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As sequencing technologies advance, there is increasing interest in the value of more comprehensive molecular profiling techniques. Our laboratory has clinically validated two next generation sequencing (NGS) assays for detecting somatic variants in solid tumors. The Sema4 Signal Solid Tumor Panel, utilizes the Thermo Fisher 161 gene Oncomine Comprehensive v3 assay and the Sema4 Signal Whole Exome/Transcriptome Sequencing (WES/WTS) assay consists of whole transcriptome RNAseq for the tumor and whole exome sequencing for the tumor and normal DNA on the Illumina NovaSeq. We compiled a cohort of 224 breast tumors from 211 patients encompassing 10 histological subtypes (BRCA (102), IDC (74), ILC (21), BRCANOS (11), BRCNOS (4), IBC (1), ACBC (1), IMMC (1), MBC (2), MDLC (2), Other (5)). A total of 209 specimens were analyzed by our Solid Tumor panel, and 15 were analyzed by WES/WTS. ER/PR/HER2 status was available for 212 tumors with 36 triple-negative, 123 ER(+)/PR(+)/HER2(-), and 23 ER(+)/PR(+)/HER2(+). Treatment history was available for 151 patients. Of the 118 patients who self-reported racial identity, 8 identified as Asian, 33 identified as Black or African American, 77 identified as white. Sequencing with either the 161 gene panel or WES/WTS detected clinically significant variants in 95% (213/224) of the tumors sequenced. Targetable PIK3CA variants were reported in 36% (80/224) of tumors, as well as CCND1 amplification (16%, 26/224) and ESR1 resistance variants (10%, 24/224). Tier 1 drugs were reported in 35% (79/224) of tumors, as well as tier 2 drugs (4%, 10/224). At least 2 patients had tumor specimens obtained at time points spanning 18 or more months and acquired ESR1 p.Y537S and p.Y537C resistance mutations in the later specimens. A patient with triple-negative specimens, obtained in 2018 and 2020, had no change in clinically significant variants but had loss of variants of unknown significance in the later sample. Panel sequencing and WES/WTS are comparable in terms of known variant detection, but panel-based testing has considerable limitations for assessing disease progression and genomic complexity. For the 15 WES/WTS specimens, we were able to determine additional molecular features, including complex copy number state, which has relevance to prognostic information, tumor mutation burden, homologous recombination deficiency, and differential gene expression. Moreover, WES of the tumor and normal specimens resolved multiple germline variants that had been reported as somatic variants in a tumor-only report from a patient with triple-negative breast cancer. Our findings support the adoption of tumor/normal WES/WTS as a standard diagnostic tool for breast cancer patients. Citation Format: Kimberly S. Cole, Zhiqiang Li, Amy Tiersten, Wanying Zhang, Hussam Al-Kateb, Feras M. Hantash, Michael R. Rossi, Scott Newman, Xiang Zhou, Eric E. Schadt, Rong Chen, William K. Oh, Marc Y. Fink. Value-added clinical tumor/normal whole exome and whole transcriptome sequencing versus a DNA and RNA tumor only gene panel for managing breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-15.

Introduction Historically, high rates of actionable driver mutations have been reported in never-smokers with lung adenocarcinoma (ADC). In the era of modern, comprehensive cancer mutation sequencing, this relationship necessitates a more detailed analysis. Methods All Mount Sinai patients between January 1, 2015, and June 1, 2020, with a diagnosis of ADC of any stage with known smoking status who received genomic testing were included. Most patients were analyzed using the Sema4 hotspot panel or the Oncomine Comprehensive Assay version 3 next-generation sequencing (NGS) panel conducted at Sema4. Patients were considered fully genotyped if they were comprehensively analyzed for alterations in EGFR, KRAS, MET, ALK, RET, ROS1, BRAF, NTRK1-3, and ERBB2, otherwise they were considered partially genotyped. Results Two hundred and thirty-six never-smokers and 671 smokers met the above criteria. Of the never-smokers, 201 (85%) had a driver mutation with 167 (71%) considered actionable (ie, those with US Food and Drug Administration-approved agents). Among smokers, 439 (65%) had an identified driver mutation with 258 (38%) actionable (P < .0001). When comprehensively sequenced, 95% (70/74) of never-smokers had a driver mutation with 78% (58/74) actionable; whereas, for smokers, 75% (135/180) had a driver with only 47% (74/180) actionable (P < .0001). Within mutations groups, EGFR G719X and KRAS G12Cs were more common to smokers. For stage IV patients harboring EGFR-mutant tumors treated with EGFR-directed therapies, never-smokers had significantly improved OS compared to smokers (hazard ratio = 2.71; P = .025). In multivariable analysis, Asian ancestry and female sex remained significant predictors of (1) OS in stage IV patients and (2) likelihood of harboring a receptor of fusion-based driver. Conclusion Comprehensive NGS revealed driver alterations in 95% of never-smokers, with the majority having an associated therapy available. All efforts should be exhausted to identify or rule out the presence of an actionable driver mutation in all metastatic lung ADC.

We previously described mutation rates of BRAFSingle-center retrospective cohort examining thyroidectomy tissue blocks from consecutive pediatric WDTC patients between 2001 and 2015. Tissues were analyzed at Quest Diagnostics for BRAF, RAS mutations, RET-PTC and PAX8-PPARγ, and additional fusions, using standalone and NGS tests. WDTC included papillary (PTC), follicular (FTC) and follicular-variant PTC (FVPTC).We genotyped 46 samples (36 females). Mean age at diagnosis was 14.7 years and the cohort comprised of mostly Hispanic (60.9%) and Caucasian (26.1%) patients. Mean follow-up was 3.5 years. Genetic alterations (GA) were noted in 69.6%, with BRAFBRAF

Purpose With prospective clinical sequencing of tumors emerging as a mainstay in cancer care, an urgent need exists for a clinical support tool that distills the clinical implications associated with specific mutation events into a standardized and easily interpretable format. To this end, we developed OncoKB, an expert-guided precision oncology knowledge base. Methods OncoKB annotates the biologic and oncogenic effects and prognostic and predictive significance of somatic molecular alterations. Potential treatment implications are stratified by the level of evidence that a specific molecular alteration is predictive of drug response on the basis of US Food and Drug Administration labeling, National Comprehensive Cancer Network guidelines, disease-focused expert group recommendations, and scientific literature. Results To date, > 3,000 unique mutations, fusions, and copy number alterations in 418 cancer-associated genes have been annotated. To test the utility of OncoKB, we annotated all genomic events in 5,983 primary tumor samples in 19 cancer types. Forty-one percent of samples harbored at least one potentially actionable alteration, of which 7.5% were predictive of clinical benefit from a standard treatment. OncoKB annotations are available through a public Web resource ( http://oncokb.org ) and are incorporated into the cBioPortal for Cancer Genomics to facilitate the interpretation of genomic alterations by physicians and researchers. Conclusion OncoKB, a comprehensive and curated precision oncology knowledge base, offers oncologists detailed, evidence-based information about individual somatic mutations and structural alterations present in patient tumors with the goal of supporting optimal treatment decisions.

Background: Individuals with prediabetes mellitus (PreDM) and low circulating 25-hydroxyvitamin D [25(OH)D] are at increased risk of type 2 diabetes mellitus (T2DM). Objective: We aimed to determine whether low 25(OH)D concentrations are associated with defects in insulin action and insulin secretion in persons with PreDM. Methods: In this cross-sectional study, we stratified 488 nondiabetic subjects as having PreDM or normal fasting glucose (NFG) and a 25(OH)D concentration ≤20 ng/mL (deficient) or >20 ng/mL (sufficient). We determined insulin resistance by steady state plasma glucose (SSPG) concentration and homeostasis model assessment of insulin resistance (HOMA-IR) and insulin secretion by homeostasis model assessment of β-cell function (HOMA-β). We compared insulin resistance and secretion measures in PreDM and NFG groups; 25(OH)D-deficient and 25(OH)D-sufficient groups; and PreDM-deficient, PreDM-sufficient, NFG-deficient, and NFG-sufficient subgroups, adjusting for age, sex, race, body mass index, multivitamin use, and season. Results: In the PreDM group, mean SSPG concentration and HOMA-IR were higher and mean HOMA-β was lower than in the NFG group (P < 0.001 for all comparisons). In the 25(OH)D-deficient group, mean SSPG concentration was higher (P < 0.001), but neither mean HOMA-IR nor HOMA-β was significantly different from that in the 25(OH)D-sufficient group. In the PreDM-deficient subgroup, mean (95% CI) SSPG concentration was higher (P < 0.01) than in the PreDM-sufficient, NFG-deficient, and NFG-sufficient subgroups [192 (177–207) mg/dL vs. 166 (155–177) mg/dL, 148 (138–159) mg/dL, and 136 (127–144) mg/dL, respectively]. Despite greater insulin resistance, mean HOMA-β was not significantly higher in the PreDM-deficient subgroup than in the PreDM-sufficient, NFG-deficient, and NFG-sufficient subgroups [98 (85–112) vs. 91 (82–101), 123 (112–136), and 115 (106–124), respectively]. Conclusion: Subjects with PreDM and low circulating 25(OH)D concentrations are the subgroup of nondiabetic individuals who are the most insulin resistant and have impaired β-cell function, attributes that put them at enhanced risk of T2DM.

There is substantial epidemiological evidence that low plasma concentrations of 25-hydroxyvitamin D (25(OH)D) are associated with increased risk of developing hypertension,1,2 although not all studies agree.3 Furthermore, analysis of data from the 2001–2004 National Health and Nutrition Examination Survey with linked mortality data through 2006 indicated that concentrations of 25(OH)D were inversely associated with all-cause and cardiovascular disease (CVD) mortality in the 2,609 participants with hypertension.4 There is also a coherent rationale for vitamin D deficiency to be associated with hypertension because it has been well documented that low vitamin D levels upregulate the renin-angiotensin-aldosterone system, increase inflammation, and cause endothelial dysfunction.5–7 However, thus far the results of interventional studies suggest that treatment with vitamin D neither substantially lowers blood pressure (BP) nor uniformly prevents CVD.8–10 The extensive literature is summarized in recent reviews.11,12 One possible explanation for this somewhat paradoxical situation is that considerable heterogeneity in CVD risk exists in patients with hypertension.13–15 More specifically, approximately 50% of patients with hypertension, treated or untreated, are insulin resistant and have the CVD risk factors usually associated with this abnormality.13 To the best of our knowledge, there is no available information regarding the relationship between 25(OH)D concentrations, specific measures of insulin action, and CVD risk factors in patients with hypertension. This study examines the relationships among these variables with the goal of providing new and clinically relevant information concerning the impact of insulin resistance and 25(OH)D concentrations on CVD risk in patients with hypertension.

BackgroundWell-differentiated thyroid cancer (WDTC) incidence in pediatrics is rising, most being papillary thyroid carcinoma (PTC). The objective of the study was to assess the prevalence of different mutations in pediatric WDTC and correlate the genotype with the clinical phenotype.MethodsThis is a single-center retrospective study. Thyroid tissue blocks from 42 consecutive pediatric WDTC patients who underwent thyroidectomy between 2001 and 2013 were analyzed at Quest Diagnostics for BRAF(V600E), RAS mutations (N,K,H), and RET/PTC and PAX8/PPARγ rearrangements, using validated molecular methods. Thyroid carcinomas included PTC, follicular thyroid carcinoma (FTC), and follicular variant of PTC (FVPTC).ResultsThirty-nine samples (29 females) were genotyped. The mean age at diagnosis was 14.7 years (range 7.9-18.4 years), and most were Hispanic (56.4%) or Caucasian (35.9%). The mean follow-up period was 2.9 years. Mutations were noted in 21/39 (53.8%), with both BRAF(V600E) (n = 9), and RET/PTC (n = 6) detected only in PTC. Mutations were detected in 2/5 FTC (PAX8/PPARγ and NRAS) and 3/6 FVPTC cases (PAX8/PPARγ). Of 28 PTC patients, 57.1% had mutations: 32.1% with BRAF(V600E), 21.4% with RET/PTC, and 3.6% with NRAS. Of patients with BRAF(V600E), 77.8% were Hispanic and 88.9% were >15 years, while all RET/PTC-positive patients were ≤15 years (p = 0.003). Tumor size, lymph node involvement, and distant metastasis at diagnosis (or soon after (131)I ablation) did not vary significantly based on the mutation.ConclusionsBRAF(V600E) was the most common mutation, especially in older and Hispanic adolescents. A larger, ethnically diverse pediatric cohort followed long term will enable the genotypic variability, clinical presentation, and response to therapy to be better assessed.

Fragile X syndrome is caused by expansion and methylation of a CGG tract in the 5' untranslated region of the FMR1 gene. The estimated frequency of expanded alleles (≥55 repeats) in the United States is 1:257-1:382, but these estimates were not calculated from unbiased populations. We sought to determine the frequency of fragile X syndrome premutation (55-200 repeats) and full mutation (200 repeats) alleles in nonselected, unbiased populations undergoing routine carrier screening for other diseases.A previously validated laboratory-developed test using triplet-primed polymerase chain reaction was used to detect premutation and full mutation alleles in an unselected series of 11,759 consecutive cystic fibrosis carrier screening samples and 2011 samples submitted for screening for genetic diseases prevalent among the Ashkenazi Jewish population.Premutations were identified in 48 cystic fibrosis screening samples (1:245) and 15 samples (1:134) from the Ashkenazi Jewish population. Adjusted for the ethnic mix of the US population and self-reported ethnicity in our screening population, the estimated female premutation carrier frequency in the United States was 1:178. The calculated frequency of full mutation alleles was 1:3335 overall, and the calculated premutation frequency in males was 1:400. Based on frequency of larger, ≥70 repeat alleles, and reported penetrance, the calculated fragile X-associated tremor and ataxia syndrome, and fragile X-associated primary ovarian insufficiency frequencies is 1:4848 and 1:3560, respectively.Our calculated fragile X syndrome carrier rate is higher than previous estimates for the US population and warrants further consideration of population-based carrier screening.

Recently, DNA rearrangements in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been described with increasing frequency. These large DNA rearrangements are not detected using conventional methods of DNA sequencing, single-strand conformational polymorphism, or denaturing high-performance liquid chromatography. We and others have described methods to detect such rearrangements in the CFTR gene. With one exception, all rearrangements reported thus far are single or multiple exon deletions, whereas only one report has described a large duplication. We describe here the detection and characterization of a novel large duplication in the CFTR gene. This duplication, referred to as gIVS6a + 415_IVS10 + 2987Dup26817bp, was detected in a classic CF female patient whose other mutation was DeltaF508. The duplication was inherited paternally. The duplication encompassed exons 6b to 10 and occurred on the IVS8-11TG/IVS8-7T/G1540 haplotype. This large duplication is predicted to result in the production of a truncated CFTR protein lacking the terminal part of NBD1 domain and beyond and thus can be considered a null allele. The combination of the DeltaF508 and gIVS6a + 415_IVS10 + 2987Dup26817bp mutation probably causes the severe CF phenotype in this patient. We designed a simple polymerase chain reaction test to detect the duplication, and we further detected the same duplication from another independent laboratory. The duplication breakpoint is identical in all three patients, suggesting a likely founder mutation.

Purpose: To develop a high-throughput, automated, accurate method suitable for population-based carrier detection of fragile X syndrome. Methods: We developed a new method called capillary Southern analysis that allows automated high-throughput screening for expanded fragile X mental retardation 1 (FMR1) alleles. Initially samples are analyzed by a multiplex polymerase chain reaction that contains an internal control to establish gender. All females heterozygous for two normal alleles are reported as normal without further analysis. All females homozygous at the FMR1 locus (24% of all analysis) are then analyzed by capillary Southern analysis. Theoretically this method can detect expansion as high as 2000 CGG repeats, although in our series the largest nonmosaic FMR1 present was 950 CGG repeats. After assay development, we performed capillary Southern analysis on 995 female and 557 male samples submitted for fragile X syndrome testing by polymerase chain reaction and Southern blot. Results: The polymerase chain reaction/capillary Southern analysis technique identified 100% of six female premutation carriers, seven full mutation carrier females, one premutation male, and five affected males. There was only one discrepancy between analysis by polymerase chain reaction/Southern blot and analysis by polymerase chain reaction/capillary Southern analysis. A single female sample appeared to be heterozygous for a premutation allele by polymerase chain reaction/capillary Southern analysis but was negative by Southern blot. It is possible this patient is a mosaic for the premutation allele, but because samples were deidentified, we were unable to determine whether this was a true false positive. Conclusion: We have developed an automated, high-throughput technique capable of detecting carriers of fragile X syndrome with 100% sensitivity and at least 99.5% specificity. This should allow population-based carrier detection for the most commonly inherited form of mental retardation.

Insulin resistance has been associated with higher plasma amino acid (AA) concentrations, but majority of studies have used indirect measures of insulin resistance. Our main objective was to define the relationship between plasma AA concentrations and a direct measure of insulin resistance in women and men. This was a cross-sectional study of 182 nondiabetic individuals (118 women and 64 men) who had measurement of 24 AAs and steady-state plasma glucose (SSPG) concentration (insulin resistance) using the insulin suppression test. Fourteen out of 24 AA concentrations were significantly (P

Molecular profiling of tumor mutations has expedited molecular targeted therapy for cancer patients. Additionally, tumor mutation load and DNA microsatellite instability (MSI) status can help predict patient’s response to immunotherapy. We analytically validated a gene test targeting clinically actionable cancer mutations of all 4 major types: single nucleotide variations (SNVs); insertion/deletions (INDELs); whole gene copy number variations (CNVs); and structural rearrangements (translocations) as well as the MSI status. This test interrogates all coding exons from 49 core cancer genes, introns for a subset of genes selected for detection of prevalent gene rearrangements, and the TERT (telomerase reverse transcriptase) promoter region. MSI status is determined from a set of 5 intronic mono-nucleotide repeats collectively associated with microsatellite instability. Targeted DNA regions are captured by in-solution hybridization with complementary biotinylated RNA baits and sequenced on the Illumina NextSeq®500 platform. Paired formalin-fixed, paraffin-embedded (FFPE) tumor samples and whole blood samples are analyzed simultaneously for the detection of SNV, INDEL, CNV, translocation and MSI status. FFPE samples can also be analyzed alone if the whole blood sample is not available, eliminating the reporting of MSI status. A minimum of 50 ng FFPE DNA and 100 ng of whole blood DNA are required for this test. A total of 123 FFPE, 2 FFPE FNA samples, 19 paired FFPE and whole blood paired samples covering lung cancer, colorectal cancer, melanoma and breast cancer were included in this validation study. Analytical validation of assay performance demonstrated that, on average, 700-fold read depth was achieved across all targeted regions with >95% of these regions covered by a minimum of 300 unique reads. Analytical sensitivity was ≥5% mutation frequency for SNV and INDEL and ≥20% for translocation and CNV. Thirty-eight unique variants were confirmed between this test and orthogonal methodologies: 22 SNVs, 6 INDELs, 5 translocations, and 5 CNV. Fifty-eight out of 59 (98% concordance) paired FFPE/blood samples achieved the same MSI status result with this targeted sequencing approach compared to the reference National Comprehensive Cancer Network (NCCN) Bethesda PCR test. MSI-positive samples, on average, were determined to have 5-fold higher mutation count compared to MSI-negative samples, consistent with the previously reported higher mutation burden. In conclusion, we have developed and analytically validated a core cancer gene test employing NGS technology with demonstrated high analytical sensitivity and specificity. Coupled with clinical interpretation, this test will facilitate clinical decision-making for molecular targeted therapy and immunotherapy. Citation Format: Lin Ma, Michael Hua, Steven Rivera, Anna Gerasimova, Quoclinh Nguyen, Sirisha Sunkara, Robert Lagier, Alla Smolgovsky, David Wolfson, Jared F. Taylor, Frederick Racke, Charles Strom, Andrew Grupe, Joseph Catanese, Feras Hantash. Validation of a clinically actionable cancer core gene test for solid tumors facilitating targeted molecular therapy and immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5356. doi:10.1158/1538-7445.AM2017-5356

We thank Dr Giral and colleagues for their comments on our recent study of lipoprotein(a) [Lp(a)].1 The quantitative assessment of Lp(a) has historically proven challenging in part because of the (1) highly heterogeneous number of Kringle-IV type-2 repeats (and thus molecular weight), and (2) lack of standardized methodology and calibration across published studies. A 2002 National Heart, Lung, and Blood Institute consensus workshop that recommended use of isoform size-independent assays that express Lp(a) concentrations in terms of particle number (nmol/L) rather than total mass (mg/dL) and provided control samples for calibration served as a major advance in the field.2 In accordance with these standards, our study reported Lp(a) concentrations in …

The cytosolic proteins EntE, EntF, and EntB/G, which are Escherichia coli enzymes necessary for the final stage of enterobactin synthesis, are released by osmotic shock. Here, consistent with the idea that cytoplasmic proteins found in shockates have an affinity for membranes, a small fraction of each was found in membrane preparations. Two procedures demonstrated that the enzymes were enriched in a minor membrane fraction of buoyant density intermediate between that of cytoplasmic and outer membranes, providing indirect support for the notion that these proteins have a role in enterobactin excretion as well as synthesis.

Background— Lipoprotein(a) [Lp(a)] is a low-density lipoprotein–like particle largely independent of known risk factors and predictive of cardiovascular disease. Statins may offset the risk associated with elevated Lp(a), but it is unknown whether Lp(a) is a determinant of residual risk in the setting of low low-density lipoprotein cholesterol after potent statin therapy. Methods and Results— Baseline and on-treatment Lp(a) concentrations were assessed in 9612 multiethnic participants in the JUPITER trial (Justification for the Use of Statins in Prevention: An Intervention Trial Evaluating Rosuvastatin) before and after random allocation to rosuvastatin 20 mg/d or placebo, with outcomes reported for whites (n=7746). Lp(a) concentrations (median [25th–75th percentile], in nmol/L) were highest in blacks (60 [34–100]), then Asians (38 [18–60]), Hispanics (24 [11–46]), and whites (23 [10–50]; P P P =0.02). Similarly, on-statin Lp(a) concentrations were associated with residual risk of cardiovascular disease (adjusted hazard ratio, 1.27; 95% confidence interval, 1.01–1.59; P =0.04), which was independent of low-density lipoprotein cholesterol and other factors. Rosuvastatin significantly reduced incident cardiovascular disease among participants with baseline Lp(a) greater than or equal to the median (hazard ratio, 0.62; 95% confidence interval, 0.43–0.90) and Lp(a) less than the median (hazard ratio, 0.46; 95% confidence interval, 0.30–0.72), with no evidence of interaction. Similar results were obtained when analyses included nonwhites. Conclusion— Among white JUPITER participants treated with potent statin therapy, Lp(a) was a significant determinant of residual risk. The magnitude of relative risk reduction with rosuvastatin was similar among participants with high or low Lp(a). Clinical Trials Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT00239681.

Purpose: Fragile X syndrome is caused by expansion and subsequent methylation of a CGG trinucleotide repeat in the FMR1 5′-untranslated region. Southern blot analysis is typically required to determine expansion size for triplet repeat lengths >200. We describe a triplet-primed polymerase chain reaction-based method using automated capillary electrophoresis detection for qualitative assessment of expanded CGG repeats. Methods: The assay uses triplet-primed polymerase chain reaction in combination with GC-melting reagents and substitution of 7-deaza-2-deoxyGTP for dGTP. Amplicons are resolved by capillary electrophoresis. Results: A distinctive pattern of tapering or “stutter” polymerase chain reaction amplification was evident on capillary electrophoresis in male and female patients harboring all expanded allele lengths examined (up to 2000 CGG repeats) and could be used to differentiate normal, intermediate, premutation, and full mutation alleles. Full mutation alleles exhibited an additional late-migrating amplicon on capillary electrophoresis. Mixing experiments demonstrated sensitivity as low as 1% for detection of the full mutation allele. In a 1275-sample concordance study against our existing polymerase chain reaction platform (with Southern blot analysis for repeat lengths ≥55), the triplet-primed polymerase chain reaction method exhibited 100% concordance for normal, intermediate, expanded, and full mutation alleles. This method also detected the full mutation alleles in DNA isolated from blood spots. Conclusion: This assay provides an accurate assessment of FMR1 repeat status and holds promise for use in carrier and newborn screening. The method distinguishes normal homozygous females from full mutation carrying females. Although the method is not useful for accurate sizing, it supplements the classic polymerase chain reaction method and results in significant reduction in the number of Southern blot analyses required to be performed in the laboratory to accurately assess the FMR1 genotype in all individuals.

Patients develop cystic fibrosis because of a variety of homozygous recessive mutations, including single nucleotide polymorphisms, insertions, and deletions, in the cystic fibrosis transmembrane regulator (CFTR) gene, or because of compound heterozygosity for two mutations in the CFTR gene. A false determination of homozygosity for a particular CFTR mutation could negatively affect both carrier screens for a patient's family as well as researchers' ability to study the physiological implications of a particular mutation. We argued previously that homozygosity for rare or novel mutations in the CFTR gene could result from a mutation on one allele and the presence of a large deletion encompassing the same sequence region on the second allele. We present here a patient with classic cystic fibrosis who has a novel microdeletion in exon 7 on one allele and a large deletion encompassing exon 7 on the second allele. These data highlight the need to prevent misdiagnosis of homozygous mutations, which can lead to misinterpretation of mutation penetrance and its effects on protein function.

Two mutations in the MCOLN1 mucolipidosis IV (ML IV) gene represent approximately 95% of the mutations in Ashkenazi-Jewish patients with ML IV. The mutations, a splice site mutation (IVS3-2AG) and an approximately 6.4-kb deletion (511del6434), account for 72% and 23% of ML IV alleles in this population, respectively. An automated high-throughput assay was developed using the 5'-nuclease (TaqMan) method for the simultaneous detection of both mutations in a single reaction. Three fluorescent probes specifically detected wild-type, IVS3-2AG, and 511del6434 alleles in each reaction real-time. Data collected were automatically analyzed, and genotype results were uploaded into a laboratory information management system. The assay was validated using genomic controls, demonstrating high robustness and accuracy. Carrier screening of 10,527 samples revealed 77 heterozygote carriers of IVS3-2AG, 25 heterozygote carriers of 511del6434, and two compound heterozygote of both mutant alleles. The frequency of mutated alleles was 0.73% for IVS3-2AG and 0.24% for 511del6434. The combined carrier frequency was 1:103 with predicted disease incidence of 1:42,436 individuals in this population, slightly lower than previously described frequencies. This automated high-throughput assay is labor saving, because two mutations can be detected in a single reaction. The method has potential for use in other assays requiring simultaneous detection of two mutations.

Purpose: Most cystic fibrosis mutation screening methods do not detect large exon deletions or duplications in the cystic fibrosis transmembrane regulator gene. We looked for such mutations in congenital bilateral absence of the vas deferens patients in whom routine screening assays had identified only one or no cystic fibrosis transmembrane regulator gene mutations. Methods: DNA samples from 48 men with congenital bilateral absence of the vas deferens were tested for exonic deletions and duplications in the cystic fibrosis transmembrane regulator gene using a laboratory-developed semiquantitative fluorescent PCR assay. Results: Semi-quantitative fluorescent PCR identified a large deletion in one (2%) of the 48 patients. This patient, previously characterized as carrying only the IVS8-5T mutation, was found to have a deletion of exons 22-24 of the cystic fibrosis transmembrane regulator gene. In a second patient with the IVS8-5T mutation, we identified a one-base pair insertion in exon 17b that disrupted the reading frame. Conclusions: Analysis of the cystic fibrosis transmembrane regulator gene for exon deletions and duplications should be included for complete study of CBAVD patients, especially those considering assisted reproduction.

Background: Positive control materials for clinical diagnostic molecular genetic testing are in critically short supply. High-quality DNA that closely resembles DNA isolated from patient specimens can be obtained from Epstein–Barr virus (EBV)–transformed peripheral blood lymphocyte cell lines. Here we report the development of a process to (a) recover residual blood samples with clinically important mutations detected during routine medical care, (b) select samples likely to provide viable lymphocytes for EBV transformation, (c) establish stable cell lines and confirm the reported mutation(s), and (d) validate the cell lines for use as positive controls in clinical molecular genetic testing applications.Methods: A network of 32 genetic testing laboratories was established to obtain anonymous, residual clinical samples for transformation and to validate resulting cell lines for use as positive controls. Three panel meetings with experts in molecular genetic testing were held to evaluate results and formulate a process that could function in the context of current common practices in molecular diagnostic testing.Results: Thirteen laboratories submitted a total of 113 residual clinical blood samples with mutations for 14 genetic disorders. Forty-one EBV-transformed cell lines were established. Thirty-five individual point and deletion mutations were shown to be stable after 20 population doublings in culture. Thirty-three cell lines were characterized for specific mutations and validated for use as positive controls in clinical diagnostic applications.Conclusions: A process for producing and validating positive control cell lines from residual clinical blood samples has been developed. Sustainable implementation of the process could help alleviate the current shortage of positive control materials.

Cystic fibrosis (CF) is one of the most common monogenic diseases affecting Caucasians and has an incidence of approximately 1:3,300 births. Currently recommended screening panels for mutations in the responsible gene (CF transmembrane regulator gene, CFTR) do not detect all disease-associated mutations. Our laboratory offers extensive sequencing of the CFTR (ABCC7) gene (including the promoter, all exons and splice junction sites, and regions of selected introns) as a clinical test to detect mutations which are not found with conventional screening. The objective of this report is to summarize the findings of extensive CFTR sequencing from our first 157 consecutive patient samples. In most patients with classic CF symptoms (18/24, 75%), extensive CFTR sequencing confirmed the diagnosis by finding two disease-associated mutations. In contrast, only 5 of 75 (7%) patients with atypical CF had been identified with two CFTR mutations. A diagnosis of CF was confirmed in 10 of 17 (58%) newborns with either positive sweat chloride readings or positive immunoreactive trypsinogen (IRT) screen results. We ascertained ten novel sequence variants that are potentially disease-associated: two deletions (c.1641AG>T, c.2949_2853delTACTC), seven missense mutations (p.S158T, p.G451V, p.K481E, p.C491S, p.H949L, p.T1036N, p.F1099L), and one complex allele ([p.356_A357del; p.358I]). We ascertained three other apparently novel complex alleles. Finally, several patients were found to carry partial CFTR gene deletions. In summary, extensive CFTR gene sequencing can detect rare mutations which are not found with other screening and diagnostic tests, and can thus establish a definitive diagnosis in symptomatic patients with previously negative results. This enables carrier detection and prenatal diagnosis in additional family members.

Purpose: To determine the carrier frequency of the 3199del6 cystic fibrosis (CF) mutation in individuals heterozygous for I148T in a large-scale CF testing population. Methods: DNA samples from 439 consecutive I148T-heterozygous individuals were screened for the 3199del6 mutation using a laboratory-developed test. Results: Genotyping revealed four samples heterozygous for the 3199del6 mutation (0.9%). The four samples positive for 3199del6 had an IVS 8 genotype of 7T/9T. The 3199del6 mutation was not observed after genotyping of 348 random, anonymous samples. Conclusion: The 3199del6 mutation occurs in 0.9% of individuals positive for the I148T mutation and < 0.07% of chromosomes that are wild type for the ACMG panel mutations.

Background: The recommendation for population- based cystic fibrosis (CF) carrier screening by the American College of Medical Genetics for the 25 most prevalent mutations and 6 polymorphisms in the CF transmembrane regulatory gene has greatly increased clinical laboratory test volumes. We describe the development and technical validation of a DNA chip in a 96-well format to allow for high-throughput genotype analysis. Methods: The CF Portrait™ chip contains an 8 × 8 array of capture probes and controls to detect all requisite alleles. Single-tube multiplex PCR with 15 biotin-labeled primer pairs was used to amplify sequences containing all single-nucleotide polymorphisms to be interrogated. Detection of a thin-film signal created by hybridization of multiplex PCR-amplified DNA to complementary capture probes was performed with an automated image analysis instrument, NucleoSight™. Allele classification, data formatting, and uploading to a laboratory information system were fully automated. Results: The described platform correctly classified all mutations and polymorphisms and can screen ∼1300 patient samples in a 10-h shift. Final validation was performed by two separate 1000-sample comparisons with Roche CF Gold line probe strips and the Applera CF OLA, Ver 3.0. The CF Portrait Biochip made no errors during this validation, whereas the Applera assay made seven miscalls of the IVS-8 5T/7T/9T polymorphism Conclusions: The CF Portrait platform is an automated, high-throughput, DNA chip-based assay capable of accurately classifying all CF mutations in the recommended screening panel, including the IVS-8 5T/7T/9T polymorphism.

BACKGROUND: ALK, ROS1, and RET translocations are frequently detected in NSCLC patients. Crizotinib, a tyrosine kinase inhibitor (TKI), was approved by the FDA in 2011 to treat NSCLC in patients harboring ALK translocations as detected by an FDA-approved assay. However, the FDA-approved ALK FISH assay is technically challenging, with failures due to pre-analytic variables. Another approach, intragenic differential expression (IDE), detects translocations by comparing expression levels of the 5′ end with the 3′ end of target gene transcripts. In this study we developed and evaluated a rapid IDE assay to screen for ALK, ROS1, and RET translocations, independent of the fusion partner. METHODS: A total of 419 samples (408 randomly-selected NSCLC clinical samples, ALK positive and ROS1 positive cell lines (2 each), and 7 previously-tested RET-positive clinical samples) were used to develop and evaluate performance characteristics of the IDE assays. To determine IDE scores, levels of ALK, ROS1, and RET expression were first determined by quantitative RT-PCR measurement of the 5′- and 3′- ends of the respective transcripts. The differences in expression levels were calculated as ΔCt (Ct5′ - Ct3′). High ΔCt values indicate presumptive presence of gene translocations. 212/408 NSCLC samples were analyzed by ALK FISH and EML4-ALK RT-PCR, and 196/408 samples were analyzed by EML4-ALK RT-PCR. RESULTS: Thirty-one of the 408 (7.6%) clinical samples tested positive for ALK rearrangements by IDE. Among them, 20 were confirmed by FISH and/or EML4-ALK (true positive, 64.5%), while 11 were negative by FISH and/or EML4-ALK (false positive, 35.5%). One of 10 ALK FISH positive samples tested negative by both ALK IDE and EML4-ALK RT-PCR analysis (false negative), while one of 202 FISH-negative sample tested positive by both EML4-ALK and ALK IDE. ALK IDE exhibited 94.5% (189/200) concordance with ALK FISH and 96.0% (356/371) concordance with the EML4-ALK assay. For ROS1, both ROS1-positive cell lines and 4/408 (1.0%) NSCLC samples tested positive for ROS1 by IDE. Among the 4 IDE-positive NSCLC samples, 1 was confirmed by ROS1 FISH. For RET, all 7 known positives and 10/408 (2.5%) NSCLC samples tested positive by IDE. Three of six RET IDE positive NSCLC samples were confirmed by RET FISH. Overall, ALK, ROS1, and RET translocations were mutually exclusive in NSCLC patients. The lung IDE assay had a failure rate of 3.7%. CONCLUSION: These findings demonstrate the feasibility of using IDE to detect ALK, ROS1, and RET gene translocations. These assays may have potential as a screening tool to select patients for further confirmation by FISH for TKI-targeted therapy. The IDE concept can be applied to a wide range of somatic translocations. Citation Format: Shih-Min Cheng, Cindy Barlan, Feras Hantash, Heather R. Sanders, Patricia H. Chan, Vladimira Sulcova, Marc A. Sanidad, Kevin Qu, Joann C. Kelly, Fatih Z. Boyar, Anthony D. Sferruzza, Frederic M. Waldman. Detection of ALK, ROS1, and RET translocations in non-small cell lung cancer (NSCLC) patients by intragenic differential expression analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4675. doi:10.1158/1538-7445.AM2014-4675

The terminal reactions in the synthesis of the siderophore enterobactin (Ent) by Escherichia coli require the EntD, E, F and B/G polypeptides. The idea that these molecules form a complex (Ent synthase) that is membrane-associated was re-evaluated. In vitro results provided no evidence in support of the proposal: (i) Ent synthase activity occurred normally under conditions where membrane was either absent or disrupted by high concentrations of neutral detergents, and (ii) immunoprecipitation experiments conducted on extracts engaged in Ent synthesis failed to detect any association among the Ent polypeptides. However, Western blot analyses showed that EntE, F and B/G were released from cells by osmotic shock and freeze/thaw treatment but not by conversion of cells to spheroplasts. These results demonstrated that EntE, F and B/G belong to the Beacham group D class of proteins. The shockability of a given group D Ent protein was unaffected by the absence of either EntB/G or EntD and, for EntB/G, the N-terminus was sufficient for release by osmotic shock. The behaviour of group D proteins is generally attributed to their association (partial, loose or transient) with cytoplasmic membrane; therefore, the results are indirect evidence that Ent synthase interacts with membrane in vivo. At the very least, the data indicate that EntE, F and B/G are compartmentalized in E. coli and, because other biosynthetic enzymes for siderophores and surfactants are related to these Ent proteins, suggest that this entire protein class may be sequestered in vivo.

The alpha globin gene cluster contains two highly homologous alpha globin genes, HBA1 and HBA2, that code for identical proteins. Mutations in the alpha globin gene cluster are predominantly large deletions causing the loss of either one copy of the alpha-globin gene (e.g. -α3.7 and -α4.2 deletions) or both copies (e.g. --THAI, --FIL or --MED). A few large deletions encompassing the regulatory HS-40 region have also been described in alpha-thalassaemia patients. Point mutations and small base pair insertions or deletions have also been detected in HBA1 and HBA2 genes. Seven common large deletions in the alpha globin gene cluster are detected by a gapped-PCR assay. These common mutations and some other types of rearrangements can be detected by Southern blot, a laborious and time consuming method. However, these methods may not accurately identify the total number of copies of alpha globin like genes. We designed a single-tube alpha globin gene dosage assay (αGDA) using semi-quantitative fluorescent PCR (SQF PCR) for detecting the total number of alpha globin genes. Primers that amplify specific fragments from HBA1 and HBA2 genes, a fragment between the alpha globin pseudogenes, and three fragments flanking and including the HS-40 regulatory region were included in a single PCR reaction together with primers that amplify fragments from 3 different normalization genes. Using the αGDA, we were able to detect in patient samples varying copy numbers of alpha globin genes and to identify the nature of DNA rearrangements between HBA1 and HBA2. We also identified novel alpha globin conversion events that were verified by DNA sequencing. We also designed a complimentary comprehensive DNA sequencing assay to detect point mutations and small base pair insertions or deletions in the HBA1 and HBA2 genes. Using this method, and in combination with cation exchange HPLC and agarose gel electrophoresis, novel mutations in alpha globins were identified and submitted to the globin gene server, including Hb Linwood (α2 40 Lys>Gln), Hb Creve Coeur (α2 24 Tyr>Asp), and Hb Westborough (α-3.7 130 Ala>Val). The simplicity of αGDA will allow the replacement of the laborious Southern blot analysis to detect large deletions in the alpha globin gene cluster and to provide accurate information of total a-globin gene dosage, while the DNA sequencing assay will allow the detection of known and novel variants.

Because standard techniques used to detect mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene do not detect single or multiple exonic rearrangements, the importance of such rearrangements may be underestimated. Using an in-house developed, single-tube, semi-quantitative fluorescent PCR (SQF PCR) assay, we analyzed 36 DNA samples submitted for extensive CFTR sequencing and identified ten samples with rearrangements. Of 36 patients with classic CF, 10 (28%) harbored various deletions in the CFTR gene, accounting for 14% of CF chromosomes. A deletion encompassing the CFTR promoter and exons 1 and 2 was detected in a sample from one proband, and in the maternal DNA as well. In another family, a deletion of the promoter and exon 1 was detected in three siblings. In both of these cases, the families were African American and the 3120+1GA splice site mutation was also identified. These promoter deletions have not been previously described. In a third case, a deletion of exons 17a, 17b, and 18 was identified in a Caucasian female and the same mutation was detected in the paternal DNA. In the other seven cases, we identified the following deletions: exons 2 and 3 (n = 2); exons 4, 5, and 6a; exons 17a and 17b; exons 22 and 23; and exons 22, 23, and 24 (n = 2). In our series, the frequency of CFTR rearrangements in classic CF patients, when only one mutation was identified by extensive DNA sequencing, was60% (10/16). Screening for exon deletions and duplications in the CFTR gene would be beneficial in classic CF cases, especially when only one mutation is identified by standard methodologies.