Screening of CYP21 gene mutations in 129 French patients affected by steroid 21-hydroxylase deficiency

The frequency of 12 different mutations of the steroid 21-hydroxylase gene (CYP21) was investigated in 129 French patients affected by congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase deficiency. Eighty-nine percent of the CAH chromosomes were characterized. The most frequent mutations were a C-G substitution in intron 2, the deletion of the CYP21 gene and a T-A substitution in exon 4 in the severe form of the disease, and a G-T substitution in exon 7 in the nonclassic form. The correlation between the genotypes and the clinical forms of the disease showed marked variation in the phenotype from a single genotype, suggesting that individual variation and undetected additional mutations on the same CAH chromosome accounted for the phenotype. In 65 informative meioses of CAH families, no de novo mutation was found. © Wiley-Liss, Inc.     

Structural characterisation of the distal 5' flanking region of the human interleukin-10 gene

Interleukin-10 (IL-10) is an important immunoregulatory cytokine. The recent characterisation of the proximal 5' flanking region of IL-10 led to the identification of the promoter region. Two polymorphic dinucleotide repeats and 10 single nucleotide polymorphisms (SNPs) have been identified and suggested to be useful genetic markers in several diseases. We have sequenced a further 5275 bp from -9296 to -4021 of the distal part of the 5' flanking region of the human IL-10 gene from the cosmid clone pWE15-4/11. Our sequence analysis reveals a high density of Alu-repeats within the IL-10 gene locus, including three novel, related structures which we term Alu-IL10 (A-C). Using three overlapping PCR products spanning 5110 bp of this distal part of the IL-10 gene the following single base pair substitutions were identified: at -8571 C/T, -8531 G/A, -6752 A/T, -6208 G/C, -5402 C/G. In addition a heterozygous three base pair deletion at -7400 was observed. The SNPs at -8571 C/T and -8531 G/A are contained within an Alu-repeat. These data should further the understanding of how the IL-10 gene is controlled in man and how its function may vary between individuals.     

Structural analysis of the chimeric CYP21P/CYP21 gene in steroid 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase (CYP21) gene. More than 90% of CAH cases are caused by mutations of the CYP21 gene. Approximately 75% of the defective CYP21 genes are generated through intergenic recombination, termed "apparent gene conversion," from the neighboring CYP21Ppseudogene. A chimeric CYP21P/CYP21gene with its 5' end corresponding to CYP21P and 3' end corresponding to CYP21 has been identified. This type of gene is nonfunctional because it produces a truncated protein. We found two distinct chimeric genes in CAH patients. Both genes had a sequence with -300 nucleotides of the 5' head as the CYP21P gene. The coding region consisted of a fusion molecule with the CYP21P gene in two different regions. One of the junctions was located in the chi-like sequence of GCTGGGC in the third intron and the other was in the minisatellite consensus TGGCAGGAGG of exon 5 of the CYP21P gene. In addition, analysis of restriction fragment length polymorphism for these two 3.3-kb chimeric molecules showed that these sequences arose as a consequence of unequal crossover between the CYP21Pand CYP21 genes. It is plausible that both consensus sequences are responsible for the gene conversion of these two chimeric genes.     

Sequence variants in the 5' flanking region of the leptin gene are associated with obesity in women

Few mutations have been found in the human leptin gene and the relationship between leptin gene sequence variation and human overweight is uncertain. To determine whether sequence variation within the leptin gene and its regulatory elements contribute to extreme obesity, we screened ∼3 kb of the 5' flanking region and the three exons in 125 unrelated extremely obese (BMI ≥ 40 kg/m²) and 86 average weight women (BMI < 27 kg/m²). Within the protein coding regions only one heterozygous silent mutation was found (codon 102; AAC/AAT). Within the 5' flanking region, six frequent sequence variants were detected ( q > 0.10), and the allele frequencies of three of these variants differed between obese and average weight Caucasian women (+19, χ²= 4.46, p = 0.035; −1823, χ²= 4.36, p = 0.037; −2548, χ²= 5.73, p = 0.017). Nine infrequent sequence variants were detected ( q < 0.05) but they did not occur more often among obese women compared with those of average-weight. For extremely obese women, three polymorphisms (+19, −188, and −633) predicted the degree of obesity. Allelic variants may influence the regulation of the leptin gene and thereby influence body weight, particularly among extremely obese women. However, given the low variability in coding regions and the high variability in the 5' flanking region, discerning the functional significance of each variant is likely to be difficult.     

Polymorphism in the 5' flanking region of the human insulin gene: a genetic marker for non-insulin-dependent diabetes

We sought to determine whether differences in the human insulin gene or its immediate flanking sequences could be found in diabetes. Peripheral leukocyte DNA from 217 unrelated persons, including blacks, whites, and Pima Indians, was analyzed by restriction-enzyme digestion, blotting to nitrocellulose filters, and hybridization to cloned [32P]insulin-gene probes. A region of length variation including deletions (0.1 to 0.2 kilobase pairs) or insertions (0.6 to 5.5 kb) of DNA was found only in the immediate 5' flanking region in 33 per cent of the genes examined. A 1.6-kb insertion accounted for 80 per cent of the polymorphism. This variant was found more often in subjects with non-insulin-dependent diabetes than in nondiabetics, regardless of race (P = 0.011). Length polymorphism in the 5' flanking region of the insulin gene may provide a genetic marker for non-insulin-dependent diabetes.     

CYP21 and CYP21P variability in steroid 21-hydroxylase deficiency patients and in the general population in the Netherlands

Steroid 21-hydroxylase deficiency is caused by defectiveness of the CYP21 gene. Such defects have presumably originated from interactions with the nearby CYP21P pseudogene during evolution. We studied these mechanisms by comparing the genetic variability of CYP21, CYP21P, and CYP21P/CYP21 hybrids (resulting from large-scale rearrangements) at eight mutation sites in a group of Dutch steroid 21-hydroxylase deficiency patients, their family members, and controls. The most common CYP21 defect in patients with salt-losing steroid 21-hydroxylase deficiency was a splice junction mutation in intron 2. The most common defect in the simple virilising form of the disease was ile72 --> asn. CYP21P showed considerable sequence variation in its central and 3' sections; the 5' section was constant. A single nucleotide (T) insert in exon 7 was found in all CYP21P genes. During the course of evolution, this was probably the third defect introduced into CYP21P after the splice junction mutation in intron 2 and the 8 bp deletion in exon 3. Gene conversions introducing CYP21-like sequences contribute to CYP21P variability. Such an event has occurred de novo in one family. A comparison of CYP21 and CYP21P mutations on the same chromosome shows that at least some of the small-scale gene conversions that supposedly transfer defects to CYP21 involve interaction between homologous chromosomes. The majority of the putative CYP21P-CYP21 transitions in hybrid genes appears to occur in a distinct zone that lies 5' of nucleotide 2108, which is further downstream than previously hypothesised. The other transitions lie upstream of nucleotide 999. Apparent 'large-scale' CYP21-CYP21P gene conversions lead to hybrid genes that are very similar to those found in CYP21 deletions, so these haplotypes have probably resulted from a meiotic double unequal crossover.     

Identification of two SNPs in the 5' flanking region of the ACP1 gene and evaluation of disequilibrium among polymorphic sites

We have studied the 5' flanking region of the ACP1 gene looking for new polymorphisms. Two SNPs, Dde I and Fok I restricted, have been found in this region. We determined the genotype of Dde I and Fok I SNPs, as well as of three other known SNPs, codon 43 ( Cfo I restricted), codon 41 and codon 105 ( Taq I restricted), located respectively in exons 3, 4 and 6 in 62 unrelated subjects from the Italian population. Haplotype distribution for the ten possible pairs of loci were determined by a maximum likelihood procedure. Overall, statistically significant deviations from expected frequencies assuming equilibrium have been observed for the following pairs: Fok I/codon 41, Fok I/ Taq I, codon 41/ Taq I (complete association), Dde I/ Fok I, Dde I/codon 41 and Dde I/ Taq I. The data suggest that the Fok I area could include sequences operating in strict functional association with sequences included in the codon 41/ Taq I area, possibly in order to regulate the F/S isoforms ratio of the A* and *B alleles. Since the ratio between the concentration of the two F and S isoforms is different for the three ACP1 alleles, we suggest that the ACP1 locus has been subjected to strong selective pressure to obtain an optimal alternative splicing mechanism of the *A and *B alleles. The *C variant, on the other hand, seems completely independent from sequences in the Fok I/codon 41/ Taq I areas, resulting in an inverted F/S ratio compared to that found for *A and *B alleles.     

Use of DNA probes from the 5' flanking region of the HLA-B gene to examine polymorphism at the HLA-B locus

Two DNA probes isolated from the region 5' to the HLA-B gene are described. One of these, pCH6, detects a TaqI polymorphism which is correlated with serological alleles of HLA-B. Both probes are used to show a high level of DNA sequence homology between the 5' flanking region of HLA-B and HLA-C.     

CK13基因5′旁侧的初步功能分析

目的　探讨细胞角蛋白 13(cytokeratin13,CK13)基因表达调控的机理 ,研究 CK13基因 5′旁侧不同基序对其转录活性的影响。　方法　采用分子克隆结合报告基因分析的方法 ,构建 CK 13基因 5′旁侧 5 13bp内不同基序与氯霉素乙酰转移酶 (chloramphenicol acetyltransferase,CAT)报告基因增强子载体p CAT的重组体 ,通过脂质体介导的转染技术导入 He L a细胞 ,检测各报告基因载体 CAT的相对活性。　结果　 CK13基因 5′旁侧起始密码子 ATG上游 - nt.32 5～ - nt.2 0 7间 119bp中具有某种抑制子元件 ,-nt.2 0 6～ - nt.94间 113bp中具有某种增强子元件。　结论　 CK13基因 5′旁侧 5 13bp内存在促进及抑制CK13基因表达的反应元件 ,进一步定位这些顺式反应元件并研究与之相互作用的反式作用因子 ,可望阐明 CK13基因表达调控及组织特异性表达的详细机理。     

Duplication of 111 bases in exon 1 of the CYP21 gene is combined with deletion of CYP21P-C4B genes in steroid 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase (CYP21) gene. A 9.3-kb fragment generated by NdeI and AseI digestion by Southern blot analysis indicated that a consequence of deletion of the C4-CYP21 repeat module was the production of a distinct chimeric CYP21P/CYP21 molecule. In the present study, we report a novel CYP21 genotype in two CAH families in which the gene appeared as 9.4- and 3.3-kb fragments by TaqI digestion, rather than as a chimeric gene. From the analysis of PCR amplification patterns and DNA sequencing, we found that there was a duplication of 111 bases from codons 21 to 57 inserted at codon 58 in exon 1 of the CYP21 gene. In addition, codon 21 in the repeated sequence changed from TGG to AGG. Furthermore, this novel CYP21 gene present in both CAH families showed no mutations at IVS2-12A/C>G, 707-714delGAGACTAC, and P30L. Interestingly, the 5' end region of these two CYP21 genes showed the sequence of the CYP21P gene at nucleotides (nt) -103, -110, -123, and thereafter. Our data suggest that these two CYP21 genes are caused by deletion of the CYP21P, XA, RP2, and C4B genes. Possibly, the additional 111-base duplicated coding sequence may be generated by multiple intergenic recombinations, while there seems to be no relationship with deletion of the CYP21P-C4B regions.     

Identification of non-amplifying CYP21 genes when using PCR-based diagnosis of 21-hydroxylase deficiency in congenital adrenal hyperplasia (CAH) affected pedigrees

Steroid 21-hydroxylase deficiency is among the most common inborn errors of metabolism in man. Characterization of mutations in the 21- hydroxylase gene (CYP21) has permitted genetic diagnosis, facilitated by the polymerase chain reaction (PCR). The most common mutation is conversion of an A or C at nt656 to a G in the second intron causing aberrant splicing of mRNA. Homozygosity for nt656G is associated with profoundly deficient adrenal cortisol and aldosterone synthesis, secondary hypersecretion of adrenal androgens, and a severe form of congenital adrenal hyperplasia (CAH) characterized by ambiguous genitalia and/or sodium wasting in newborns. During the course of genetic analysis of CYP21 mutations in CAH families, we and others have noticed a number of relatives genotyped as nt656G homozygotes, yet showing no clinical signs of disease. A number of lines of evidence have led us to propose that the putative asymptomatic nt656G/G individuals are incorrectly typed due to dropout of one haplotype during PCR amplification of CYP21. For prenatal diagnosis, we recommend that microsatellite typing be used as a supplement to CYP21 genotyping in order to resolve ambiguities at nt656.      

The chimeric<Emphasis Type="Italic"> CYP21P/CYP21</Emphasis> gene and 21-hydroxylase deficiency

The chimeric CYP21P/CYP21 gene is a consequence of a 26- or 32-kb deletion in the C4-CYP21 repeat module of CYP21P, tenascin A (XA), serine/threonine nuclear protein kinase (RP2), and the C4B and CYP21 genes in congenital adrenal hyperplasia (CAH) with steroid 21-hydroxylase deficiency. To date, there have been three distinct chimeras found in CAH patients in ethnic Chinese. Initiation for production of these molecules is proposed to be chi-like sequences and a minisatellite consensus existing in several noncoding regions in CYP21 genes. These molecules have the 5 end of the CYP21P-specific sequence in common but differ in the 3 end of CYP21-specific genes. In addition, there appears to be a 3.2-kb fragment generated by Taq I digestion, which leads to allele dropout in PCR amplification for detecting the aberrant splicing site of the IVS2 –12A/C>G mutation at nucleotide (nt) 655 in the CYP21 gene. Therefore, the chimeric CYP21P/CYP21 cannot be detected by conventional methods. It has been demonstrated that a PCR product amplified with allele-specific primers covering tenascin B (TNXB) to the 5 end of the CYP21 gene combined with Southern analysis by Ase I and Nde I digestion may be used for identifying the chimera in the CYP21 gene.     

A TNFR2 3' flanking region polymorphism in systemic lupus erythematosus

The role of pro-inflammatory cytokines in systemic lupus erythematosus (SLE) remains somewhat controversial. Several studies have shown increased production of TNF alpha and IL-6 in patients with SLE. Increased production of IL-6, TNF alpha, and IL-1 soluble receptors have also been reported. This finding is provocative because the soluble receptors have the capacity to act as antagonists. Several other inflammatory disorders are also associated with increased production of soluble TNF alpha receptors suggesting that this may be a general compensatory mechanism designed to down-regulate inflammation. The recent identification of an SLE disease susceptibility locus near the TNFR2 locus (TNFR p75) suggested the hypothesis that genetically driven differences in soluble TNFR2 production could play a role in the genetic susceptibility to SLE. We therefore characterized the frequency of a genetic polymorphism in the 3' untranslated region of the TNFR2 gene in Caucasoid SLE patients and geographically matched controls. No difference in the gene frequency of the two base-pair polymorphism in SLE patients compared to controls was found, nor was there any association with any particular clinical phenotype.