scholarly journals A Novel Semiquantitative Polymerase Chain Reaction/Enzyme Digestion-Based Method for Detection of Large Scale Deletions/Conversions of the CYP21 Gene and Mutation Screening in Turkish Families with 21-Hydroxylase Deficiency

2003 ◽  
Vol 88 (12) ◽  
pp. 5893-5897 ◽  
Author(s):  
T. Tukel ◽  
O. Uyguner ◽  
J. Q. Wei ◽  
M. Yuksel-Apak ◽  
N. Saka ◽  
...  
Keyword(s):  
Southern Blot ◽  
Blot Analysis ◽  
Southern Blot Analysis ◽  
Large Scale ◽  
Point Mutations ◽  
Enzyme Digestion ◽  
Allele Frequencies ◽  
Hydroxylase Deficiency ◽  
Cyp21 Gene ◽  
21 Hydroxylase Deficiency

Abstract 21-Hydroxylase deficiency is a recessively inherited disorder resulting from mutations in the CYP21 gene. The CYP21 gene is located along with the CYP21P pseudogene in the human leukocyte antigen major histocompatibility complex region on chromosome 6. Molecular diagnosis is difficult due to the 98% similarity of CYP21 and CYP21P genes and the fact that almost all frequently reported mutations reside on the pseudogene. Allele-specific PCR for the 8 most frequently reported point mutations was performed in 31 Turkish families with at least a single 21-hydroxylase-deficient individual. The allele frequencies of the point mutations were as follows: P30L, 0%; IVS2 (AS,A/C-G,-13), 22.5%; G110Δ8nt, 3.2%; I172N, 11.4%; exon 6 cluster (I236N, V237E, M239K), 3.2%; V281L, 0%; Q318X, 8%; and R356W, 9.6%. Large deletions and gene conversions were detected by Southern blot analysis, and the allele frequencies were 9.6% and 22.5%, respectively. Sequence analysis of the gene, performed on patients with only 1 mutated allele, revealed 2 missense mutations (R339H and P435S). A novel semiquantitative PCR/enzyme digestion-based method for the detection of large scale deletions/conversions of the gene was developed for routine diagnostic purposes, and its accuracy was shown by comparison with the results of Southern blot analysis.

scholarly journals CYP21 Gene Mutation Analysis in 198 Patients with 21-Hydroxylase Deficiency in The Netherlands: Six Novel Mutations and a Specific Cluster of Four Mutations

2003 ◽  
Vol 88 (8) ◽  
pp. 3852-3859 ◽  
Author(s):  
Nike M. M. L. Stikkelbroeck ◽  
Lies H. Hoefsloot ◽  
Ilse J. de Wijs ◽  
Barto J. Otten ◽  
Ad R. M. M. Hermus ◽  
...  
Keyword(s):  
The Netherlands ◽  
Point Mutations ◽  
Phenotype Correlation ◽  
Novel Mutations ◽  
Hydroxylase Deficiency ◽  
Salt Wasting ◽  
Cyp21 Gene ◽  
Genotype Phenotype Correlation ◽  
Single Allele ◽  
21 Hydroxylase Deficiency

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is one of the most common autosomal recessive disorders. The aim of this study was to assess the frequencies of CYP21 mutations and to study genotype-phenotype correlation in a large population of Dutch 21-hydroxylase deficient patients. From 198 patients with 21-hydroxylase deficiency, 370 unrelated alleles were studied. Gene deletion/conversion was present in 118 of the 370 alleles (31.9%). The most frequent point mutations were I2G (28.1%) and I172N (12.4%). Clustering of pseudogene-derived mutations in exons 7 and 8 (V281L-F306 + 1nt-Q318X-R356W) on a single allele was found in seven unrelated alleles (1.9%). This cluster had been reported before in two other Dutch patients and in two patients in a study from New York, but not in other series worldwide. Six novel mutations were found: 995–996insA, 1123delC, G291R, S301Y, Y376X, and R483Q. Genotype-phenotype correlation (in 87 well documented patients) showed that 28 of 29 (97%) patients with two null mutations and 23 of 24 (96%) patients with mutation I2G (homozygous or heterozygous with a null mutation) had classic salt wasting. Patients with mutation I172N (homozygous or heterozygous with a null or I2G mutation) had salt wasting (2 of 17, 12%), simple virilizing (10 of 17, 59%), or nonclassic CAH (5 of 17, 29%). All six patients with mutation P30L, V281L, or P453S (homozygous or compound heterozygous) had nonclassic CAH. The frequency of CYP21 mutations and the genotype-phenotype correlation in 21-hydroxylase deficient patients in The Netherlands show in general high concordance with previous reports from other Western European countries. However, a cluster of four pseudogene-derived point mutations on exons 7 and 8 on a single allele, observed in almost 2% of the unrelated alleles, seems to be particular for the Dutch population and six novel CYP21 gene mutations were found.

scholarly journals Long-term engraftment of normal and post-5-fluorouracil murine marrow into normal nonmyeloablated mice [see comments]

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2566-2571 ◽  
Author(s):  
FM Stewart ◽  
RB Crittenden ◽  
PA Lowry ◽  
S Pearson-White ◽  
PJ Quesenberry
Keyword(s):  
Southern Blot ◽  
Blot Analysis ◽  
Southern Blot Analysis ◽  
Normal Male ◽  
Normal Donor ◽  
Long Term Stability ◽  
Donor Bone Marrow ◽  
Male Donor ◽  
Marrow Cells

We report the successful long-term engraftment of normal male donor bone marrow (BM) transfused into noncytoablated female mice, challenging the assumption that “niches” need to be created for marrow to engraft. We have used chromosomal banding and Southern blot analysis to identify transplanted male marrow cells, and shown the long-term stability of the chimeric marrows. Balb/C, BDF1, or CBA-J female hosts (no irradiation) received for 5 consecutive days 40 x 10(6) male cells (per day) of the same strain, and repopulation patterns were observed. Parallel studies were performed using tibia/femur equivalents of normal marrow or marrow from Balb/C mice pretreated 6 days previously with 150 mg/kg 5-fluorouracil (5-FU). Chromosome banding techniques showed that 5% to 46% of marrow cells were male 3 to 9 months posttransplant with normal donor marrow. Southern blot analysis, using the pY2 probe, showed continued engraftment at 21 to 25 months posttransplant, ranging from 15% to 42% male engrafted cells in marrow. Normal donor male marrow engrafted significantly better than 5-FU-pretreated male marrow as shown 1 to 12 months posttransplant in non-cytoablated female recipients. Percentages of male engrafted cells in BM ranged from 23% to 78% for recipients of normal donor marrow and from 0.1% to 39% for recipients of 5-FU marrow. Mean engraftment for 6 mice receiving normal marrow was 38%, whereas that for 6 mice receiving post-5-FU marrow was 8%, as assayed 1 to 3 months posttransplant. At 10 to 12 months, mean engraftment for the normal donor group was 46%, compared with 16% for the 5-FU group. The patterns of engraftment with normal and 5-FU marrow were similar for spleen and thymus. These results show that long-term chimerism can be established after transplantation of normal donor marrow to normal nonirradiated host mice and indicate that marrow spaces do not have to be created for successful engraftment. They suggest that transplanted marrow competes equally with host marrow for marrow space. Finally, these data show that post-5-FU Balb/C male marrow is markedly inferior in the repopulation of Balb/C female host marrow, spleen, and thymus, and suggest that this population of cells may not be the ideal population for gene transfer studies.

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