian inconsistent trios are removed or Mendelian inconsistent markers are treated as missing genotypes, and the magnitude of the type I error increases both with an increase in sample size and with an increase in genotyping error rate. The results also show that a simple strategy, that is, merging each rare haplotype to a most similar common haplotype, can control the type I error inflation for a wide range of genotyping error rates, and after merging rare haplotypes, the power of the test is very similar to that without merging the rare haplotypes. Therefore, we conclude that a simple strategy may make the HS-TDT robust to genotyping errors. Our simulation results also show that this strategy may also be applicable to other haplotype-based TDTs.

Algorithms↗

PubMed2003

Independent mutational events are rare in the ATM gene: haplotype prescreening enhances mutation detection rate.

Mutations in the ATM gene are responsible for the autosomal recessive disorder ataxia-telangiectasia (A-T). Many different mutations have been identified using various techniques, with detection efficiencies ranging from 57 to 85%. In this study, we employed short tandem repeat (STR) haplotypes to enhance mutation identification in 55 unrelated A-T families of Iberian origin (20 Spanish, 17 Brazilian, and 18 Hispanic-American); we were able to identify 95% of the expected mutations. Allelic sizes were standardized based on a reference sample (CEPH 1347-2). Subsequent mutation screening was performed by PTT, SSCP, and DHPLC, and abnormal regions were sequenced. Many STR haplotypes were found within each population and six haplotypes were observed across several of these populations. Single nucleotide polymorphism (SNP) haplotypes further suggested that most of these common mutations are ancestrally related, and not hot spots. However, two mutations (8977C>T and 8264_8268delATAAG) may indeed be recurring mutational events. Common haplotypes were present in 13 of 20 Spanish A-T families (65%), in 11 of 17 Brazilian A-T families (65%), and, in contrast, in only eight of 18 Hispanic-American families (44%). Three mutations were identified that would be missed by conventional screening strategies. In all, 62 different mutations (28 not previously reported) were identified and their associated haplotypes defined, thereby establishing a new database for Iberian A-T families, and extending the spectrum of worldwide ATM mutations.

Ataxia Telangiectasia↗

PubMed1992

Two missense mutations causing mild hyperphenylalaninemia associated with DNA haplotype 12.

The genetic defects responsible for most phenylketonuria (PKU) and hyperphenylalaninemia (HPA) cases are located in the phenylalanine hydroxylase (PAH) gene. Approximately 50-60 mutations have been reported in Caucasians and are reflected in a wide range of clinical severities. Most mutations are linked to specific haplotypes, as defined by eight polymorphic restriction sites in the PAH gene. We hypothesized that there is at least one mild mutation linked to haplotype 12 in the Swedish PKU/HPA population, since 7 of 8 patients carrying haplotype 12 had mild HPA. Sequence analysis revealed a C-to-G transversion at the second base of codon 322, resulting in a substitution of glycine for alanine, in four mutant haplotype 12 genes, and a G-to-A transition at the second base of codon 408, resulting in a substitution of glutamine for arginine, in another three mutant haplotype 12 genes. These mutations segregated with mutant haplotype 12 alleles in nuclear families but were not present on normal or other mutant alleles. Both mutations were tested in a eukaryotic expression system in which enzyme activities of different mutant PAH enzymes reflect the relative severities of the mutations, although these in vitro activities cannot be translated directly into in vivo hepatic activities. The A322G mutant PAH had about 75% and the R408Q mutant PAH about 55% of the wild-type PAH enzyme activity. These in vitro activities are the highest reported for mutant PAH enzymes produced in the same expression system.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Metabolism, Inborn Errors↗

PubMed2005

Linear allele-specific long-range amplification: a novel method of long-range molecular haplotyping.

Haplotypes have been repeatedly shown to be more powerful than collections of single-locus markers in gene-mapping studies. Various haplotyping methods including statistical estimation are employed but molecular haplotyping, the acquisition of information directly on physical DNA sequences, has been in demand for its accuracy and independence from family pedigrees. We investigated the allelic specificity of long-range PCR, which was successful for long-range haplotyping in recent reports, and found problems of initial mispriming and crossover amplification significantly confounded its application. Based on these observations, we designed a novel method based on linear amplification of a hemizygous DNA segment with a single phosphorothioate-modified oligonucleotide. Our results revealed, with a single nucleotide polymorphism as the discriminative marker, downstream haplotypes of 14-15 kb DNA segment could be confidently scored. With two rounds of the method and five single nucleotide polymorphisms, molecular haplotypes of 29.3 kb spanning the HCR and CDSN genes, two genes associated with the susceptibility of psoriasis, of 11 members, belonging to a CEPH family, were revealed. Clear Mendelian segregation of 35 highly heterozygous SNPs confirmed the accuracy of the method. Problems of low specificity associated with long-range PCR were not observed. The simplicity, along with long-sequence accessibility and feasibility of a single nucleotide difference as the discriminative marker indicated our method holds promise for future gene-mapping studies.

Adenomatous Polyposis Coli↗

PubMed1989

Pulsed field gel electrophoresis identifies a high degree of variability in the number of tandem 21-hydroxylase and complement C4 gene repeats in 21-hydroxylase deficiency haplotypes.

The human steroid 21-hydroxylase gene, CYP21B, and its closely homologous pseudogene, CYP21A, are each normally located centromeric to a complement C4 gene C4B and C4A respectively, in an organization suggesting tandem duplication of a CYP21 + C4 unit. Such an organization has been considered to facilitate gene deletion and addition events by unequal crossover between the tandem repeats. However, the large size (approximately 30 kb) of the individual CYP21 + C4 repeat units together with the difficulty in identifying reliable CYP21A- and CYP21B-specific markers has prevented direct monitoring of gene organization on individual haplotypes by conventional Southern analyses. In the present investigation we have sought to clarify the CYP21 and C4 gene organization in members of 32 British 21-hydroxylase deficiency families by employing additional experimental approaches, notably a long-range restriction mapping approach, which permits assessment through a VNTR type of analysis, of the number of CYP21 and C4 units on individual haplotypes. Our results show that there is a very high frequency (33%) of 21-hydroxylase deficiency haplotypes where functional CYP21B gene sequence has been removed as a consequence of CYP21 + C4 gene deletion while several haplotypes show evidence of gene addition. In each case that we have investigated the gene deletion and gene addition haplotypes differ in length from conventional haplotypes by integral multiples of approximately 30 kb, which strongly supports the involvement of unequal crossover mechanisms. Additionally, the comparatively frequent occurrence of CYP21 fusion genes which contain both CYP21A- and CYP21B-associated markers is suggested by the combined data from Southern analyses, long-range restriction mapping and characterization of selected regions of CYP21 genes which have been amplified in vitro.

Adrenal Hyperplasia, Congenital↗