High performance methylated DNA markers for detection of colon adenocarcinoma.

BACKGROUND: Colon cancer (CC) is treatable if detected in its early stages. Improved CC detection assays that are highly sensitive, specific, and available at point of care are needed. In this study, we systematically selected and tested methylated markers that demonstrate high sensitivity and specificity for detection of CC in tissue and circulating cell-free DNA. METHODS: Hierarchical analysis of 22 candidate CpG loci was conducted using The Cancer Genome Atlas (TCGA) COAD 450K HumanMethylation database. Methylation of 13 loci was analyzed using quantitative multiplex methylation-specific PCR (QM-MSP) in a training set of fresh frozen colon tissues (N = 53). Hypermethylated markers were identified that were highest in cancer and lowest in normal colon tissue using the 75th percentile in Mann-Whitney analyses and the receiver operating characteristic (ROC) statistic. The cumulative methylation status of the marker panel was assayed in an independent test set of fresh frozen colon tissues (N = 52) using conditions defined and locked in the training set. A minimal marker panel of 6 genes was defined based on ROC area under the curve (AUC). Plasma samples (N = 20 colorectal cancers, stage IV and N = 20 normal) were tested by cMethDNA assay to evaluate marker performance in liquid biopsy. RESULTS: In the test set of samples, compared to normal tissue, a 6-gene panel showed 100% sensitivity and 90% specificity for detection of CC, and an AUC of 1.00 (95% CI 1.00, 1.00). In stage IV colorectal cancer plasma versus normal, an 8-gene panel showed 95% sensitivity, 100% specificity, and an AUC of 0.996 (95% CI 0.986, 1.00) while a 5-gene subset showed 100% sensitivity, 100% specificity, and an AUC of 1.00 (95% CI 1.00, 1.00), highly concordant with our observations in tissue. CONCLUSIONS: We identified high performance methylated DNA marker panels for detection of CC. This knowledge has set the stage for development and implementation of novel, automated, self-contained CC detection assays in tissue and blood which can expeditiously and accurately detect colon cancer in both developed and underdeveloped regions of the world, enabling optimal use of limited resources in low- and middle-income countries.

Use of a Standardized Tool to Identify Women at Risk for Hereditary Breast and Ovarian.

OBJECTIVE: To increase rates of identification and genetic counseling referral for women at risk of hereditary breast and ovarian cancer (HBOC). DESIGN: Evidence-based practice improvement initiative. SETTING/LOCAL PROBLEM: Private suburban obstetric and gynecologic (OB/GYN) practice in Tennessee with no standardized process for HBOC risk assessment or referral to genetic services. PARTICIPANTS: Provider-led women's health care teams delivering well-woman care for women ages 18 years and older. INTERVENTION/MEASUREMENTS: We implemented the use of a standardized familial risk assessment tool and clinical decision-making algorithm. Preimplementation and postimplementation risk identification and genetic services referral rates were measured, as was clinicians' compliance with using the risk assessment tool. The aim of the initiative was to increase identification and referral rates by 25 percentage points. RESULTS: Women at risk of HBOC in the postimplementation group were 25.9 times more likely to be identified as being at risk (OR = 25.88, 95% confidence interval [10.78, 62.14]) and 31.5 times more likely to be offered referral to genetic counseling (OR = 31.50, 95% CI [13.37, 74.22]) compared with those in the preimplementation group. Rates of risk identification and referral to genetic counseling for women at risk of HBOC improved by 58.2 and 69.3 percentage points, respectively, surpassing the aims of this initiative and showing statistical significance of p < .001 for both indices. CONCLUSION: The use of a standardized risk assessment tool and process for HBOC risk identification and genetic referral resulted in a significant increase in the identification and referral of women at risk in this setting. Early identification of women with HBOC is a crucial first step in increasing the use of enhanced screening and interventions that can reduce HBOC-associated cancer morbidity and mortality.

Oncotype DX and Prosigna in breast cancer patients: A comparison study.

BACKGROUND: Oncotype Dx® (ODX) is the most used prognostic and predictive assay for ER + breast cancer (BCa) and is categorized into low (< 18), intermediate (18 to 30), or high (≥31) risk of recurrence. Prosigna® is a prognostic signature to estimate distant recurrence-free survival for stage I/II, ER+ cancer in postmenopausal women treated with adjuvant therapy. The goal of the study is to assess the agreement between ODX and Prosigna®. MATERIALS AND METHODS: 100 previously ODX classified peri and postmenopausal, early-stage (I or II) BCa patients were retrieved and Prosigna assay was performed on archived tumor blocks on a NanoString nCounter® DX Analysis System. RESULTS: ODX assay was assigned as follows: 57% low, 39% intermediate, and 4% high. There were 8% two-step disagreements (high to low or vice versa) between ODX and Prosigna®; and 42% one-step disagreement (low to intermediate or vice versa). 78% were classified by Prosigna as luminal A and 22% as luminal B. The majority of luminal A cases (67/78; 85.9%) had low ROR score whereas ODX classified almost two-thirds (50/78~ 64%) as low RS. An insignificant percentage of luminal B cases (1/22 - 4.5%) were classified as high RS by ODX, and a modest percentage were classified as high ROR by Prosigna (15/22 ~68%). According to our follow up results, recurrence was detected in three cases. In all three cases; Prosigna was a better indicator of recurrence. CONCLUSIONS: The agreement between ODX and Prosigna® is low, and this has management implications, especially when chemotherapy is needed.

Genetic Analysis of Pediatric Pancreatoblastoma: A Case Report.

ABSTRACT: Pediatric pancreatoblastoma (PBL) is a rare disease, and the treatment of which is diverse. The molecular alteration in pancreatoblastoma is not very clear. A 7-year-old female who presented with intermittent abdominal pain, anorexia, and abdominal mass was admitted in our hospital. Pancreaticoduodenectomy, cholecystectomy, and retroperitoneal lymph node dissection were conducted. Immunohistochemistry results after surgery showed creatine kinase +, clusters differentiation 56 -, clusters differentiation 99 +, carcinoembryonic antigen -, periodic acid-Schiff +, B- catenin +, Ki-67 + 70%, progesterone receptor +, neuron-specific enolase -, vimentin -, and insulin -. According to the cell shape and the results of immunohistochemistry, the patient was diagnosed with PBL. The tumor tissue, adjacent tissue, and blood were collected. Mutation profiles were detected using next-generation sequencing technique with a panel of 704 genes. The child recovered well without complications postoperatively. There were 261 genes mutated in her plasma or tumor tissue (mutant frequency ≥1%). The adjacent tissue and plasma harbored the echinoderm microtubule-associated protein-like 4 gene-anaplastic lymphoma kinase fusion and tumor tissue harbored proto-oncogene receptor tyrosine kinase 1-solute carrier family 34 member A2 fusion. The gene alteration profiles of PBL patients warrant further investigations, which may provide new insight into the treatment of this disease.

Case-Based Review and Clinical Guidance on the Use of Genomic Assays for Early-Stage Breast Cancer: Breast Cancer Therapy Expert Group (BCTEG).

In addition to classical clinicopathologic factors, such as hormone receptor positivity, human epidermal growth factor receptor 2 (HER2) status, and tumor size, grade, and lymph node status, a number of commercially available genomic tests may be used to help inform treatment decisions for early breast cancer patients. Although these tests improve our understanding of breast cancer and help to individualize treatment decisions, clinicians face challenges when deciding on the most appropriate test to order, and the advantages, if any, of one test over another. The Breast Cancer Therapy Expert Group (BCTEG) recently convened a roundtable meeting to discuss issues surrounding the use of genomic testing in early breast cancer, with the goal of providing practical guidance on the use of these tests by the community oncologist, for whom breast cancer may be only one of many tumor types they treat. The group recognizes that genomic testing can provide important prognostic (eg, risk for recurrence), and in some cases predictive, information (eg, benefit of chemotherapy, or extended adjuvant endocrine therapy), which can be used to help guide treatment decisions in breast cancer. The available tests differ in the types of information they provide, and in the patient populations and clinical trials that were conducted to validate them. We summarize the discussion of the BCTEG on this topic, and we also consider several patient cases and clinical scenarios in which genomic testing may, or may not, be useful to guide treatment decisions for the practicing community oncologist.

Whole Exome Sequencing Identifies Novel Genetic Alterations in Patients with Pheochromocytoma/Paraganglioma.

BACKGROUND: Pheochromocytoma and paragangliomas (PPGL) are known as tumors with the highest level of heritability, approximately 30% of all cases. Clinical practice guidelines of PPGL recommend genetic testing for germline variants in all patients. In this study, we used whole exome sequencing to identify novel causative variants associated with PPGL to improve the detection of rare genetic variants in our cohort. METHODS: Thirty-six tested negative for pathogenic variants in previous Sanger sequencing or targeted gene panel testing for PPGL underwent whole exome sequencing. Whole exome sequencing was performed using DNA samples enriched using TruSeq Custom Enrichment Kit and sequenced with MiSeq (Illumina Inc.). Sequencing alignment and variant calling were performed using SAMtools. RESULTS: Among previously mutation undetected 36 patients, two likely pathogenic variants and 13 variants of uncertain significance (VUS) were detected in 32 pheochromocytoma-related genes. SDHA c.778G>A (p.Gly260Arg) was detected in a patient with head and neck paraganglioma, and KIF1B c.2787-2A>C in a patient with a bladder paraganglioma. Additionally, a likely pathogenic variant in BRCA2, VUS in TP53, and VUS in NFU1 were detected. CONCLUSION: Exome sequencing further identified genetic alterations by 5.6% in previously mutation undetected patients in PPGL. Implementation of targeted gene sequencing consisted of extended genes of PPGL in routine clinical screening can support the level of comprehensive patient assessment.

Integrated routine workflow using next-generation sequencing and a fully-automated platform for the detection of KRAS, NRAS and BRAF mutations in formalin-fixed paraffin embedded samples with poor DNA quality in patients with colorectal carcinoma.

BACKGROUND: KRAS and NRAS mutations are identified resistance mutations to anti-epidermal growth factor receptor monoclonal antibodies in patients with metastatic colorectal cancer. BRAF status is also routinely assessed for its poor prognosis value. In our institute, next-generation sequencing (NGS) is routinely used for gene-panel mutations detection including KRAS, NRAS and BRAF, but DNA quality is sometimes not sufficient for sequencing. In our routine practice, Idylla platform is used for the analysis of samples that don't reach sufficient quality criteria for NGS assay. METHODS: In this study, data from mCRC samples analyzed from May 2017 to 2018 were retrospectively collected. All samples with a poor DNA quality for sequencing have been assessed using Idylla platform. First, KRAS Idylla assay cartridge has been used for the determination of KRAS mutational status. All KRAS wild-type samples have then been analyzed using NRAS-BRAF assay. Among 669 samples, 67 samples failed the DNA quality control and have been assessed on Idylla KRAS mutation test. RESULTS: Among 67 samples, 50 (75%) samples had a valid result with Idylla KRAS mutation test including 22 carrying a KRAS mutation. For 28 samples, NRAS and BRAF mutational statuses have been assessed using Idylla NRAS-BRAF mutation test. Among 28 samples, 27 (96%) had a valid result including 2 samples bearing a NRAS mutation and 3 samples bearing a BRAF mutation. CONCLUSIONS: Our study shows that an integrated workflow using NGS and Idylla platform allows the determination of KRAS, NRAS and BRAF mutational statuses of 651/669 (97.3%) samples and retrieve 49/67 (73.1%)samples that don't reach DNA quality requirements for NGS.

Interinstitutional variation in predictive value of the ThyroSeq v2 genomic classifier for cytologically indeterminate thyroid nodules.

BACKGROUND: The ThyroSeq v2 next-generation sequencing assay estimates the probability of malignancy in indeterminate thyroid nodules. Its diagnostic accuracy in different practice settings and patient populations is not well understood. METHODS: We analyzed 273 Bethesda III/IV indeterminate thyroid nodules evaluated with ThyroSeq at 4 institutions: 2 comprehensive cancer centers (n = 98 and 102), a multicenter health care system (n = 60), and an academic medical center (n = 13). The positive and negative predictive values of ThyroSeq and distribution of final pathologic diagnoses were analyzed and compared with values predicted by Bayes theorem. RESULTS: Across 4 institutions, the positive predictive value was 35% (22%-43%) and negative predictive value was 93% (88%-100%). Predictive values correlated closely with Bayes theorem estimates (r2 = 0.84), although positive predictive values were lower than expected. RAS mutations were the most common molecular alteration. Among 84 RAS-mutated nodules, malignancy risk was variable (25%, range 10%-37%) and distribution of benign diagnoses differed across institutions (adenoma/hyperplasia 12%-85%, noninvasive follicular thyroid neoplasm with papillary-like nuclear features 5%-46%). CONCLUSION: In a multi-institutional analysis, ThyroSeq positive predictive values were variable and lower than expected. This is attributable to differences in the prevalence of malignancy and variability in pathologist interpretations of noninvasive tumors. It is important that clinicians understand ThyroSeq performance in their practice setting when evaluating these results.

Rapid preimplantation genetic screening using a handheld, nanopore-based DNA sequencer.

OBJECTIVE: To determine if a handheld, nanopore-based DNA sequencer can be used for rapid preimplantation genetic screening (PGS). DESIGN: Laboratory study. SETTING: Academic medical center. PATIENT(S): Amplified genomic DNA from euploid and aneuploid trophectoderm biopsy samples (n=9) that was also tested using traditional next generation sequencing (NGS). INTERVENTION(S): Short-read DNA library preparation and nanopore-based sequencing using a hand-held MinION sequencer. MAIN OUTCOME MEASURE(S): Comparison of cytogenetic testing result from NGS and nanopore-based sequencing and the time required for library preparation and sequencing. RESULT(S): Multiplexed short-read DNA library preparation was completed in 45 minutes. Sequencing on a single sample was completed within 20 minutes and 5 samples were simultaneously sequenced in under 2 hours. Whole-chromosome aneuploidy screening results obtained from nanopore-based sequencing were identical to those obtained using NGS. CONCLUSION(S): Here we report the first application of nanopore-based sequencing for PGS on trophectoderm biopsy samples using a novel rapid multiplxed short-read nanopore sequencing library preparation protocol. Sequencing for aneuploidy screening could be performed on a single sample in 20 minutes and on 5 samples, simultaneously, within 2 hours. Overall, nanopore sequencing is a promising tool to perform rapid PGS onsite, enabling same day testing and embryo transfer, thus obviating the need for complex, large and expensive DNA sequencers or embryo freezing.

[A novel quantitative JAK2V617F detection kit: prospective clinical performance study comparing MPN patients and healthy subjects].

The JAK2V617F mutation is the commonest major genetic mutation of myeloproliferative neoplasms (MPNs) and has been defined in the WHO diagnostic criteria for MPNs. However, there is still no approved in vitro diagnostic test kit available in Japan. We evaluated a JAK2V617F allele quantification kit (test method) in a prospective, multicenter clinical performance study involving patients with MPNs who were diagnosed with polycythemia vera, essential thrombocythemia, and primary myelofibrosis; healthy volunteers were also included in the analysis. Good correlation was observed between the allele burden determined using the test method vs. that determined using next-generation sequencing (NGS) in the patient group (r=0.998, y=1.071x-0.069; n=156). Furthermore, all allele burdens in the healthy group (n=54) were below the lower limit of the measurement range of the test method (0.042%). Our results confirmed that the test method could quantitatively measure the JAK2V617F allele burden in patients with MPN. Thus, the novel JAK2V617F allele quantification kit can be considered useful for the diagnosis of MPNs.

Minimally invasive skin tape strip RNA sequencing identifies novel characteristics of the type 2-high atopic dermatitis disease endotype.

BACKGROUND: Expression profiling of skin biopsy specimens has established molecular features of the skin in patients with atopic dermatitis (AD). The invasiveness of biopsies has prevented their use in defining individual-level AD pathobiological mechanisms (endotypes) in large research studies. OBJECTIVE: We sought to determine whether minimally invasive skin tape strip transcriptome analysis identifies gene expression dysregulation in AD and molecular disease endotypes. METHODS: We sampled nonlesional and lesional skin tape strips and biopsy specimens from white adult patients with AD (18 male and 12 female patients; age [mean ± SE], 36.3 ± 2.2 years) and healthy control subjects (9 male and 16 female subjects; age [mean ± SE], 34.8 ± 2.2 years). AmpliSeq whole-transcriptome sequencing was performed on extracted RNA. Differential expression, clustering/pathway analyses, immunostaining of skin biopsy specimens, and clinical trait correlations were performed. RESULTS: Skin tape expression profiles were distinct from skin biopsy profiles and better sampled epidermal differentiation complex genes. Skin tape expression of 29 immune and epidermis-related genes (false discovery rate < 5%) separated patients with AD from healthy subjects. Agnostic gene set analyses and clustering revealed 50% of patients with AD exhibited a type 2 inflammatory signature (type 2-high endotype) characterized by differential expression of 656 genes, including overexpression of IL13, IL4R, CCL22, CCR4 (log2 fold change = 5.5, 2.0, 4.0, and 4.1, respectively) and at a pathway level by TH2/dendritic cell activation. Both expression and immunostaining of skin biopsy specimens indicated this type 2-high group was enriched for inflammatory, type 2-skewed dendritic cells expressing FcεRI. The type 2-high endotype group exhibited more severe disease by using both the Eczema Area and Severity Index score and body surface area covered by lesions. CONCLUSION: Minimally invasive expression profiling of nonlesional skin reveals stratification in AD molecular pathology by type 2 inflammation that correlates with disease severity.

Review of Clinical Next-Generation Sequencing.

CONTEXT: - Next-generation sequencing (NGS) is a technology being used by many laboratories to test for inherited disorders and tumor mutations. This technology is new for many practicing pathologists, who may not be familiar with the uses, methodology, and limitations of NGS. OBJECTIVE: - To familiarize pathologists with several aspects of NGS, including current and expanding uses; methodology including wet bench aspects, bioinformatics, and interpretation; validation and proficiency; limitations; and issues related to the integration of NGS data into patient care. DATA SOURCES: - The review is based on peer-reviewed literature and personal experience using NGS in a clinical setting at a major academic center. CONCLUSIONS: - The clinical applications of NGS will increase as the technology, bioinformatics, and resources evolve to address the limitations and improve quality of results. The challenge for clinical laboratories is to ensure testing is clinically relevant, cost-effective, and can be integrated into clinical care.

The first Canadian experience with the Afirma® gene expression classifier test.

BACKGROUND: Thyroid nodules are common and often benign, although prove to be malignant upon surgical pathology in 5-15% of cases. When assessed with ultrasound-guided fine-needle aspiration (USFNA), 15-30% of the nodules yield an indeterminate result. The Afirma® gene expression classifier (AGEC) was developed to improve management of indeterminate thyroid nodules (ITNs) by classifying them as "benign" or "suspicious." Objectives were (1) to assess the performance of the AGEC in two Canadian academic medical centres (2), to search for inter-institutional variation and (3) to compare AGEC performance in Canadian versus American institutions. METHODS: We undertook a retrospective cohort study of patients with indeterminate cytopathology (Bethesda Class III or IV) as per USFNA who underwent AGEC testing. We reviewed patient demographics, cytopathological results, AGEC data and, if the patient underwent surgery, results from their final pathology. RESULTS: In total, we included 172 patients with Bethesda Class III or IV thyroid nodules underwent AGEC testing, 109 in Montreal, Quebec and 63 in St. John's, Newfoundland, in this study. Among the nodules sent for testing, 55% (60/109) in Montreal and 46% (29/63) in St. John's returned as "benign." None of these patients underwent surgery. On the other hand, 45% (49/109) nodules in Montreal and 54% (34/63) in St. John's were found to be "suspicious," for a total of 83 specimens. Seventy seven of these patients underwent surgery. Both in Montreal and St. John's, the final pathology yielded malignant thyroid disease in approximately 50% of the specimens categorized as "suspicious." Since 2013, no patient diagnosed with a benign nodule as per AGEC testing was found to harbor a malignant thyroid nodule on follow-up. CONCLUSIONS: Molecular analysis is increasingly used in the management of indeterminate thyroid nodules. This study highlights the experience of two Canadian centres with AGEC testing. We found inter-institutional variability in the rate of nodules returning as "benign," however we found similar rates of confirmed malignancy in nodules returning as "suspicious." According the literature, results for AGEC testing in two Canadian institutions align with results reported in American centres.

Validation and optimization of the Ion Torrent S5 XL sequencer and Oncomine workflow for BRCA1 and BRCA2 genetic testing.

In this study, we validated the analytical performance of BRCA1/2 sequencing using Ion Torrent's new bench-top sequencer with amplicon panel with optimized bioinformatics pipelines. Using 43 samples that were previously validated by Illumina's MiSeq platform and/or by Sanger sequencing/multiplex ligation-dependent probe amplification, we amplified the target with the Oncomine™ BRCA Research Assay and sequenced on Ion Torrent S5 XL (Thermo Fisher Scientific, Waltham, MA, USA). We compared two bioinformatics pipelines for optimal processing of S5 XL sequence data: the Torrent Suite with a plug-in Torrent Variant Caller (Thermo Fisher Scientific), and commercial NextGENe software (Softgenetics, State College, PA, USA). All expected 681 single nucleotide variants, 15 small indels, and three copy number variants were correctly called, except one common variant adjacent to a rare variant on the primer-binding site. The sensitivity, specificity, false positive rate, and accuracy for detection of single nucleotide variant and small indels of S5 XL sequencing were 99.85%, 100%, 0%, and 99.99% for the Torrent Variant Caller and 99.85%, 99.99%, 0.14%, and 99.99% for NextGENe, respectively. The reproducibility of variant calling was 100%, and the precision of variant frequency also showed good performance with coefficients of variation between 0.32 and 5.29%. We obtained highly accurate data through uniform and sufficient coverage depth over all target regions and through optimization of the bioinformatics pipeline. We confirmed that our platform is accurate and practical for diagnostic BRCA1/2 testing in a clinical laboratory.

Resequencing array for gene variant detection in malignant hyperthermia and butyrylcholinestherase deficiency.

Malignant hyperthermia (MH) and butyrylcholinestherase (BCHE) deficiency are two relevant pharmacogenetic disorders in anesthetic practice linked with sequence variants, the former in the RyR1 and CACNA1S genes, the latter in the BCHE gene. Genotyping for known pathogenic variants in these genes is useful to help identify susceptible individuals, and others may exist but remain unknown, because full-length sequence of these genes is, in general, not investigated. To facilitate this task, we developed a resequencing DNA array, the perioperative patient safety (POPS) array, to be able to screen the entire coding sequences of the RyR1, CACNA1S and BCHE genes. MH-susceptible individuals (n = 121) identified with the in vitro contracture test, the standard diagnostic tool for MH susceptibility, were genotyped with the arrays. Compared with capillary sequencing, call rates with the arrays could achieve 100% at maximal sensitivity, although to reduce false positive rates, sensitivity was adjusted to 0.85, 0.87 and 0.66 for RyR1, CACNA1S and BCHE respectively, with overall base call specificity exceeding 99%. Detection of 29 predetermined RyR1 variants in 44 individuals was successful in 97% of the cases, among them all 16 variants of established diagnostic value. In a trial application of the arrays, 21 MH-susceptible subjects with no known RyR1 or CACNA1S variants were screened, resulting in the discovery of new variants, all confirmed by capillary sequencing. In conclusion, arrays offer an efficient high-throughput alternative for diagnostic genotyping of candidate genes affecting MH susceptibility, BCHE deficiency and other neuromuscular disorders, simultaneously enabling a comprehensive search for rare variants in these genes.

Interaction of osmium(ii) redox probes with DNA: insights from theory.

In the course of developing ultrasensitive and quantitative electrochemical point-of-care analytical tools for genetic detection of infectious diseases, osmium(ii) metallointercalators were revealed to be suitable and efficient redox probes to monitor the in vitro DNA amplification [Defever etal, Anal. Chem., 2011, 83, 1815-1821]. In this work, we thus propose a complete computational protocol in order to evaluate the affinity between Os(ii) complexes with double-stranded DNA. This protocol is based on molecular dynamics, with the parametrization of the GAFF force field for the Os(ii) complexes presenting an octahedral environment with polypyridine ligands, and QM/QM' calculations to evaluate the binding energy. For three Os(ii) probes and different binding sites, molecular dynamics simulations and interaction energies calculated at the QM/QM' level are successively discussed and compared to experimental data in order to identify the most stable binding sites. The computational protocol we propose should then be used to design more efficient Os(ii) metallointercalators.

Maternal Plasma DNA and RNA Sequencing for Prenatal Testing.

Cell-free DNA (cfDNA) testing has recently become indispensable in diagnostic testing and screening. In the prenatal setting, this type of testing is often called noninvasive prenatal testing (NIPT). With a number of techniques, using either next-generation sequencing or single nucleotide polymorphism-based approaches, fetal cfDNA in maternal plasma can be analyzed to screen for rhesus D genotype, common chromosomal aneuploidies, and increasingly for testing other conditions, including monogenic disorders. With regard to screening for common aneuploidies, challenges arise when implementing NIPT in current prenatal settings. Depending on the method used (targeted or nontargeted), chromosomal anomalies other than trisomy 21, 18, or 13 can be detected, either of fetal or maternal origin, also referred to as unsolicited or incidental findings. For various biological reasons, there is a small chance of having either a false-positive or false-negative NIPT result, or no result, also referred to as a "no-call." Both pre- and posttest counseling for NIPT should include discussing potential discrepancies. Since NIPT remains a screening test, a positive NIPT result should be confirmed by invasive diagnostic testing (either by chorionic villus biopsy or by amniocentesis). As the scope of NIPT is widening, professional guidelines need to discuss the ethics of what to offer and how to offer. In this review, we discuss the current biochemical, clinical, and ethical challenges of cfDNA testing in the prenatal setting and its future perspectives including novel applications that target RNA instead of DNA.

[The application of genetic risk score in genetic studies of complex human diseases].

Complex diseases such as cardiovascular disease, type 2 diabetes, essential hypertension, asthma, obesity and cancer have spread across the globe and become the predominant cause of death. There are growing concerns over the role of genetic susceptibility in pathogenesis of complex diseases. However, the related susceptibility genes and sequence variations are still unknown. To elucidate the genetic basis of complex diseases, researchers have identified a large number of genetic variants associated with complex diseases through genome-wide association studies (GWAS) and candidate gene studies recently. The identification of these causal and/or associated variants promotes the development of approaches for complex diseases prediction and prevention. Genetic risk score (GRS), an emerging method for exploring correlation between single nucleotide polymorphisms (SNPs) and clinical phenotypes of complex diseases, integrates weak effects of multiple SNPs and dramatically enhances predictability of complex diseases by gene polymorphisms. This method has been applied successfully in genetic studies of many complex diseases. Here we focus on the introduction of the computational methods and evaluation criteria of GRS, enumerate a series of achievements through GRS application, discuss some limitations during application, and finally prospect the future of GRS.

Development and Validation of a Next-Generation Sequencing Assay for BRCA1 and BRCA2 Variants for the Clinical Laboratory.

The objective of this study was to design and validate a next-generation sequencing assay (NGS) to detect BRCA1 and BRCA2 mutations. We developed an assay using random shearing of genomic DNA followed by RNA bait tile hybridization and NGS sequencing on both the Illumina MiSeq and Ion Personal Gene Machine (PGM). We determined that the MiSeq Reporter software supplied with the instrument could not detect deletions greater than 9 base pairs. Therefore, we developed an alternative alignment and variant calling software, Quest Sequencing Analysis Pipeline (QSAP), that was capable of detecting large deletions and insertions. In validation studies, we used DNA from 27 stem cell lines, all with known deleterious BRCA1 or BRCA2 mutations, and DNA from 67 consented control individuals who had a total of 352 benign variants. Both the MiSeq/QSAP combination and PGM/Torrent Suite combination had 100% sensitivity for the 379 known variants in the validation series. However, the PGM/Torrent Suite combination had a lower intra- and inter-assay precision of 96.2% and 96.7%, respectively when compared to the MiSeq/QSAP combination of 100% and 99.4%, respectively. All PGM/Torrent Suite inconsistencies were false-positive variant assignments. We began commercial testing using both platforms and in the first 521 clinical samples MiSeq/QSAP had 100% sensitivity for BRCA1/2 variants, including a 64-bp deletion and a 10-bp insertion not identified by PGM/Torrent Suite, which also suffered from a high false-positive rate. Neither the MiSeq nor PGM platform with their supplied alignment and variant calling software are appropriate for a clinical laboratory BRCA sequencing test. We have developed an NGS BRCA1/2 sequencing assay, MiSeq/QSAP, with 100% analytic sensitivity and specificity in the validation set consisting of 379 variants. The MiSeq/QSAP combination has sufficient performance for use in a clinical laboratory.