Combined use of MLPA and nonfluorescent multiplex PCR analysis by high performance liquid chromatography for the detection of genomic rearrangements

Large genomic rearrangements are recognized as playing a pathogenic role in an increasing number of human genetic diseases. It is important to develop efficient methods for the routine detection and confirmation of these germline defects. Multiplex ligation-dependent probe amplification (MLPA) is considered an early step for molecular diagnosis of several genetic disorders. However, artifacts might hamper the interpretation of MLPA analysis, especially when rearrangements involve a single exon. Therefore, rearrangements must be verified by two independent methods. In this study, we developed nonfluorescent multiplex-PCR coupled to high-performance liquid chromatography (NFMP-HPLC) and analyzed whether the use of this method combined with MLPA could be helpful in the detection and confirmation of gross MSH2 and MLH1 genomic rearrangements. A total of nine nonfluorescent multiplex-PCRs were developed to analyze the 16 MSH2 and 19 MLH1 exons. Reliable multiplex amplifications and nonfluorescent peak quantitation were obtained with a limited number of cycles (< or = 25) using a denaturing HPLC (DHPLC) instrument under nondenaturing conditions. The results obtained by NFMP-HPLC were highly reproducible. The combined use of MLPA and NFMP-HPLC identified and independently confirmed putative MLH1 and MSH2 deletions in eight out of 50 unrelated patients with hereditary nonpolyposis colorectal cancer (HNPCC). In five cases, the deletions affected a single exon and in three cases multiple contiguous exons. These results were in agreement with breakpoint and complementary DNA (cDNA) analyses. Considering that MLPA and NFMP-HPLC are unlikely to be affected by the same artifacts, their combined use could also provide a robust and cost-effective strategy for routine screening and confirmation of putative rearrangements in other genes, especially when a single exon is involved or a precise characterization of breakpoints is not achieved.     

Stability of BAT26 in tumours of hereditary nonpolyposis colorectal cancer patients with MSH2 intragenic deletion

Colon cancers arising in most patients with hereditary nonpolyposis colorectal cancer (HNPCC) show microsatellite instability (MSI). BAT26, a quasimonomorphic polyA stretch located just 3' of MSH2 exon 5, is considered the most sensitive and specific marker of MSI. A total of 10 HNPCC families with large intragenic MSH2 deletions, encompassing exon 5 and intron 5, identified by multiplex ligation-dependent probe amplification (MLPA) were included in this study. The deletions under study were del1-16, del1-8, del1-7, del1-6, and del3-6, detected in 3, 1, 2, 3, and 1 families, respectively. Although all patients examined from these 10 families developed unstable tumours, 13/19 MSI-H tumours (68 %) surprisingly showed stability of BAT26. By MLPA and MSH2 sequence analyses of the BAT26-stable tumours, we demonstrated that the wild-type MSH2 allele was somatically inactivated by an identical large deletion, with complete loss of intron 5/BAT26 sequences at the tumour DNA level. We could infer that the apparent stability of BAT26 was due to the complete absence of target BAT26 sequences in the tumour sample, which results in exclusive amplification of contaminant normal DNA, containing a single copy of a wild-type stable BAT26 sequence. Identification of a subset of MSH2-related unstable tumours that are not recognized by analysis of BAT26 instability indicates that this marker should never be used alone for rapid MSI screening of HNPCC tumours. Moreover, our findings indicate that BAT26 stability in the context of MSI is strongly suggestive of the presence of a large intragenic MSH2 deletion.     

Genomic deletions in MSH2 or MLH1 are a frequent cause of hereditary non-polyposis colorectal cancer: identification of novel and recurrent deletions by MLPA

Gene dosage abnormalities account for a significant proportion of the mutations in genes tested in DNA diagnostic laboratories. Detection of these changes has proved a challenge as the methods available to date are time consuming or unreliable. The multiplex ligation-dependent probe assay (MLPA) is a new technique allowing relative quantification of up to 40 different nucleic acid sequences in a single reaction tube. We have evaluated MLPA for potential use in the diagnostic setting against the following criteria: accuracy, reagent cost, hands-on time, reliability, and retests required. A total of 215 UK patients referred for genetic testing on the basis of a family history consistent with autosomal dominant hereditary non-polyposis colorectal cancer (HNPCC or Lynch syndrome) were tested by MLPA. Of these, 12 cases with deletions of one or more exons were identified, six with MLH1 deletions and six with MSH2 deletions. Test failure rates were less than 5% and overall mutation detection sensitivity in this series was increased by approximately 50% by the inclusion of MLPA for an additional testing cost of about 10%. Two novel mutations in MSH2 and 10 novel point mutations in MLH1 were also identified during the course of this study. We conclude that MLPA is a cost effective and robust gene dosage method that can be readily adopted by diagnostic services. Comprehensive mutation scanning for MSH2 and MLH1 is incomplete without gene dosage analysis.     

中国人家族性结直肠癌错配修复基因大片段变异分析

目的分析中国人家庭性结直肠癌错配修复基因大片段变异的特点。方法 采用多重连接探针扩增技术，分析32例具有家庭史结直肠癌、20例散发性结直肠癌患者错配修复基因MSH2的16个外显子、MLH1的19个外显子及7个其它基因外显子的拷贝数。研究工作包括：（1）双盲法分析阴性和阳性对照样本，完成方法学可靠性检验；（2）分析结直肠癌患者外周血细胞DNA，筛查MSH2和MLH1基因大片段变异。结果 多重连接探针扩增技术分析系统稳定检出阳性对照样本的DNA大片段缺失；在3/32（9.4%）具有家庭聚集性结直肠癌患者中检出遗传性MSH2基因DNA大片段缺失。而在20例散发性结直肠癌患者未检出这类突变。结论 中国人家族性结直肠癌患者中错配修复基因的大片段变异是频发事件，对此类患者的遗传检测应包含错配修复基因大片段变异的筛查。     

Genetic analysis of dystrophin gene for affected male and female carriers with Duchenne/Becker muscular dystrophy in Korea

Duchenne and Becker muscular dystrophy (DMD/BMD) are X-linked recessive disorders caused by mutation in dystrophin gene. We analyzed the results of a genetic test in 29 DMD/BMD patients, their six female relatives, and two myopathic female patients in Korea. As the methods developed, we applied different procedures for dystrophin gene analysis; initially, multiplex polymerase chain reaction was used, followed by multiplex ligation-dependent probe amplification (MLPA). Additionally, we used direct DNA sequencing for some patients who had negative results using the above methods. The overall mutation detection rate was 72.4% (21/29) in DMD/BMD patients, identifying deletions in 58.6% (17/29). Most of the deletions were confined to the central hot spot region between exons 44 and 55 (52.9%, 7/19). The percentage of deletions and duplications revealed by MLPA was 45.5% (5/11) and 27.2% (3/11), respectively. Using the MLPA method, we detected mutations confirming their carrier status in all female relatives and symptomatic female patients. In one patient in whom MLPA revealed a single exon deletion of the dystrophin gene, subsequent DNA sequencing analysis identified a novel nonsense mutation (c.4558G > T; Gln1520X). The MLPA assay is a useful quantitative method for detecting mutation in asymptomatic or symptomatic carriers as well as DMD/BMD patients.     

Multiplex ligation-dependent probe amplification analysis to screen for deletions and duplications of the LDLR gene in patients with familial hypercholesterolaemia

The most common genetic defect in patients with autosomal dominant hypercholesterolaemia is a mutation of the low-density lipoprotein receptor ( LDLR ) gene. An estimate of the frequency of major rearrangements has been limited by the availability of an effective analytical method and testing of large cohorts. We present data from a cohort of 611 patients referred with suspected heterozygous familial hypercholesterolaemia (FH) from five UK lipid clinics, who were initially screened for point mutations in LDLR and the common APOB and PCSK9 mutations. The 377 cases in whom no mutation was found were then screened for large rearrangements by multiplex ligation-dependent probe amplification (MLPA) analysis. A rearrangement was identified in 19 patients. This represents 7.5% of the total detected mutations of the cohort. Of these, the majority of mutations (12/19) were deletions of more than one exon, two were duplications of more than one exon and five were single exon deletions that need interpreting with care. Five rearrangements (26%) are previously unreported. We conclude that MLPA analysis is a simple and rapid method for detecting large rearrangements and should be included in diagnostic genetic testing for FH.     

Deletions of NF1 gene and exons detected by multiplex ligation-dependent probe amplification

To estimate the contribution of single and multi-exon NF1 gene copy-number changes to the NF1 mutation spectrum, we analysed a series of 201 Italian patients with neurofibromatosis type 1 (NF1). Of these, 138 had previously been found, using denaturing high-performance liquid chromatography or protein truncation test, to be heterozygous for intragenic NF1 point mutations/deletions/insertions, and were excluded from this analysis. The remaining 63 patients were analysed using multiplex ligation-dependent probe amplification (MLPA), which allows detection of deletions or duplications encompassing >or=1 NF1 exons, as well as entire gene deletions. MLPA results were validated using real-time quantitative PCR (qPCR) or fluorescent in situ hybridisation. MLPA screening followed by real-time qPCR detected a total of 23 deletions. Of these deletions, six were single exon, eight were multi-exon, and nine were of the entire NF1 gene. In our series, deletions encompassing >or=1 NF1 exons accounted for approximately 7% (14/201) of the NF1 gene mutation spectrum, suggesting that screening for these should now be systematically included in genetic testing of patients with NF1.     

应用多重PCR和MLPA技术检测DMD患者和携带者的基因突变及产前诊断

目的 应用多重PCR和多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测Duchenne/Becker肌营养不良症(Duchenne/Becker muscular dystrophy,DMD/BMD)患者、携带者并应用于产前诊断.方法 首先采用多重PCR对临床诊断为DMD/BMD的患者检测DMD基因的26个外显子,未查到缺失突变者和可能的携带者采用MLPA检测全部79个外显子是否有缺失或重复突变.对产前诊断病例,用PCR法检测缺失突变,用MLPA法检测重复突变.结果 多重PCR对22例患者的DMD基因的26个外显子检测.13例有缺失突变.未查到常见缺失突变的9例患者经MLPA检测DMD基因的全部79个外显子,3例为重复突变、1例为单个第18外显子缺失、其他5例未查到缺失和重复突变.16例携带者中,3例有家族史,其中2例检出突变;13例为检测到突变的散发病例患儿的母亲,有8例检测到突变.产前诊断9个胎儿(其中双胎1例),2例胎儿有突变,引产后核实无误;7例胎儿未检测到突变,现均已分娩.结论 多重PCR可检出92.86%的缺失突变并可用于缺失突变的产前诊断,因其简便、可靠、价廉可作为临床上DMD/BMD基因诊断的初选.MLPA可用于多重PCR未检测到缺失突变的患者及携带者的检查.     

应用多重PCR和MLPA技术检测DMD患者和携带者的基因突变及产前诊断

目的 应用多重PCR和多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测Duchenne/Becker肌营养不良症(Duchenne/Becker muscular dystrophy,DMD/BMD)患者、携带者并应用于产前诊断.方法 首先采用多重PCR对临床诊断为DMD/BMD的患者检测DMD基因的26个外显子,未查到缺失突变者和可能的携带者采用MLPA检测全部79个外显子是否有缺失或重复突变.对产前诊断病例,用PCR法检测缺失突变,用MLPA法检测重复突变.结果 多重PCR对22例患者的DMD基因的26个外显子检测.13例有缺失突变.未查到常见缺失突变的9例患者经MLPA检测DMD基因的全部79个外显子,3例为重复突变、1例为单个第18外显子缺失、其他5例未查到缺失和重复突变.16例携带者中,3例有家族史,其中2例检出突变;13例为检测到突变的散发病例患儿的母亲,有8例检测到突变.产前诊断9个胎儿(其中双胎1例),2例胎儿有突变,引产后核实无误;7例胎儿未检测到突变,现均已分娩.结论 多重PCR可检出92.86%的缺失突变并可用于缺失突变的产前诊断,因其简便、可靠、价廉可作为临床上DMD/BMD基因诊断的初选.MLPA可用于多重PCR未检测到缺失突变的患者及携带者的检查.     

应用多重PCR和MLPA技术检测DMD患者和携带者的基因突变及产前诊断

目的 应用多重PCR和多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测Duchenne/Becker肌营养不良症(Duchenne/Becker muscular dystrophy,DMD/BMD)患者、携带者并应用于产前诊断.方法 首先采用多重PCR对临床诊断为DMD/BMD的患者检测DMD基因的26个外显子,未查到缺失突变者和可能的携带者采用MLPA检测全部79个外显子是否有缺失或重复突变.对产前诊断病例,用PCR法检测缺失突变,用MLPA法检测重复突变.结果 多重PCR对22例患者的DMD基因的26个外显子检测.13例有缺失突变.未查到常见缺失突变的9例患者经MLPA检测DMD基因的全部79个外显子,3例为重复突变、1例为单个第18外显子缺失、其他5例未查到缺失和重复突变.16例携带者中,3例有家族史,其中2例检出突变;13例为检测到突变的散发病例患儿的母亲,有8例检测到突变.产前诊断9个胎儿(其中双胎1例),2例胎儿有突变,引产后核实无误;7例胎儿未检测到突变,现均已分娩.结论 多重PCR可检出92.86%的缺失突变并可用于缺失突变的产前诊断,因其简便、可靠、价廉可作为临床上DMD/BMD基因诊断的初选.MLPA可用于多重PCR未检测到缺失突变的患者及携带者的检查.     

应用多重PCR和MLPA技术检测DMD患者和携带者的基因突变及产前诊断

目的 应用多重PCR和多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测Duchenne/Becker肌营养不良症(Duchenne/Becker muscular dystrophy,DMD/BMD)患者、携带者并应用于产前诊断.方法 首先采用多重PCR对临床诊断为DMD/BMD的患者检测DMD基因的26个外显子,未查到缺失突变者和可能的携带者采用MLPA检测全部79个外显子是否有缺失或重复突变.对产前诊断病例,用PCR法检测缺失突变,用MLPA法检测重复突变.结果 多重PCR对22例患者的DMD基因的26个外显子检测.13例有缺失突变.未查到常见缺失突变的9例患者经MLPA检测DMD基因的全部79个外显子,3例为重复突变、1例为单个第18外显子缺失、其他5例未查到缺失和重复突变.16例携带者中,3例有家族史,其中2例检出突变;13例为检测到突变的散发病例患儿的母亲,有8例检测到突变.产前诊断9个胎儿(其中双胎1例),2例胎儿有突变,引产后核实无误;7例胎儿未检测到突变,现均已分娩.结论 多重PCR可检出92.86%的缺失突变并可用于缺失突变的产前诊断,因其简便、可靠、价廉可作为临床上DMD/BMD基因诊断的初选.MLPA可用于多重PCR未检测到缺失突变的患者及携带者的检查.     

应用多重PCR和MLPA技术检测DMD患者和携带者的基因突变及产前诊断

目的 应用多重PCR和多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测Duchenne/Becker肌营养不良症(Duchenne/Becker muscular dystrophy,DMD/BMD)患者、携带者并应用于产前诊断.方法 首先采用多重PCR对临床诊断为DMD/BMD的患者检测DMD基因的26个外显子,未查到缺失突变者和可能的携带者采用MLPA检测全部79个外显子是否有缺失或重复突变.对产前诊断病例,用PCR法检测缺失突变,用MLPA法检测重复突变.结果 多重PCR对22例患者的DMD基因的26个外显子检测.13例有缺失突变.未查到常见缺失突变的9例患者经MLPA检测DMD基因的全部79个外显子,3例为重复突变、1例为单个第18外显子缺失、其他5例未查到缺失和重复突变.16例携带者中,3例有家族史,其中2例检出突变;13例为检测到突变的散发病例患儿的母亲,有8例检测到突变.产前诊断9个胎儿(其中双胎1例),2例胎儿有突变,引产后核实无误;7例胎儿未检测到突变,现均已分娩.结论 多重PCR可检出92.86%的缺失突变并可用于缺失突变的产前诊断,因其简便、可靠、价廉可作为临床上DMD/BMD基因诊断的初选.MLPA可用于多重PCR未检测到缺失突变的患者及携带者的检查.     

应用多重PCR和MLPA技术检测DMD患者和携带者的基因突变及产前诊断

目的 应用多重PCR和多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测Duchenne/Becker肌营养不良症(Duchenne/Becker muscular dystrophy,DMD/BMD)患者、携带者并应用于产前诊断.方法 首先采用多重PCR对临床诊断为DMD/BMD的患者检测DMD基因的26个外显子,未查到缺失突变者和可能的携带者采用MLPA检测全部79个外显子是否有缺失或重复突变.对产前诊断病例,用PCR法检测缺失突变,用MLPA法检测重复突变.结果 多重PCR对22例患者的DMD基因的26个外显子检测.13例有缺失突变.未查到常见缺失突变的9例患者经MLPA检测DMD基因的全部79个外显子,3例为重复突变、1例为单个第18外显子缺失、其他5例未查到缺失和重复突变.16例携带者中,3例有家族史,其中2例检出突变;13例为检测到突变的散发病例患儿的母亲,有8例检测到突变.产前诊断9个胎儿(其中双胎1例),2例胎儿有突变,引产后核实无误;7例胎儿未检测到突变,现均已分娩.结论 多重PCR可检出92.86%的缺失突变并可用于缺失突变的产前诊断,因其简便、可靠、价廉可作为临床上DMD/BMD基因诊断的初选.MLPA可用于多重PCR未检测到缺失突变的患者及携带者的检查.     

应用多重PCR和MLPA技术检测DMD患者和携带者的基因突变及产前诊断

目的 应用多重PCR和多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测Duchenne/Becker肌营养不良症(Duchenne/Becker muscular dystrophy,DMD/BMD)患者、携带者并应用于产前诊断.方法 首先采用多重PCR对临床诊断为DMD/BMD的患者检测DMD基因的26个外显子,未查到缺失突变者和可能的携带者采用MLPA检测全部79个外显子是否有缺失或重复突变.对产前诊断病例,用PCR法检测缺失突变,用MLPA法检测重复突变.结果 多重PCR对22例患者的DMD基因的26个外显子检测.13例有缺失突变.未查到常见缺失突变的9例患者经MLPA检测DMD基因的全部79个外显子,3例为重复突变、1例为单个第18外显子缺失、其他5例未查到缺失和重复突变.16例携带者中,3例有家族史,其中2例检出突变;13例为检测到突变的散发病例患儿的母亲,有8例检测到突变.产前诊断9个胎儿(其中双胎1例),2例胎儿有突变,引产后核实无误;7例胎儿未检测到突变,现均已分娩.结论 多重PCR可检出92.86%的缺失突变并可用于缺失突变的产前诊断,因其简便、可靠、价廉可作为临床上DMD/BMD基因诊断的初选.MLPA可用于多重PCR未检测到缺失突变的患者及携带者的检查.     

应用多重PCR和MLPA技术检测DMD患者和携带者的基因突变及产前诊断

目的 应用多重PCR和多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测Duchenne/Becker肌营养不良症(Duchenne/Becker muscular dystrophy,DMD/BMD)患者、携带者并应用于产前诊断.方法 首先采用多重PCR对临床诊断为DMD/BMD的患者检测DMD基因的26个外显子,未查到缺失突变者和可能的携带者采用MLPA检测全部79个外显子是否有缺失或重复突变.对产前诊断病例,用PCR法检测缺失突变,用MLPA法检测重复突变.结果 多重PCR对22例患者的DMD基因的26个外显子检测.13例有缺失突变.未查到常见缺失突变的9例患者经MLPA检测DMD基因的全部79个外显子,3例为重复突变、1例为单个第18外显子缺失、其他5例未查到缺失和重复突变.16例携带者中,3例有家族史,其中2例检出突变;13例为检测到突变的散发病例患儿的母亲,有8例检测到突变.产前诊断9个胎儿(其中双胎1例),2例胎儿有突变,引产后核实无误;7例胎儿未检测到突变,现均已分娩.结论 多重PCR可检出92.86%的缺失突变并可用于缺失突变的产前诊断,因其简便、可靠、价廉可作为临床上DMD/BMD基因诊断的初选.MLPA可用于多重PCR未检测到缺失突变的患者及携带者的检查.     

应用多重PCR和MLPA技术检测DMD患者和携带者的基因突变及产前诊断

目的 应用多重PCR和多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测Duchenne/Becker肌营养不良症(Duchenne/Becker muscular dystrophy,DMD/BMD)患者、携带者并应用于产前诊断.方法 首先采用多重PCR对临床诊断为DMD/BMD的患者检测DMD基因的26个外显子,未查到缺失突变者和可能的携带者采用MLPA检测全部79个外显子是否有缺失或重复突变.对产前诊断病例,用PCR法检测缺失突变,用MLPA法检测重复突变.结果 多重PCR对22例患者的DMD基因的26个外显子检测.13例有缺失突变.未查到常见缺失突变的9例患者经MLPA检测DMD基因的全部79个外显子,3例为重复突变、1例为单个第18外显子缺失、其他5例未查到缺失和重复突变.16例携带者中,3例有家族史,其中2例检出突变;13例为检测到突变的散发病例患儿的母亲,有8例检测到突变.产前诊断9个胎儿(其中双胎1例),2例胎儿有突变,引产后核实无误;7例胎儿未检测到突变,现均已分娩.结论 多重PCR可检出92.86%的缺失突变并可用于缺失突变的产前诊断,因其简便、可靠、价廉可作为临床上DMD/BMD基因诊断的初选.MLPA可用于多重PCR未检测到缺失突变的患者及携带者的检查.     

应用多重PCR和MLPA技术检测DMD患者和携带者的基因突变及产前诊断

目的 应用多重PCR和多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测Duchenne/Becker肌营养不良症(Duchenne/Becker muscular dystrophy,DMD/BMD)患者、携带者并应用于产前诊断.方法 首先采用多重PCR对临床诊断为DMD/BMD的患者检测DMD基因的26个外显子,未查到缺失突变者和可能的携带者采用MLPA检测全部79个外显子是否有缺失或重复突变.对产前诊断病例,用PCR法检测缺失突变,用MLPA法检测重复突变.结果 多重PCR对22例患者的DMD基因的26个外显子检测.13例有缺失突变.未查到常见缺失突变的9例患者经MLPA检测DMD基因的全部79个外显子,3例为重复突变、1例为单个第18外显子缺失、其他5例未查到缺失和重复突变.16例携带者中,3例有家族史,其中2例检出突变;13例为检测到突变的散发病例患儿的母亲,有8例检测到突变.产前诊断9个胎儿(其中双胎1例),2例胎儿有突变,引产后核实无误;7例胎儿未检测到突变,现均已分娩.结论 多重PCR可检出92.86%的缺失突变并可用于缺失突变的产前诊断,因其简便、可靠、价廉可作为临床上DMD/BMD基因诊断的初选.MLPA可用于多重PCR未检测到缺失突变的患者及携带者的检查.     

应用多重PCR和MLPA技术检测DMD患者和携带者的基因突变及产前诊断

目的 应用多重PCR和多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测Duchenne/Becker肌营养不良症(Duchenne/Becker muscular dystrophy,DMD/BMD)患者、携带者并应用于产前诊断.方法 首先采用多重PCR对临床诊断为DMD/BMD的患者检测DMD基因的26个外显子,未查到缺失突变者和可能的携带者采用MLPA检测全部79个外显子是否有缺失或重复突变.对产前诊断病例,用PCR法检测缺失突变,用MLPA法检测重复突变.结果 多重PCR对22例患者的DMD基因的26个外显子检测.13例有缺失突变.未查到常见缺失突变的9例患者经MLPA检测DMD基因的全部79个外显子,3例为重复突变、1例为单个第18外显子缺失、其他5例未查到缺失和重复突变.16例携带者中,3例有家族史,其中2例检出突变;13例为检测到突变的散发病例患儿的母亲,有8例检测到突变.产前诊断9个胎儿(其中双胎1例),2例胎儿有突变,引产后核实无误;7例胎儿未检测到突变,现均已分娩.结论 多重PCR可检出92.86%的缺失突变并可用于缺失突变的产前诊断,因其简便、可靠、价廉可作为临床上DMD/BMD基因诊断的初选.MLPA可用于多重PCR未检测到缺失突变的患者及携带者的检查.     

Large BRCA1 gene deletions are found in 3% of German high-risk breast cancer families

We have tested for large BRCA1 gene rearrangements in German high-risk breast and ovarian cancer families previously screened negative for point mutations by dHPLC and sequencing. Using the novel MLPA method, two deletions of exons 1A, 1B and 2 and exon 17, respectively, were detected in four out of 75 families investigated in Southern Germany. An identical exon 17 deletion with the same breakpoints and a deletion of exons 1A, 1B and 2 were found by fluorescent multiplex PCR in two out of 30 families investigated in Northern Germany. Combining both populations, genomic rearrangements were found in 6% of the mutation-negative families and 3% of all high-risk families and account for 8% of all BRCA1 mutations. Our data indicate that the exon 17 deletion may be a founder mutation in the German population. The prevalence of BRCA1 gene deletions or duplications in our patients is similar to previous reports from Germany and France. Genomic quantification by MLPA is a useful method for molecular diagnostics in high-risk breast cancer families.