Analysis of Heterozygous BRCA1 5382ins Founder Mutation in a Cohort of Egyptian Breast Cancer Female Patients Using Pyrosequencing Technique

Background: Up to half of the heritable mutations in breast cancer (BC) are attributed to BRCA1 and BRCA2 genes. The mutation prevalence is variable based on ethnicity and may be influenced by founder mutations. The aim of this pilot study is to determine for the first time, the prevalence of BRCA1 5382insC founder mutation in a cohort of Egyptian familial breast cancer patients (FBC). Methods: Female patients were selected to have familial type of breast cancer. Twenty healthy females were included as a control group. Peripheral blood samples were withdrawn from all studied females and were analyzed for BRCA1 5382insC founder mutation detection using pyrosequencing technique. Results: Eighty Egyptian FBC females were eligible to be enrolled in the study with a mean age of 48.31 ± 10.97years.We found a BRCA1 5382insC mutation carrier frequency of 5% of total studied FBC patients (4 out of 80 patients) with 95% confidence interval (1.61-12.99). There was a high statistical significant difference between carriers and non-carriers concerning the number of affected family members by BC, (p=0.001).  Conclusion: BRCA1 5382insC founder mutation is not uncommon among Egyptian FBC females. The carrier frequency is comparable to that reported worldwide; however it is lower than those from previous Egyptian studies using different molecular techniques. The strong association between the mutation and the number of affected family members suggest wider screening of the mutation among high risk families using the reliable pyrosequencing technique.     

Quantitative methylation analysis of resection margins and lymph nodes in oral squamous cell carcinoma

Resection is the preferred treatment for oral squamous cell carcinoma, and pathological staging of the resected specimen is crucial. The role of molecular biology in the diagnosis of minimal residual disease has not been fully investigated and may improve staging. Multiple adjacent specimens were taken from the tumour, the invasive front, the surgical margin, and the lymph nodes of 20 specimens from patients with oral cancer. Bisulphite-treated DNA from these specimens was assayed quantitatively with pyrosequencing methylation assays (PMA) of CpG islands within the gene promoters of the p16 and CYGB genes. Results were recorded with histopathological results, and compared with clinical outcome. Biological and technical replicates confirmed the reliability of the techniques. PMA upgraded 13 of the 20 surgical margins, 6 of which subsequently had a recurrent tumour. Not all of these recurrences were predicted and the effects of adjuvant treatment make firm conclusions difficult.     

Changes of saliva microbiota in nasopharyngeal carcinoma patients under chemoradiation therapy

ObjectiveA growing body of evidence has implicated human oral microbiota in the aetiology of oral and systemic diseases. Nasopharyngeal carcinoma (NPC), an epithelial-originated malignancy, has a complex aetiology not yet fully understood. Chemoradiation therapy of NPC can affect oral microbiota and is usually accompanied by plaque accumulation. Thus, the study aimed to understand the diversity, divergence and development of the oral microbiota in NPC patients and their associated treatment, which might provide useful insights into disease aetiology and treatment side effects.DesignA longitudinal study was designed that included three Chinese adults with NPC. Saliva samples were collected at three time points: prior to the chemoradiation treatment (carcinoma baseline, or CB), 7 months post-treatment (carcinoma-after-therapy phase 1 or CA1) and 12 months post-treatment (carcinoma-after-therapy phase 2 or CA2). Pyrosequencing of the bacterial 16S ribosomal DNA (rDNA) V1–V3 hypervariable region was employed to characterise the microbiota. Saliva samples of three healthy subjects from our former study were employed as healthy controls. Principal coordinates analysis (PCoA), Metastats and random forest prediction models were used to reveal the key microbial members associated with NPC and its treatment programme.Results(1) In total, 412 bacterial species from at least 107 genera and 13 phyla were found in the saliva samples of the NPC patients. (2) PCoA revealed that not only were the microbiota from NPC patients distinct from those of healthy controls (p < 0.001) but also that separation was found on the saliva microbiota between pre- and post-therapy (p < 0.001) in the NPC samples. (3) At the genus level and the operational taxonomic unit (OTU) level, Streptococcus was found with lower abundance in NPC samples. (4) Chemoradiation therapy did not incur similar changes in microbiota structure among the three NPC patients; the microbiota in one of them stayed largely steady, while those in the other two showed significant alteration.ConclusionsThis is the first study employing culture-independent techniques to interrogate the phylogenetic diversity, divergence and temporal development of oral microbiota in NPC patients. Our results indicated that certain bacterial taxa might be associated with NPC and that oral microbiota of NPC patients might respond to the chemoradiation therapy in a host-specific manner. Further investigation with larger sample size should help to validate the links between oral microbiota and NPC.     

The mutation rates of EGFR in non-small cell lung cancer and KRAS in colorectal cancer of Chinese patients as detected by pyrosequencing using a novel dispensation order

Background

The purpose of this study was to develop a cost-effective approach for the determination of EGFR and KRAS mutations in formalin-fixed paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) samples from Chinese patients based on a sensitive pyrosequencing (PS) technique.

Methods

The NSCLC and CRC cell lines were tested to determine the limitation of detection and reproducibility of the PS method. In addition, 494 NSCLC and 1099 CRC patient samples were assayed by PS to evaluate the EGFR or KRAS mutation patterns according to the clinicopathological features.

Results

The PS assay was able to reproducibly detect as few as 2 % mutant alleles with excellent linearity. EGFR mutations were detected in 35.63 % of the NSCLC samples, and KRAS mutations were detected in 39.76 % of the CRC samples. EGFR mutations were more frequently observed to be significant by multivariate analysis in NSCLC patients who were 65 years old or younger (OR = 2.51), had a nonsmoking history (OR = 3.63), and adenocarcinoma (OR = 3.57), but not in females (OR = 0.64). KRAS mutations were more frequently detected in CRC patients who were female (OR = 1.64) and 50 years old or older (OR = 4.17), and had adenocarcinoma (OR = 2.41).

Conclusions

This is the first extensive validation of PS on FFPE samples using the detection of EGFR exons 18–21 mutations and KRAS exon 2 mutations. Our results demonstrate the utility of PS analysis for the detection of somatic EGFR and KRAS mutations in clinical samples and provide important clinical and molecular characteristics of NSCLC and CRC from Chinese patients.

Electronic supplementary material

The online version of this article (doi:10.1186/s13046-015-0179-9) contains supplementary material, which is available to authorized users.     

Prognostic Role of Methylation Status of the MGMT Promoter Determined Quantitatively by Pyrosequencing in Glioblastoma Patients

Objective

This study investigated whether pyrosequencing can be used to determine the methylation status of the MGMT promoter as a clinical biomarker using relatively old archival tissue samples of glioblastoma. We also examined other prognostic factors for survival of glioblastoma patients.

Methods

The available study set included formalin-fixed paraffin-embedded (FFPE) tissue from 104 patients at two institutes from 1997 to 2012, all of which were diagnosed histopathologically as glioblastoma. Clinicopathologic data were collected by review of medical records. For pyrosequencing analysis, the PyroMark Q96 CpG MGMT kit (Qiagen, Hilden, Germany) was used to detect the level of methylation at exon 1 positions 17–39 of the MGMT gene, which contains 5 CpGs.

Results

Methylation of the MGMT promoter was detected in 43 (41.3%) of 104 samples. The average percentage methylation was 14.0±16.8% overall and 39.0±14.7% for methylated cases. There was no significant pattern of linear increase or decrease according to the age of the FFPE block (p=0.687). In multivariate analysis, age, performance status, extent of surgery, method of adjuvant therapy, and methylation status estimated by pyrosequencing were independently associated with overall survival. Additionally, patients with a high level of methylation survived longer than those with low methylation (p=0.016).

Conclusion

In this study, the status and extent of methylation of the MGMT promoter analyzed by pyrosequencing were associated with overall survival in glioblastoma patients. Pyrosequencing is a quantitative method that overcomes the problems of MSP and a simple technique for accurate analysis of DNA sequences.     

Single nucleotide polymorphism (SNP)-based differentiation of Shigella isolates by pyrosequencing

Analysis of single nucleotide polymorphisms (SNPs) is an important genetic tool that provides molecular markers for rapid differentiation of closely related strains. We have applied SNP discovery and analysis for distinguishing each of the four Shigella serogroups (Boydii, Dysenteriae, Flexneri, and Sonnei) and for discriminating individual strains within the same serogroup by using 24 SNPs selected from nine genes. Five SNPs were identified from sequence analysis of two housekeeping genes (gapA and thrB) used previously in our lab to differentiate Shigella isolates into distinct lineages. The remaining 19 SNPs were identified by in silico analyses of eight Shigella genomes and are within the genes lpxC, sanA, yaaH, ybaP, ygaZ, yhbO, and ynhA. A total of 118 Shigella strains comprising 20 Boydii, 29 Dysenteriae, 42 Flexneri, and 27 Sonnei isolates were analyzed using the SNP typing scheme reported here. The combination of the 24 SNPs resulted in the identification of 26 SNP genotypes among the four Shigella serogroups and also provided some discriminatory resolution among individual strains within the same serogroup. The SNPs presented here should prove useful in identifying Shigella using PCR amplification and rapid sequence typing strategies.     

Evaluation of PyroMark Q24 pyrosequencing as a method for the identification of mycobacteria

We evaluated PyroMark Q24 (QIAGEN) pyrosequencing as a method for the identification of mycobacteria, with potential application in clinical practice. Sequence data from the hypervariable region A of the 16S rRNA gene (43 and 35 bp sequences) were obtained using PyroMark Q24, and a similarity search was performed automatically with PyroMark IdentiFire software. Of the 148 mycobacterial type strains tested, 138 (93.2%) were accurately identified to single or clade species level, including complex level. From the remaining 10 strains, 3 (Mycobacterium gilvum, Mycobacterium goodi, and Mycobacterium thermoresistible) showed poor sequencing quality of homopolymers. For 6 other strains (Mycobacterium cosmeticum, Mycobacterium flavescens, Mycobacterium pallens, Mycobacterium hodleri, Mycobacterium xenopi, and Mycobacterium crocinum), the sequences were unreadable from the middle, and Sanger sequencing indicated biallelic site. Finally, a 40 bp sequence for Mycobacterium gordonae could not be obtained despite repeated attempts. PyroMark Q24 provided accurate identification of multiple mycobacterial strains isolated from common clinical settings, but additional gene sequencing is required to distinguish species identified as a group or complex.     

A comparison of methylation levels in HPV18, HPV31 and HPV33 genomes reveals similar associations with cervical precancers

BackgroundHigh risk human papillomavirus (HR-HPV) infection is common and only a small minority of infections become persistent and lead to cervical cancers. Women positive for HR-HPV usually require a second test to avoid unnecessary colposcopies and over treatment. Elevated DNA methylation of HR-HPV L1 and L2 genes in high grade disease has emerged as a promising molecular triage tool.ObjectivesOur aim was to accurately measure methylation levels at selected CpG positions in the HPV18, HPV31 and HPV33 genomes. We focused on the L2, L1, URR and E6 regions because these were previously shown to be interesting areas for study.Study designPyrosequencing was used to measure methylation in 208 HPV18, 207 HPV31, and 126 HPV33 positive women selected from a London colposcopy referral population.ResultsAfter adjustment for multiple testing, at FDR 5%, elevated methylation was significantly associated with cervical intraepithelial neoplasia grades 2 or worse (CIN2+) in all investigated CpGs in HPV18 L2 and L1. Two of 6 L2 and 12 of 15 L1 sites in HPV31 and 6 of 8 L2 and 3 of 13 L1 sites in HPV33 showed significantly elevated methylation in CIN2+. Methylation of CpG sites in the URR and E6 region of the HPV types was low and most differences were not significant.ConclusionElevated methylation of CpG sites in the L1 and L2 regions of HPV18, HPV31 and HPV33 is associated with CIN2+ and a panel test may be useful for triage of women with HR-HPV infections.     

拉米夫定治疗慢性乙型肝炎中变异病毒株的动态变化

目的:建立定量检测及监测乙肝病毒DNA的rtM204I/V(YMDD)及rtL180M变异的方法.方法:以克隆、测序鉴定及构建乙肝病毒DNA YMDD及变异(YIDD和YVDD)质粒作为标准,4例接受拉米夫定治疗(100 mg/d,疗程48 wk以上)的慢性乙型肝炎患者血清标本为对象,用焦磷酸测序的方法检测变异位点核苷酸的频率,并计算变异株的含量比例,通过检测不同变异株类型的标准质粒,确定测序峰图的背景信号.结果:通过检测标准质粒后,确定背景信号为5%,变异位点调整后的核苷酸频率转化为变异病毒株的比例后,4例患者治疗前均检测出变异的病毒株(4.5%-33%),并且随治疗时间延长呈增多的趋势.病毒载量及ALT分析提示基因型耐药发生后,将发生病毒学反跳及临床耐药.结论:焦磷酸测序可以定量检测及动态监测变异病毒株的含量.拉米夫定耐药突变株在拉米夫定治疗前已存在,并随治疗时间的增加而增长,出现病毒学反弹及临床耐药.     

Equine herpesvirus 1 (EHV-1) nucleotide polymorphism determination using formalin fixed tissues in EHV-1 induced abortions and myelopathies with real-time PCR and pyrosequencing

Equine herpesvirus-1 (EHV-1) strains with a single point mutation at the 2254 nucleotide position with a G₂₂₅₄ constitution within the DNA polymerase gene are associated strongly with equine myeloencephalopathies. Infections with non-neuropathogenic EHV-1 strains without the G₂₂₅₄ nucleotide but with an A₂₂₅₄ nucleotide are associated less frequently with equine neurologic disease. A retrospective study utilizing DNA extracted from formalin fixed paraffin embedded tissues was conducted with real time PCR and pyrosequencing, to determine the infecting EHV-1 strains. Infection with EHV-1 A₂₂₅₄ and or G₂₂₅₄ strain was detected with real time PCR, and was confirmed with a rapid pyrosequencing technique. Pyrosequencing was useful in at least 2 cases where real time PCR was equivocal in determining the infecting EHV-1 strain type. The strain with G₂₂₅₄ mutation was detected in 9.4% of 21 studied abortion cases, and in 86.6% of 15 neurologic cases.