Noninvasive Detection of Fetal Subchromosome Abnormalities via Deep Sequencing of Maternal Plasma

The purpose of this study was to determine the deep sequencing and analytic conditions needed to detect fetal subchromosome abnormalities across the genome from a maternal blood sample. Cell-free (cf) DNA was isolated from the plasma of 11 pregnant women carrying fetuses with subchromosomal duplications and deletions, translocations, mosaicism, and trisomy 20 diagnosed by metaphase karyotype. Massively parallel sequencing (MPS) was performed with 25-mer tags at approximately 10⁹ tags per sample and mapped to reference human genome assembly hg19. Tags were counted and normalized to fixed genome bin sizes of 1 Mb or 100 kb to detect statistically distinct copy-number changes compared to the reference. All seven cases of microdeletions, duplications, translocations, and the trisomy 20 were detected blindly by MPS, including a microdeletion as small as 300 kb. In two of these cases in which the metaphase karyotype showed additional material of unknown origin, MPS identified both the translocation breakpoint and the chromosomal origin of the additional material. In the four mosaic cases, the subchromosomal abnormality was not demonstrated by MPS. This work shows that in nonmosaic cases, it is possible to obtain a fetal molecular karyotype by MPS of maternal plasma cfDNA that is equivalent to a chromosome microarray and in some cases is better than a metaphase karyotype. This approach combines the advantage of enhanced fetal genomic resolution with the improved safety of a noninvasive maternal blood test.     

Identification of Hepatotropic Viruses from Plasma Using Deep Sequencing: A Next Generation Diagnostic Tool

We conducted an unbiased metagenomics survey using plasma from patients with chronic hepatitis B, chronic hepatitis C, autoimmune hepatitis (AIH), non-alcoholic steatohepatitis (NASH), and patients without liver disease (control). RNA and DNA libraries were sequenced from plasma filtrates enriched in viral particles to catalog virus populations. Hepatitis viruses were readily detected at high coverage in patients with chronic viral hepatitis B and C, but only a limited number of sequences resembling other viruses were found. The exception was a library from a patient diagnosed with hepatitis C virus (HCV) infection that contained multiple sequences matching GB virus C (GBV-C). Abundant GBV-C reads were also found in plasma from patients with AIH, whereas Torque teno virus (TTV) was found at high frequency in samples from patients with AIH and NASH. After taxonomic classification of sequences by BLASTn, a substantial fraction in each library, ranging from 35% to 76%, remained unclassified. These unknown sequences were assembled into scaffolds along with virus, phage and endogenous retrovirus sequences and then analyzed by BLASTx against the non-redundant protein database. Nearly the full genome of a heretofore-unknown circovirus was assembled and many scaffolds that encoded proteins with similarity to plant, insect and mammalian viruses. The presence of this novel circovirus was confirmed by PCR. BLASTx also identified many polypeptides resembling nucleo-cytoplasmic large DNA viruses (NCLDV) proteins. We re-evaluated these alignments with a profile hidden Markov method, HHblits, and observed inconsistencies in the target proteins reported by the different algorithms. This suggests that sequence alignments are insufficient to identify NCLDV proteins, especially when these alignments are only to small portions of the target protein. Nevertheless, we have now established a reliable protocol for the identification of viruses in plasma that can also be adapted to other patient samples such as urine, bile, saliva and other body fluids.     

扩增子测序技术应用于乳腺癌循环游离DNA无创性检测的研究

目的:应用扩增子测序方法检测乳腺癌循环游离DNA肿瘤相关突变。方法:军事医学科学院附属医院的10例HER2阳性晚期乳腺癌患者血样入组,应用扩增子测序技术检测血细胞和血浆中的循环游离DNA(cfDNA),筛选出肿瘤相关基因突变,即循环肿瘤DNA(ctDNA)。结果:检测10例晚期乳腺癌患者外周循环血50个基因,共检出11个突变基因,其中PIK3CA、ERBB4的阳性检出率均达到100%,AKT、TP53的阳性检出率为90%。结论:检测覆盖度及深度均良好,结果提示11个基因的热点突变区域出现单核苷酸的多态性基因突变,突变基因与PI3K-AKT-m TOR通路、Ras-Raf-MEK-ERK通路明显相关。扩增子测序技术和筛选方法可以用于乳腺癌ctDNA的无创性检测。     

A Method to Quantify Cell-Free Fetal DNA Fraction in Maternal Plasma Using Next Generation Sequencing: Its Application in Non-Invasive Prenatal Chromosomal Aneuploidy Detection

Objective

The fraction of circulating cell-free fetal (cff) DNA in maternal plasma is a critical parameter for aneuploidy screening with non-invasive prenatal testing, especially for those samples located in equivocal zones. We developed an approach to quantify cff DNA fractions directly with sequencing data, and increased cff DNAs by optimizing library construction procedure.

Methods

Artificial DNA mixture samples (360), with known cff DNA fractions, were used to develop a method to determine cff DNA fraction through calculating the proportion of Y chromosomal unique reads, with sequencing data generated by Ion Proton. To validate our method, we investigated cff DNA fractions of 2,063 pregnant women with fetuses who were diagnosed as high risk of fetal defects. The z-score was calculated to determine aneuploidies for chromosomes 21, 18 and 13. The relationships between z-score and parameters of pregnancies were also analyzed. To improve cff DNA fractions in our samples, two groups were established as follows: in group A, the large-size DNA fragments were removed, and in group B these were retained, during library construction.

Results

A method to determine cff DNA fractions was successfully developed using 360 artificial mixture samples in which cff DNA fractions were known. A strong positive correlation was found between z-score and fetal DNA fraction in the artificial mixture samples of trisomy 21, 18 and 13, as well as in clinical maternal plasma samples. There was a positive correlation between gestational age and the cff DNA fraction in the clinical samples, but no correlation for maternal age. Moreover, increased fetal DNA fractions were found in group A compared to group B.

Conclusion

A relatively accurate method was developed to determine the cff DNA fraction in maternal plasma. By optimizing, we can improve cff DNA fractions in sequencing samples, which may contribute to improvements in detection rate and reliability.     

Exome Sequencing of Cell-Free DNA from Metastatic Cancer Patients Identifies Clinically Actionable Mutations Distinct from Primary Disease

The identification of the molecular drivers of cancer by sequencing is the backbone of precision medicine and the basis of personalized therapy; however, biopsies of primary tumors provide only a snapshot of the evolution of the disease and may miss potential therapeutic targets, especially in the metastatic setting. A liquid biopsy, in the form of cell-free DNA (cfDNA) sequencing, has the potential to capture the inter- and intra-tumoral heterogeneity present in metastatic disease, and, through serial blood draws, track the evolution of the tumor genome.In order to determine the clinical utility of cfDNA sequencing we performed whole-exome sequencing on cfDNA and tumor DNA from two patients with metastatic disease; only minor modifications to our sequencing and analysis pipelines were required for sequencing and mutation calling of cfDNA. The first patient had metastatic sarcoma and 47 of 48 mutations present in the primary tumor were also found in the cell-free DNA. The second patient had metastatic breast cancer and sequencing identified an ESR1 mutation in the cfDNA and metastatic site, but not in the primary tumor. This likely explains tumor progression on Anastrozole. Significant heterogeneity between the primary and metastatic tumors, with cfDNA reflecting the metastases, suggested separation from the primary lesion early in tumor evolution. This is best illustrated by an activating PIK3CA mutation (H1047R) which was clonal in the primary tumor, but completely absent from either the metastasis or cfDNA. Here we show that cfDNA sequencing supplies clinically actionable information with minimal risks compared to metastatic biopsies. This study demonstrates the utility of whole-exome sequencing of cell-free DNA from patients with metastatic disease. cfDNA sequencing identified an ESR1 mutation, potentially explaining a patient’s resistance to aromatase inhibition, and gave insight into how metastatic lesions differ from the primary tumor.     

Identification of MicroRNAs in Meloidogyne incognita Using Deep Sequencing

MicroRNAs play important regulatory roles in eukaryotic lineages. In this paper, we employed deep sequencing technology to sequence and identify microRNAs in M. incognita genome, which is one of the important plant parasitic nematodes. We identified 102 M. incognita microRNA genes, which can be grouped into 71 nonredundant miRNAs based on mature sequences. Among the 71 miRANs, 27 are known miRNAs and 44 are novel miRNAs. We identified seven miRNA clusters in M. incognita genome. Four of the seven clusters, miR-100/let-7, miR-71-1/miR-2a-1, miR-71-2/miR-2a-2 and miR-279/miR-2b are conserved in other species. We validated the expressions of 5 M. incognita microRNAs, including 3 known microRNAs (miR-71, miR-100b and let-7) and 2 novel microRNAs (NOVEL-1 and NOVEL-2), using RT-PCR. We can detect all 5 microRNAs. The expression levels of four microRNAs obtained using RT-PCR were consistent with those obtained by high-throughput sequencing except for those of let-7. We also examined how M. incognita miRNAs are conserved in four other nematodes species: C. elegans, A. suum, B. malayi and P. pacificus. We found that four microRNAs, miR-100, miR-92, miR-279 and miR-137, exist only in genomes of parasitic nematodes, but do not exist in the genomes of the free living nematode C. elegans. Our research created a unique resource for the research of plant parasitic nematodes. The candidate microRNAs could help elucidate the genomic structure, gene regulation, evolutionary processes, and developmental features of plant parasitic nematodes and nematode-plant interaction.     

Detection of Clonal and Subclonal Copy-Number Variants in Cell-Free DNA from Patients with Breast Cancer Using a Massively Multiplexed PCR Methodology

We demonstrate proof-of-concept for the use of massively multiplexed PCR and next-generation sequencing (mmPCR-NGS) to identify both clonal and subclonal copy-number variants (CNVs) in circulating tumor DNA. This is the first report of a targeted methodology for detection of CNVs in plasma.Using an in vitro model of cell-free DNA, we show that mmPCR-NGS can accurately detect CNVs with average allelic imbalances as low as 0.5%, an improvement over previously reported whole-genome sequencing approaches. Our method revealed differences in the spectrum of CNVs detected in tumor tissue subsections and matching plasma samples from 11 patients with stage II breast cancer. Moreover, we showed that liquid biopsies are able to detect subclonal mutations that may be missed in tumor tissue biopsies. We anticipate that this mmPCR-NGS methodology will have broad applicability for the characterization, diagnosis, and therapeutic monitoring of CNV-enriched cancers, such as breast, ovarian, and lung cancer.     

Combined Targeted DNA Sequencing in Non-Small Cell Lung Cancer (NSCLC) Using UNCseq and NGScopy,and RNA Sequencing Using UNCqeR for the Detection of Genetic Aberrations in NSCLC

The recent FDA approval of the MiSeqDx platform provides a unique opportunity to develop targeted next generation sequencing (NGS) panels for human disease, including cancer. We have developed a scalable, targeted panel-based assay termed UNCseq, which involves a NGS panel of over 200 cancer-associated genes and a standardized downstream bioinformatics pipeline for detection of single nucleotide variations (SNV) as well as small insertions and deletions (indel). In addition, we developed a novel algorithm, NGScopy, designed for samples with sparse sequencing coverage to detect large-scale copy number variations (CNV), similar to human SNP Array 6.0 as well as small-scale intragenic CNV. Overall, we applied this assay to 100 snap-frozen lung cancer specimens lacking same-patient germline DNA (07–0120 tissue cohort) and validated our results against Sanger sequencing, SNP Array, and our recently published integrated DNA-seq/RNA-seq assay, UNCqeR, where RNA-seq of same-patient tumor specimens confirmed SNV detected by DNA-seq, if RNA-seq coverage depth was adequate. In addition, we applied the UNCseq assay on an independent lung cancer tumor tissue collection with available same-patient germline DNA (11–1115 tissue cohort) and confirmed mutations using assays performed in a CLIA-certified laboratory. We conclude that UNCseq can identify SNV, indel, and CNV in tumor specimens lacking germline DNA in a cost-efficient fashion.     

肺癌病人肿瘤组织DNA高甲基化片段的筛选

关于DNA甲基化在肿瘤中的作用的研究,大多集中在研究已知的抑癌基因启动子区的异常高甲基化。而一些未知的参与肿瘤发生的基因也可能受甲基化调控,寻找这些与肿瘤相关的基因,对深入了解肿瘤发生的机制具有重要意义。利用甲基化敏感性随机引物PCR(Methyrlation-Sensitive Arbitrarily Primed PCR,MS-AP-PCR),检查了肺癌组织中基因组范围内CpG岛高甲基化情况,分离到8个高甲基化片段(hypermethylated DNA fragment．HMDF)。通过克隆、测序和Blast、NewCpGseek软件分析,发现所有的片段均为典型的CpG岛,有4个片段与人2、7、9、10号染色体上的同源性为99％～100％,但只有1个是已知的基因。进一步利用Neural Network Promoter Prediction、TSSG和TSSW等软件对其余7个片段可能的生物学意义进行了分析,结果有4个片段是候选的启动子区,提示它们可能源于新基因。所获得的高甲基化片段可能是中国人肺癌发生过程中特有的表遗传学改变。     

Detection of Cytosine Methylation in Ancient DNA from Five Native American Populations Using Bisulfite Sequencing

While cytosine methylation has been widely studied in extant populations, relatively few studies have analyzed methylation in ancient DNA. Most existing studies of epigenetic marks in ancient DNA have inferred patterns of methylation in highly degraded samples using post-mortem damage to cytosines as a proxy for cytosine methylation levels. However, this approach limits the inference of methylation compared with direct bisulfite sequencing, the current gold standard for analyzing cytosine methylation at single nucleotide resolution. In this study, we used direct bisulfite sequencing to assess cytosine methylation in ancient DNA from the skeletal remains of 30 Native Americans ranging in age from approximately 230 to 4500 years before present. Unmethylated cytosines were converted to uracils by treatment with sodium bisulfite, bisulfite products of a CpG-rich retrotransposon were pyrosequenced, and C-to-T ratios were quantified for a single CpG position. We found that cytosine methylation is readily recoverable from most samples, given adequate preservation of endogenous nuclear DNA. In addition, our results indicate that the precision of cytosine methylation estimates is inversely correlated with aDNA preservation, such that samples of low DNA concentration show higher variability in measures of percent methylation than samples of high DNA concentration. In particular, samples in this study with a DNA concentration above 0.015 ng/μL generated the most consistent measures of cytosine methylation. This study presents evidence of cytosine methylation in a large collection of ancient human remains, and indicates that it is possible to analyze epigenetic patterns in ancient populations using direct bisulfite sequencing approaches.     

Molecular Barcode Sequencing of the Whole Ligand Binding Domain of the ESR1 Gene in Cell-Free DNA from Patients with Metastatic Breast Cancer

ESR1 mutations in breast cancer are known as one of the mechanisms of resistance to aromatase inhibitors. These mutations often occur in the hotspot regions in the ligand binding domain (LBD), but comprehensive mutational analysis has shown that mutations are observed throughout the whole LBD. We previously developed a molecular barcode sequencing (MB-NGS) technique to detect ESR1 hotspot mutations in plasma with high sensitivity. In this study, we have developed a multiplex MB-NGS assay that covers the whole LBD of ESR1. The assay demonstrated that the background errors in the plasma DNA of 10 healthy controls were below 0.1%; thus, the limit of detection was set at 0.1%. We analyzed the plasma DNA of 54 patients with estrogen receptor–positive metastatic breast cancer. Seventeen mutations were detected in 13 patients (24%), with variant allele frequencies ranging from 0.13% to 10.67%, including six rare mutations with a variant allele frequency <1.0% and a novel nonhotspot mutation (A312V). Three patients had double mutations located in the same amplicons, and it was revealed that the double mutations were located in different alleles. ESR1 hotspot mutations were associated with a longer duration of aromatase inhibitor treatment under metastatic conditions and to liver metastasis. The multiplex MB-NGS assay is useful for the sensitive and comprehensive detection of mutations throughout the whole LBD of ESR1. Our assay can be applied to any specific target region of interest using tailor-made primers and can result in minimized sequencing volume and cost.     

Comparative Screening of K-ras Mutations in Colorectal Cancer and Lung Cancer Patients Using a Novel Real-Time PCR with ADx-K-ras Kit and Sanger DNA Sequencing

We determined frequency/types of K-ras mutations in colorectal/lung cancer. ADx-K-ras kit (real-time/double-loop probe PCR) was used to detect somatic tumor gene mutations compared with Sanger DNA sequencing using 583 colorectal and 244 lung cancer paraffin-embedded clinical samples. Genomic DNA was used in both methods; mutation rates at codons 12/13 and frequency of each mutation were detected and compared. The data show that 91.4% colorectal and 59.0% lung carcinoma samples were detected conclusively by DNA sequencing, whereas 100% colorectal and lung samples were detected by ADx-K-ras kit. K-ras gene mutations were detected in 32.9–27.4% colorectal samples using kit and sequencing methods, respectively. Whereas 10.6–8.3% lung cancer samples were positively detected by kit and sequencing methods, respectively. Notably, 172/677 showed mutations and 467/677 showed wild type by both methods; 38 samples showed mutations with kit but wild type with sequencing. Mutations in colorectal samples were as follows: GGT → GAT/codon-12 (35.1%); GGC → GAC/codon-13 (26.6%); GGT → GTT/codon-12 (18.2%); and GGT → GCT/codon-12 (1.6%). Mutations in lung samples were as follows: GGT > GTT/codon-12 (40.9%) and GGT > GCT/codon-12 (4.5%). In conclusion, K-ras mutations involved 32.2% colorectal and 10.6% lung samples among this cohort. ADx-K-ras real-time PCR showed higher detection rates (P < 0.05). The kit method has good clinical applicability as it is simple, fast, less prone to contamination and hence can be used effectively and reliably for clinical screening of somatic tumor gene mutations.     

Quantification of Cell-Free mSHOX2 Plasma DNA for Therapy Monitoring in Advanced Stage Non-Small Cell (NSCLC) and Small-Cell Lung Cancer (SCLC) Patients

PurposeMost patients suffering from advanced lung cancer die within a few months. To exploit new therapy regimens we need better methods for the assessment of a therapy response.ResultsAccording to the re-staging of 31 first-line patients, 19 patients were classified as non-responders while 12 patients were in the responder group. We observed a tight correlation between radiological data and the change of plasma mSHOX2 level as the equivalent for a therapy response. A ROC analysis showed a high discriminatory power for both patient groups already one week after therapy start (AUC 0.844). Additionally, a Kaplan-Meier and Cox Proportional Hazards analyses revealed a strong relationship between survival and plasma mSHOX2 value p≤0.001 (hazard ratio 11.08) providing some evidence for mSHOX2 also being a predictive marker.ConclusionThe longitudinal measurement of extracellular plasma mSHOX2 DNA yields information about the response to cytotoxic treatment and allows an early assessment of treatment response for lung cancer patients. If confirmed in a larger study this would be a valuable tool for selecting and guiding a cytotoxic treatment.     

应用PCR产物直接银染测序技术检测大肠癌p53基因点突变

应用PCR-SSCP结合PCR产物直接银染测序技术对24例大肠癌p53基因第5-7外显子进行点突变的研究。结果检出5例(26.7%)阳性,均为错义突变;其中3例为碱基GC到 AT的转换, 1例为GC到TA的颠换,另1例为AT到CG的颠换,后者尚未见报道。突变位点分布在p53基因第141、175、245、248和258位密码子,其中4例发生在CpG位点。本文对p53基因点突变谱的分析为大肠癌的病因学研究提供了科学依据, 并讨论了PCR产物直接银染测序技术的优越性。 Abstract:Mutations in exon 5~7 of p53 were screened in 24 cases of colorectal carcinoma by a combination of PCR-SSCP and PCR-product DNA silver sequencing.The results showed that all 5(26.7%) cases of point mutations detected were missense mutations,including 3 cases of GC to AT transitions,1 case of GC to TA transversion and another case of AT to CG transversion.The latter has not been reported before.The mutations occurred at codons 141,175,245,248 and 258 respectively,and 4 cases of these five mutations occurred at CpG dinucleotides.The analysis of p53 mutation spectra can provide clues to the etiology of colorectal carcinoma.The advantages of DNA silver sequencing are also discussed.     

Validation of an Ion Torrent Sequencing Platform for the Detection of Gene Mutations in Biopsy Specimens from Patients with Non-Small-Cell Lung Cancer

Background

Treatment for patients with advanced non-small cell lung cancer (NSCLC) is often determined by the presence of biomarkers that predict the response to agents targeting specific molecular pathways. Demands for multiplex analysis of the genes involved in the pathogenesis of NSCLC are increasing.

Methods

We validated the Ion Torrent Personal Genome Machine (PGM) system using the Ion AmpliSeq Cancer Hotspot Panel and compared the results with those obtained using the gold standard methods, conventional PCR and Sanger sequencing. The cycleave PCR method was used to verify the results.

Results and Conclusion

The Ion Torrent PGM resulted in a similar level of accuracy in identifying multiple genetic mutations in parallel, compared with conventional PCR and Sanger sequencing; however, the Ion Torrent PGM was superior to the other sequencing methods in terms of increased ease of use, even when taking into account the small amount of DNA that was obtained from formalin-fixed paraffin embedded (FFPE) biopsy specimens.     

Mrassf1a-Pap,a Novel Methylation-Based Assay for the Detection of Cell-Free Fetal DNA in Maternal Plasma

Objectives

RASSF1A has been described to be differentially methylated between fetal and maternal DNA and can therefore be used as a universal sex-independent marker to confirm the presence of fetal sequences in maternal plasma. However, this requires highly sensitive methods. We have previously shown that Pyrophosphorolysis-activated Polymerization (PAP) is a highly sensitive technique that can be used in noninvasive prenatal diagnosis. In this study, we have used PAP in combination with bisulfite conversion to develop a new universal methylation-based assay for the detection of fetal methylated RASSF1A sequences in maternal plasma.

Methods

Bisulfite sequencing was performed on maternal genomic (g)DNA and fetal gDNA from chorionic villi to determine differentially methylated regions in the RASSF1A gene using bisulfite specific PCR primers. Methylation specific primers for PAP were designed for the detection of fetal methylated RASSF1A sequences after bisulfite conversion and validated.

Results

Serial dilutions of fetal gDNA in a background of maternal gDNA show a relative percentage of ∼3% can be detected using this assay. Furthermore, fetal methylated RASSF1A sequences were detected both retrospectively as well as prospectively in all maternal plasma samples tested (n = 71). No methylated RASSF1A specific bands were observed in corresponding maternal gDNA. Specificity was further determined by testing anonymized plasma from non-pregnant females (n = 24) and males (n = 21). Also, no methylated RASSF1A sequences were detected here, showing this assay is very specific for methylated fetal DNA. Combining all samples and controls, we obtain an overall sensitivity and specificity of 100% (95% CI 98.4%–100%).

Conclusions

Our data demonstrate that using a combination of bisulfite conversion and PAP fetal methylated RASSF1A sequences can be detected with extreme sensitivity in a universal and sex-independent manner. Therefore, this assay could be of great value as an addition to current techniques used in noninvasive prenatal diagnostics.     

Identification of BRCA1/2 Founder Mutations in Southern Chinese Breast Cancer Patients Using Gene Sequencing and High Resolution DNA Melting Analysis

Background

Ethnic variations in breast cancer epidemiology and genetics have necessitated investigation of the spectra of BRCA1 and BRCA2 mutations in different populations. Knowledge of BRCA mutations in Chinese populations is still largely unknown. We conducted a multi-center study to characterize the spectra of BRCA mutations in Chinese breast and ovarian cancer patients from Southern China.

Methodology/Principal Findings

A total of 651 clinically high-risk breast and/or ovarian cancer patients were recruited from the Hong Kong Hereditary Breast Cancer Family Registry from 2007 to 2011. Comprehensive BRCA1 and BRCA2 mutation screening was performed using bi-directional sequencing of all coding exons of BRCA1 and BRCA2. Sequencing results were confirmed by in-house developed full high resolution DNA melting (HRM) analysis. Among the 451 probands analyzed, 69 (15.3%) deleterious BRCA mutations were identified, comprising 29 in BRCA1 and 40 in BRCA2. The four recurrent BRCA1 mutations (c.470_471delCT, c.3342_3345delAGAA, c.5406+1_5406+3delGTA and c.981_982delAT) accounted for 34.5% (10/29) of all BRCA1 mutations in this cohort. The four recurrent BRCA2 mutations (c.2808_2811delACAA, c.3109C>T, c.7436_7805del370 and c.9097_9098insA) accounted for 40% (16/40) of all BRCA2 mutations. Haplotype analysis was performed to confirm 1 BRCA1 and 3 BRCA2 mutations are putative founder mutations. Rapid HRM mutation screening for a panel of the founder mutations were developed and validated.

Conclusion

In this study, our findings suggest that BRCA mutations account for a substantial proportion of hereditary breast/ovarian cancer in Southern Chinese population. Knowing the spectrum and frequency of the founder mutations in this population will assist in the development of a cost-effective rapid screening assay, which in turn facilitates genetic counseling and testing for the purpose of cancer risk assessment.