Haemophilia A is an X‐linked bleeding disorder caused by heterogeneous mutations in the F8 gene. Two inversion hotspots in intron 22 and intron 1, as well as point mutations, small insertions and deletions in the F8 gene account for causal mutations leading to severe haemophilia A. Rarely, novel molecular mechanisms lead to a haemophilia A phenotype which cannot be completely characterized by routine molecular diagnostic methods. Here, we characterized the molecular abnormality in a boy with a severe haemophilia A phenotype. On investigation by PCR and DNA sequencing, exon 18 of F8 repeatedly failed to amplify. However, analysis by multiplex ligation‐dependent probe amplification demonstrated the presence of exon 18 sequence, suggesting a more complex rearrangement than a single exon deletion. The analysis of exon 18 and its flanking regions by inverse PCR revealed a complex mutation comprising insertions of extragenic sequences from Xq28 along with a partial duplication of exon 18. Based on the successful analysis and characterization of the familial breakpoint, we developed a PCR‐based diagnostic approach to detect this defect in family members in whom no diagnostic test could be offered until this time. 相似文献
Objectives: Oral candidiasis is being frequently recognized in patients with diabetes, and is associated with multiple pathogens including Candida albicans, Candida parapsilosis, Candida glabrata and Candida tropicalis. The aim of this study was to evaluate a usefulness of a Multiplex Polymerase Chain Reaction as a rapid diagnostic tool for identification of four oral Candida pathogens in patients with diabetes.
Materials and methods: A multiplex PCR was optimized to identify four Candida species in concentrated oral rinse samples. Common reverse primer, ITS4 and four species-specific forward primers targeting ITS1 and ITS2 regions of yeast genome were used. Species-specific single amplicon were detected by agarose gel electrophoresis. Performance efficacy of multiplex PCR was compared with phenotypic identification.
Results: Out of 100 oral rinse samples, 72 were culture positive and of these 43 were at risk of oral Candida infection (>600cfu/ml). Multiple Candida species including C. albicans, C. parapsilosis and C. tropicalis were identified in 22 samples which had risk of oral Candida infection. In total, 85 patients were positive for Candida by multiplex PCR and of them 49 had multiple Candida species. All 43 colonized specimens were also positive by multiplex PCR. C. albicans was the most predominant organism (75/85) followed by C. parapsilosis (47/85), C. tropicalis (17/85) and C. glabrata (6/85). In specimens with multiple species, the two most common organisms were C. albicans and C. parapsilosis. Multiplex PCR yielded a sensitivity of 10 Candida cells/ml of oral rinse sample.
Conclusions: Multiplex PCR is found to be rapid, sensitive and specific than phenotypic identification methods in discriminating multiple Candida species in oral rinse specimens. 相似文献
Objective To explore the genetic and clinical characteristics of isodicentric Ph chromosomes [idic(Ph)] in lymphoid blast crisis of chronic myeloid leukemia (CML-BLC). Methods Bone marrow aspirates of 2 patients withCML-BLC were analyzed by R banding after 24 hours of culturing. Genomic copy number variations (CNV) wereanalyzed by single nucleotide polymorphism array (SNP array) in case 1. The results were confirmed withfluorescence in situ hybridization (FISH). Variations of acute lymphoblastic leukemia-related genes includingCDKN2A/AB and PAX5 were detected by multiplex ligation-dependent probe amplication (MLPA). Results Deletionsand duplications on derivative chromosome 9 detected by FISH were confirmed by SNP array analysis. Thedistances between the BCR/ ABL fusion signals on the idic(Ph) chromosomes in the two patients have differedgreatly. The idic(Ph) in the second patient was supposed to be formed by two Ph chromosomes joined at their qterminals, where as the idic(Ph) in the first patient have been shown to be fused at the satellite regions oftheir p arms. Conclusion The idic(Ph) chromosomes presented in CML-BLC may predict resistance to Imatinib andresponse to Dasatinib. 相似文献
Objective To explore the value of multiplex ligation-dependent probe amplification (MLPA) for thedetection of chromosome abnormalities in miscarriage tissues, and to correlate the result with ultrasoundfindings. Methods A total of 421 cases of spontaneous abortions and fetal deaths were detected with the MLPAmethod. Results Among the 421 samples, 232 (55. 11%) had an abnormal MLPA result. For the 286 cases derivedfrom < 13 weeks pregnancy, 206 (72.03%) were abnormal. For the 49 cases from 14 ~ 19 weeks pregnancy, 14(28. 57%) were abnormal. For the 86 cases derived after 20 weeks pregnancy, 12 (13. 95%) were abnormal. Amongthe 117 cases with abnormal ultrasound findings, 33 cases (28. 21%) had an abnormal MLPA result, 28 out of the33 cases were numerical chromosome abnormality, 4 cases were chromosome microdeletion and/or micro duplication, 1 case had both numerical abnormality and microduplication. For those with abnormal ultrasound findings for the neck region, fetal edematous syndrome, multiple malformations and digestive system, the detection rates forMLPA were 71. 4% , 58. 8% , 37. 8% , and 9.1% , respectively. For those with abnormal finding of cardiacsystem, nervous system, face, skeletal system and urinary system, none was found with positive results of MLPA. Conclusion Numerical chromosomal abnormalities account for the majority of cases with spontaneous abortion.With the increase of gestational age, the occurrence of chromosomal abnormalities gradually declines. Combinedultrasound and MLPA assay can improve the detection rate and accuracy for chromosomal abormalities. 相似文献
Wilson disease is a rare disorder of copper metabolism due to mutation in ATP7B gene. Proper counseling of patients with Wilson disease, and their families necessitates finding mutation in ATP7B gene. Finding mutations in ATP7B gene with 21 exons, and more than 500 mutations is expensive and time-consuming.
Objectives
The aim of this study was to provide a simple multiplex amplification refractory mutation system PCR (M-ARMS-PCR) for screening eight common mutations in ATP7B gene.
Patients and Methods
Two sets of ARMS mutant and normal specific primer pairs were designed for genotyping of p.R778L, p.R969Q, p.H1069Q, and p.3400delC mutations as Set 1 and p.W779G, c.3061-1G > A, p.I1102T, and p.N1270S mutations as Set 2. The Multiplex ARMS assay was then subsequently tested in 65 patients with Wilson disease with known and unknown ATP7B mutations.
Results
Using these two sets, we identified H1069Q mutation in four patients, c.2335T > G mutation in three, c.3061-1G > A splice site mutation in five, c.3305T > C mutation in one, and c.3809A > G mutation in two patients.
Conclusions
The Multiplex ARMS assay used in this study can be an efficient, reliable, and cost effective method as a primary screen for patients with Wilson disease. 相似文献