Characterization of mutations on the rare duplicated C4/CYP21 haplotype in steroid 21-hydroxylase deficiency

We have defined the mutations causing congenital adrenal hyperplasia in three Swedish patients carrying a rare haplotype containing two mutated steroid 21-hydroxylase genes (CYP21) in addition to one pseudogene (CYP21P). The presence of such haplotypes complicates genetic diagnosis and screening of mutations in 21-hydroxylase deficiency, and we show how these genotypes can be resolved by amplification and analysis of each gene separately. In all cases, the rare haplotype carried the same combination of disease-causing mutations; one of the genes had the splice mutation at base 659 in intron 2, and the other had the nonsense mutation at base 1999 in exon 8 (CAG to TAG). We have thus characterized the most common haplotype containing duplicated CYP21 genes. The frequency of this haplotype is low, and if additional such haplotypes are present, they are rare in this population.     

Two distinct areas of unequal crossingover within the steroid 21-hydroxylase genes produce absence of CYP21B

We mapped crossover sites in chimeric, recombinant CYP21 genes from six patients with salt-losing congenital adrenal hyperplasia (CAH). Nucleotide sequences unique to the CYP21A pseudogene or to the active CYP21B gene were mapped using gene-specific restriction sites and oligonucleotide hybridizations. Each chimeric CYP21 gene in the CYP21-deletion linked haplotypes contained sequences near the 5' end that were characteristic of CYP21A and only a single transition from sequences of CYP21A to those of CYP21B at the 3' end. The transitions all occurred within either of two discrete regions (+470 to +999 and +1375 to +1993). All eight chimeric CYP21 genes coupled with HLA-Bw47 in five unrelated patients had the CYP21A-CYP21B sequence transition within the same gene region (+1375 to +1993). One of the three other "CYP21B deletion" haplotypes (HLA-B7) had a sequence transition within this same region, while in the other two haplotypes (HLA-B61 and HLA-B18) the transition occurred between base pairs +470 and +999. By contrast, both CYP21 genes in a haplotype containing a gene conversion of CYP21B to CYP21A contained apparent transitions between sequences of CYP21A and CYP21B. We conclude that a single, unequal crossingover between the CYP21A and the CYP21B genes yields deletion of the active CYP21 gene and salt-losing CAH and that these crossingovers do not occur randomly within the CYP21 genes of our patients.     

CYP21/C4 gene organisation in Italian 21-hydroxylase deficiency families

Summary A total of 33 Italian 21-hydroxylase (21-OH) deficiency families were investigated using a combination of short and long range restriction mapping of the CYP21/C4 gene cluster. The analyses revealed that large-scale length polymorphism in this gene cluster strictly conformed to a compound variable number of tandem repeats (VNTR) plus insertion system with between one and four CYP21 + C4 units and seven BssHII restriction fragment length polymorphisms (RFLPs) (75kb, 80kb, 105kb, 110kb, 135kb, 140kb and 180kb). A total of 9/66 disease haplotypes, but only 1/61 nondisease haplotypes, showed evidence of gene addition by exhibiting three or more CYP21 + C4 repeat units. Of these, two were identified in one 21-OH deficiency patient who has a total of eight CYP21 + C4 units, being homozygous for the HLA haplotype DR2 DQ2 B5 A28. This haplotype carries four CYP21 + C4 units, three of which contain CYP21A-like genes and one of which contains a CYP21B-like gene that presumably carries a pathological point mutation. Of the other gene addition haplotypes associated with 21-OH deficiency, four show three CYP21 + C4 units flanked by HLA-DR1 and HLA-B14 markers. Although such haplotypes have commonly been associated with non-classical 21-OH deficiency, three examples in the present study are unexpectedly found in two salt-wasting patients, who are respectively homozygous or heterozygous for this haplotype. Only 7/66 disease haplotypes showed evidence of a CYP21B gene deletion.     

High-throughput analysis of informative CYP2D6 compound haplotypes

We describe a high-throughput protocol for detecting key polymorphisms in the drug-metabolizing enzyme gene CYP2D6 and a number of linked microsatellites that is both fast and relatively inexpensive to perform. This approach employs GeneScan technology to enable a researcher to determine rapidly the status of seven simple nucleotide polymorphisms in CYP2D6 and also to assay repeat number variation at five closely linked dinucleotide microsatellite loci. The method requires only three PCRs and two GeneScan runs per sample. We anticipate that this will be of value to researchers in three different ways: (1) rapid discrimination of common CYP2D6 alleles, (2) high-resolution haplotyping for association studies involving chromosome 22q13.1 using microsatellite variation, and (3) generation of compound haplotypes for investigating the evolution of CYP2D6 variation. We also report compound haplotype frequencies for an Ashkenazi Jewish and a British sample.     

Structural relationships among the H-2 D-regions of murine MHC haplotypes

The number of genes encoding functional Ag-presenting molecules in the D region of the murine MHC differs among haplotypes. For example, the H-2b D region contains a single "D/L" gene, H-2Db, whereas the d-haplotype encodes two, H-2Dd and Ld. Using D/L specific oligonucleotide probes, we have found that, as with H-2d, the q- and v-haplotypes contain two D/L genes, whereas the other haplotype examined have one. Hybridization analysis using cloned probes that map between H-2Dd and Ld revealed similar structures in each of the three haplotypes (d, q, and v) which have "duplicated" D regions. Two approaches were used to examine allelic relationships among the D/L genes. First, the 5' region of the H-2Db gene was sequenced, and found to be more similar to H-2Ld than to H-2Dd. Second, oligonucleotide probes that distinguish H-2Ld from H-2Dd revealed H-2Ld-related genes in several haplotypes, including the duplicated haplotypes H-2q and H-2v. Analogous probes specific for H-2Dd, however, did not detect similar sequences in the other haplotypes. We interpret these results to mean that the three duplicated D regions arose from a common duplication event, and share the five gene structure of the D region cluster defined in H-2d. However, subsequent events have generated sequence divergence at the D-locus.     

Extended MHC haplotypes and CYP21/C4 gene organisation in Irish 21-hydroxylase deficiency families

Summary We have analysed fifteen classical 21-hydroxylase deficiency families from throughout Southern Ireland and report the serologically defined HLA-A, HLA-B, HLA-Cw, HLA-DR, C4A and C4B polymorphisms that characterize the inferred disease haplotypes. Additionally, we have used a combination of short and long range restriction mapping procedures in order to characterize the CYP21/C4 gene organization associated with individual serologically defined haplotypes. The results obtained indicate that disease haplotypes are characterized by a high frequency (33%) of CYP21B gene deletion and 8 out of 10 such deletion haplotypes are represented by the extended haplotype HLA-DR1, C4BQo, C4A3, HLA-B40(w60), HLA-Cw3, HLA-A3. Large scale length polymorphism in the CYP21/C4 gene cluster was found to conform strictly to a variable number of tandem repeats model with 4 alleles being detected. Disease haplotypes in which defective CYP21B gene expression is inferred to result from pathological point mutations show extensive diversity of associated HLA markers and include two examples of the extended HLA haplotype HLA-DR3, B8, Cw7, A1 haplotype, which has previously been reported to be negatively associated with 21-hydroxylase deficiency. One unusual disease haplotype has two CYP21 + C4 units, both of which appear to contain CYP21B-like genes.     

Association of cytochrome P450 genes polymorphisms (CYP1A1 and CYP1A2) with the development of chronic obstructive pulmonary disease in Bashkortostan

To assess the role that polymorphisms of cytochrome P450 genes play in genetic predisposition to chronic obstructive pulmonary disease (COPD), the allele and genotype distributions of CYPIA1 (2455 A/G, 3801T/C) and CYP1A2 (-2464T/delT, -163C/A) genes were studied in Tatar and Russian COPD patients and in cases of healthy individuals (Russian, Tatar and Bashkir), residents of Bashkortostan. It was shown that the CYP1A1 and CYP1A2 genes haplotypes frequency distribution patterns do not differed between Tatars and Russians ethnic groups (chi2 = 0.973, df = 3, p = 1.00 and chi2 = 1.546, df = 3, p = 0.92, respectively). Analysis of the the CYP1A1 and CYP1A2 genes haplotypes revealed statistically significant differences in the haplotypes frequency distributions between Bashkirs versus Russians and Tatars (chi2 = 12.328, df= 3,p = 0.008; chi2 = 9.218, df=3, p = 0.034, respectively for CYP1A1 gene and (chi2 = 18.779, df=3, p = 0.0001, chi = 14.326, df=3, p = 0.003, respectively for CYP1A2 gene). The (-2467)delT allele and CYP1A2*1D haplotype of CYPIA2 gene was associated with higher risk of COPD in Tatar ethnic group (OR = 1.83, 95% CI 1.24-2.71, chi2 = 9.48, p = 0.003 and chi2 = 9.733, p = 0.0027, Pcor = 0.008; OR = 3.908, 95% CI 1.56-10.19, respectively). On the other hand the CYP1A2*1A haplotype had protective effect (chi2 = 6.319, p = 0.0127, Pcor = 0.038; OR = 0.6012, 95% CI 0.402-0.898). But at the same time we did not find any differences in the genotypes and haplotypes frequency distributions of the CYP1A2 gene within the patients and healthy groups in Russian ethnic group. We also did not find any association of CYP1A1 gene with COPD in ethnic groups of Bashkortostan.     

DNA haplotype dependency of debrisoquine 4-hydroxylase (CYP2D6) expression among extensive metabolisers

Deficient debrisoquine/sparteine type oxidation is inherited as an autosomal recessive trait. Of all Caucasians, 5–10% are poor metabolisers, due to the absence of cytochrome P4502D6. Extensive metabolisers (EMs) exhibit highly variable metabolic activity. We investigated the relationship between CYP2D6 activity and genotypes of the CYP2D locus in a large set of French Caucasian families. Genotypes concern both common mutations affecting the enzyme activity and linkedBamHI polymorphisms of the locus. We found, like other authors, that in EMs part of the heterogeneity is explained by a subgroup of individuals heterozygous for a mutant allele. However, a second level of heterogeneity was detected among individuals not carrying mutations, and this was related to a polymorphicBamHI-defined DNA haplotype. Different combinations of haplotypes are associated with differences in CYP2D6 metabolic activity. This finding might help to clarify the conflicting data on the relation between CYP2D6 activity and susceptibility to lung cancer.     

Killer Ig-like receptor haplotype analysis by gene content: evidence for genomic diversity with a minimum of six basic framework haplotypes,each with multiple subsets

Killer Ig-like receptor (KIR) genes constitute a multigene family whose genomic diversity is achieved through differences in gene content and allelic polymorphism. KIR haplotypes containing a single activating KIR gene (A-haplotypes), and KIR haplotypes with multiple activating receptor genes (B-haplotypes) have been described. We report the evaluation of KIR gene content in extended families, sibling pairs, and an unrelated Caucasian panel through identification of the presence or absence of 14 KIR genes and 2 pseudogenes. Haplotype definition included subtyping for the expressed and nonexpressed KIR2DL5 variants, for two alleles of pseudogene 3DP1, and for two alleles of 2DS4, including a novel 2DS4 allele, KIR1D. KIR1D appears functionally homologous to the rhesus monkey KIR1D and likely arose as a consequence of a 22 nucleotide deletion in the coding sequence of 2DS4, leading to disruption of Ig-domain 2D and a premature termination codon following the first amino acid in the putative transmembrane domain. Our investigations identified 11 haplotypes within 12 families. From 49 sibling pairs and 17 consanguineous DNA samples, an additional 12 haplotypes were predicted. Our studies support a model for KIR haplotype diversity based on six basic gene compositions. We suggest that the centromeric half of the KIR genomic region is comprised of three major combinations, while the telomeric half can assume a short form with either 2DS4 or KIR1D or a long form with multiple combinations of several stimulatory KIR genes. Additional rare haplotypes can be identified, and may have arisen by gene duplication, intergenic recombination, or deletions.     

Genetic sophistication of human complement components C4A and C4B and RP-C4-CYP21-TNX (RCCX) modules in the major histocompatibility complex

Human populations are endowed with a sophisticated genetic diversity of complement C4 and its flanking genes RP, CYP21, and TNX in the RCCX modules of the major histocompatibility complex class III region. We applied definitive techniques to elucidate (a) the complement C4 polymorphisms in gene sizes, gene numbers, and protein isotypes and (b) their gene orders. Several intriguing features are unraveled, including (1) a trimodular RCCX haplotype with three long C4 genes expressing C4A protein only, (2) two trimodular haplotypes with two long (L) and one short (S) C4 genes organized in LSL configurations, (3) a quadrimodular haplotype with four C4 genes organized in a SLSL configuration, and (4) another quadrimodular structure, with four long C4 genes (LLLL), that has the human leukocyte antigen haplotype that is identical to ancestral haplotype 7.2 in the Japanese population. Long-range PCR and PshAI-RFLP analyses conclusively revealed that the short genes from the LSL and SLSL haplotypes are C4A. In four informative families, an astonishingly complex pattern of genetic diversity for RCCX haplotypes with one, two, three and four C4 genes is demonstrated; each C4 gene may be long or short, encoding a C4A or C4B protein. Such diversity may be related to different intrinsic strengths among humans to defend against infections and susceptibilities to autoimmune diseases.     

Deletion of the entire cytochrome P450 CYP2D6 gene as a cause of impaired drug metabolism in poor metabolizers of the debrisoquine/sparteine polymorphism.

The debrisoquine/sparteine polymorphism is associated with a clinically important genetic deficiency of oxidative drug metabolism. From 5% to 10% of Caucasians designated as poor metabolizers (PMs) of the debrisoquine/sparteine polymorphism have a severely impaired capacity to metabolize more than 25 therapeutically used drugs. The impaired drug metabolism in PMs is due to the absence of cytochrome P450IID6 protein. The gene controlling the P450IID6 protein, CYP2D6, is located on the long arm of chromosome 22. A pseudogene CYP2D8P and a related gene CYP2D7 are located upstream from CYP2D6. This gene locus is highly polymorphic. After digestion of genomic DNA with XbaI endonuclease, restriction fragments of 11.5 kb and 44 kb represent mutant alleles of the cytochrome CYP2D6 gene locus associated with the PM phenotype. In order to elucidate the molecular mechanism of the mutant allele reflected by the XbaI 11.5-kb fragment, a genomic library was constructed from leukocyte DNA of one individual homozygous for this fragment and screened with the human IID6 cDNA. The CYP2D genes were isolated and characterized by restriction mapping and partial sequencing. We demonstrate that the mutant 11.5-kb allele results from a deletion involving the entire functional CYP2D6 gene. This result provides an explanation for the total absence of P450IID6 protein in the liver of these PMs.     

Genetic analysis of the H-2D region using a new intra-D-region recombinant mouse strain

A rare D-region recombination event which gave rise to the B10.RQDB major histocompatibility complex haplotype has been examined to ascertain the nature of the crossover and to determine which class I genes are present in the new alignment of D-region genes. Serologic analysis have shown that the B10 . RQDB major histocompatibility complex recombinant mouse inherited the H-2Dd gene from the B10.T(6R) parental line and the H-2Db gene from the B10.A(2R) parental line, representing the first example of an intra-D-region crossover resulting from an intercross. Previous molecular genetic analyses of the d and b haplotypes revealed structural diversity in the organization of their D-region gene clusters. Hence, the D region is comprised of five class I genes in the d haplotype and only one in the b haplotype. Because allelic relationships among the various D-region genes are not defined, either a homologous or nonhomologous alignment of genes has generated the RQDB crossover. Therefore, the possibility that all three D-region antigen-presenting molecules (Dd, Ld, and Db) might be encoded by the RQDB haplotype was examined. Fluorescence-activated cell sorter and cytotoxic T lymphocyte analyses revealed no detectable levels of H-2Ld cell-surface expression, confirming earlier studies with antibody-mediated cytotoxicity and immunoprecipitation. Southern blot analysis localized the recombination point to within a 1-kb region at the centromeric end of the H-2Ld gene on the B10 . T(6R) chromosome in a region of high homology to the H-2Db gene on the B10 . A(2R) chromosome. Together, these studies define the D region of the RQDB haplotype as containing the five class I genes: Dd, D2d, D3d, D4d, and Db. In addition to providing insight into rare recombination events in the D region, the B10.RQDB mouse should be a useful tool for exploring the function of D-region genes.     

Characterization of pharmacogenetically relevant CYP2D6 and ABCB1 gene polymorphisms in a Portuguese population sample

Differences in metabolism of drugs can lead to severe toxicity or therapeutic failure. In addition to cytochrome P450 2D6, which plays a critical role in drug metabolism, ABCB1 encoded P‐glycoprotein (PGP) is also an important determinant in drug bioavailability. The genes encoding these molecules are highly variable among populations and, given their clinical importance in drug therapy, determining CYP2D6 and ABCB1 allele frequencies in specific populations is very important for useful application in clinical settings. In this study the frequency of the pharmacologically relevant CYP2D6*3, *4, *5, *6 allelic variants and gene duplication, and ABCB1 C1236T and C3435T gene polymorphisms and their haplotypes was determined in a population sample of 100 Portuguese healthy subjects. CYP2D6 allele frequencies were 1.4% (*3), 13.3% (*4), 2.8% (*5), 1.8% (*6) and 6.1% (gene duplication), with 5% of the individuals classified as PM and 8.4% as UM. The frequencies obtained for the non‐functional alleles and for the CYP2D6 gene duplication are in agreement with other South European populations, and reinforce the previously suggested south/north gradient of CYP2D6 duplications. Allelic frequencies for the ABCB1 polymorphisms were 52% (3435C) and 54% (1236C) and the most common haplotype (1236C‐3435C) occurred with a frequency of 45.5%. Although allele and haplotype frequency data for ABCB1 in Southern Europe is limited, some discrepancies were found with other European populations, with possible therapeutic implications for PGP substrate drugs. Copyright © 2009 John Wiley & Sons, Ltd.     

Origin of rat beta-globin haplotypes containing three and five genes

We have reported in rat three adult beta-gene haplotypes containing either five or three genes. Detailed sequence analysis reveals that the leftmost gene is the major gene and that at the opposite end downstream lies the minor gene. All of the genes lying between them are minor-major hybrids indicating their origin by unequal crossing-over. In two haplotypes beta-globin genes were found with an L1(1) element inserted directly into IVS2. The described results allow the formulation of a pathway of mutational events leading from the ancient two-beta-gene rodent ancestor through a three-gene haplotype to five-gene haplotypes, one of which is postulated to have arisen in common laboratory strains since their capture in the wild.     

Duplication of the CYP21A2 gene complicates mutation analysis of steroid 21-hydroxylase deficiency: characteristics of three unusual haplotypes

Steroid 21-hydroxylase deficiency, the primary cause of congenital adrenal hyperplasia, is caused by defects of the CYP21A2 gene. As a complement to hormonal measurements, mutation analysis of CYP21A2 is an important tool in the diagnosis of steroid 21-hydroxylase deficiency. Contemporary mutation-detection protocols based on the polymerase chain reaction often depend on the assumption that no more than one CYP21A2 gene is present on each chromosome 6. We describe three haplotypes with two CYP21A2 genes on the same chromosome, with defects typical of salt-losing steroid 21-hydroxylase deficiency in one of those genes, but not necessarily in the other. The frequency of these haplotypes in the general population is 6/365 (1.6%), so they are no less common than other haplotypes that indeed carry steroid 21-hydroxylase deficiency. Chromosomes that carry two CYP21A2 genes therefore represent a significant pitfall in the molecular diagnosis of steroid 21-hydroxylase deficiency. We recommend that, whenever CYP21A2 mutation analysis of an individual who is not a known carrier of steroid 21-hydroxylase deficiency is performed, the overall structure of the CYP21/ C4 region (the RCCX area) is determined by haplotyping to avoid erroneous assignment of carrier status.     

Isolation and characterization of three class II MHC genomic clones from the chicken

A genomic library was constructed from sperm DNA from an individual of the inbred chicken line G-B2, MHC haplotype B6. The library was screened with a chicken class II probe (beta 2 exon specific) and three MHC class II beta chain genomic clones were isolated. The restriction maps of the three clones showed that each of the three clones was unique. The position of the beta chain sequence was located in each of the three genomic clones by Southern blot hybridization. Subclones containing the beta chain gene were produced from each of the genomic clones and the orientation of the leader peptide, beta 1, beta 2, transmembrane, and cytoplasmic exons was determined by Southern blot hybridization and nucleotide sequencing. The complete nucleotide sequence of two of the three subclones was determined. Comparison of the nucleotide and predicted amino acid sequences of the two subclones with other class II beta chain sequences showed that the B6 chicken beta chain genes are evolutionarily related to the class II beta chain genes from chickens of other MHC haplotypes, and to class II beta chain genes from other species. Analysis of Southern blots of B6 chicken DNA, as well as the isolation of the three beta chain genes, suggests that chickens of the B6 haplotype possess at least three MHC class II beta chain genes.     

Sequences associated with human iris pigmentation

To determine whether and how common polymorphisms are associated with natural distributions of iris colors, we surveyed 851 individuals of mainly European descent at 335 SNP loci in 13 pigmentation genes and 419 other SNPs distributed throughout the genome and known or thought to be informative for certain elements of population structure. We identified numerous SNPs, haplotypes, and diplotypes (diploid pairs of haplotypes) within the OCA2, MYO5A, TYRP1, AIM, DCT, and TYR genes and the CYP1A2-15q22-ter, CYP1B1-2p21, CYP2C8-10q23, CYP2C9-10q24, and MAOA-Xp11.4 regions as significantly associated with iris colors. Half of the associated SNPs were located on chromosome 15, which corresponds with results that others have previously obtained from linkage analysis. We identified 5 additional genes (ASIP, MC1R, POMC, and SILV) and one additional region (GSTT2-22q11.23) with haplotype and/or diplotypes, but not individual SNP alleles associated with iris colors. For most of the genes, multilocus gene-wise genotype sequences were more strongly associated with iris colors than were haplotypes or SNP alleles. Diplotypes for these genes explain 15% of iris color variation. Apart from representing the first comprehensive candidate gene study for variable iris pigmentation and constituting a first step toward developing a classification model for the inference of iris color from DNA, our results suggest that cryptic population structure might serve as a leverage tool for complex trait gene mapping if genomes are screened with the appropriate ancestry informative markers.     

Variant chromosomal arrangement of adult beta-globin genes in rat

The genomic organization of three haplotypes of beta-globin genes was determined to resolve the question of the number of those genes in rat. Haplotype a, found in inbred strain DA, has three genes or pseudogenes, while haplotypes b, found in AO, Y5 and Wistar strains, and c, found in Wistar strain, have five genes or pseudogenes each. In haplotypes b and c, the first gene is of beta major type and the remaining four are of beta minor type. Partial sequencing of six out of 13 genes shows that duplications of beta minor genes are causing polymorphism in a number of genes. Also, in haplotype b two beta minor genes have a 6.5-kb intron 2, while in haplotype c only one beta minor gene contains such a large intron 2. The three structurally different haplotypes described are not interconvertible by single recombination events. The results indicate that the rat has the highest number of adult beta-globin genes found in mammals so far.     

Linkage disequilibrium between phenylketonuria and RFLP haplotype 1 at the phenylalanine hydroxylase locus in Portugal

Summary RFLPs of 36 normal and 41 mutant alleles at the phenylalanine hydroxylase locus were determined in 31 Portuguese kindreds. A total of 14 haplotypes including 10 normal and 7 mutant alleles were observed. Almost 75% of all mutant alleles were confined within only two haplotypes, namely haplotype 9 (17.1%) and haplotype 1 (56.1%). This frequency of mutant haplotype 1 in Portugal is, to our knowledge, the highest for this mutant haplotype in all studies reported to date. Other mutant haplotypes were either rare (haplotype 2, 9.7%) or totally absent (haplotype 3, 0%). Only 24.5% of all mutant alleles were found to consistently carry identified mutations, particularly R261Q (9.8%), R252W (3.3%), R408W (1.6%) and I94 (3.3%). A new mutation, L249F, located in the seventh exon of the gene, accounted for 6.5% of all mutant alleles in our series. Interestingly, this mutant genotype was consistently associated with mutant haplotype 1 (P<0.01), as also observed for the R261Q mutation. It appears, therefore, that mutant haplotype 1 is genotypically heterogeneous in Portugal and that more than two mutations account for its prevalence in this country.