Multisite Verification of a Targeted CFTR Polymerase Chain Reaction/Capillary Electrophoresis Assay That Evaluates Pathogenic Variants Across Diverse Ethnic and Ancestral Groups.

CONTEXT.­: Existing targeted cystic fibrosis screening assays miss important pathogenic CFTR variants in the ethnically diverse US population. OBJECTIVE.­: To evaluate the analytic performance of a multiplex polymerase chain reaction (PCR)/capillary electrophoresis (CE) CFTR assay panel that simultaneously interrogates primary pathogenic variants of different ethnic/ancestral groups. DESIGN.­: Performance characteristic assessment and variant coverage comparison of the panel with a focus on ethnicity-specific CFTR variants were performed. Sample DNA was primarily from whole blood or cell lines. Detection of CFTR carriers was compared across several commercially available CFTR kits and recommended variant sets based on panel content. RESULTS.­: The panel interrogated 65 pathogenic CFTR variants representing 92% coverage from a recent genomic sequencing survey of the US population, including 4 variants with top 5 frequency in African or Asian populations not reflected in other targeted panels. In simulation studies, the panel represented 95% of carriers across the global population, resulting in 6.9% to 19.0% higher carrier detection rate compared with 10 targeted panels or variant sets. Precision and sensitivity/specificity were 100% concordant. Multisite sample-level genotyping accuracy was 99.2%. Across PCR and CE instruments, sample-level genotyping accuracy was 97.1%, with greater than 99% agreement for all variant-level metrics. CONCLUSIONS.­: The CFTR assay achieves 92% or higher coverage of CFTR variants in diverse populations and provides improved pan-ethnic coverage of minority subgroups of the US populace. The assay can be completed within 5 hours from DNA sample to genotype, and performance data exceed acceptance criteria for analytic metrics. This assay panel content may help address gaps in ancestry-specific CFTR genotypes while providing a streamlined procedure with rapidly generated results.

An updated meta-analysis of the primed goal-organizational behaviour relationship.

Environmental cues (e.g. achievement-related words and pictures) can prime/activate, in the absence of awareness, a mental representation of importance stored in memory. Chen et al.'s 2021 Applied Psychology: An International Review 70, 216-253. (doi:10.1111/apps.12239) meta-analysis revealed a moderate, significant overall effect for the goal priming-organizational behaviour relationship, with three moderators identified: context-specific versus a general prime, prime modality (i.e. visual versus linguistic) and experimental setting (field versus laboratory). An independent researcher found that their finding was negligibly affected by a publication bias. Shanks & Vadillo (2021), Royal Society Open Science 8, 210544. (doi:10.1098/rsos.210544) (field: k = 13, N = 683, d = 0.64), questioned Chen et al.'s conclusion regarding the effect size found in field studies (field: k = 8, N = 357, d = 0.68). In this paper, we discussed Shanks & Vadillo's selection of additional field experiments that led to their conclusion of a publication bias. We updated Chen et al.'s meta-analysis to include relevant studies conducted since that study's publication. The present meta-analysis reproduced the original findings in Chen et al. (field: k = 11, N = 534, d = 0.67). The updated findings are consistent with: (i) laboratory findings, (ii) the findings obtained in field experiments on consciously set goals and (iii) goal setting theory (Latham & Locke, 2018 In Handbook of industrial, work & organizational Psychology, vol. 1 (eds D Ones, N Anderson, C Viswesvaran, H Sinangil), pp. 103-124).

The Effect of a Dilemma on the Relationship Between Ability to Identify the Criterion (ATIC) and Scores on a Validated Situational Interview.

Four experiments were conducted to determine whether participants' awareness of the performance criterion on which they were being evaluated results in higher scores on a criterion valid situational interview (SI) where each question either contains or does not contain a dilemma. In the first experiment there was no significant difference between those who were or were not informed of the performance criterion that the SI questions predicted. Experiment 2 replicated this finding. In each instance the SI questions in these two experiments contained a dilemma. In a third experiment, participants were randomly assigned to a 2 (knowledge/no knowledge provided of the criterion) X 2 (SI dilemma/no dilemma) design. Knowledge of the criterion increased interview scores only when the questions did not contain a dilemma. The fourth experiment revealed that including a dilemma in a SI question attenuates the ATIC-SI relationship when participants must identify rather than be informed of the performance criterion that the SI has been developed to assess.

Multisite Evaluation and Validation of a Sensitive Diagnostic and Screening System for Spinal Muscular Atrophy that Reports SMN1 and SMN2 Copy Number, along with Disease Modifier and Gene Duplication Variants.

Spinal muscular atrophy is a severe autosomal recessive disease caused by disruptions in the SMN1 gene. The nearly identical SMN2 gene copy number is associated with disease severity. SMN1 duplication markers, such as c.∗3+80T>G and c.∗211_∗212del, can assess residual carrier risk. An SMN2 disease modifier (c.859G>C) can help inform prognostic outcomes. The emergence of multiple precision gene therapies for spinal muscular atrophy requires accurate and rapid detection of SMN1 and SMN2 copy numbers to enable early treatment and optimal patient outcomes. We developed and evaluated a single-tube PCR/capillary electrophoresis assay system that quantifies SMN1/2 copy numbers and genotypes three additional clinically relevant variants. Analytical validation was performed with human cell lines and whole blood representing varying SMN1/2 copies on four capillary electrophoresis instrument models. In addition, four independent laboratories used the assay to test 468 residual clinical genomic DNA samples. The results were ≥98.3% concordant with consensus SMN1/2 exon 7 copy numbers, determined using multiplex ligation-dependent probe amplification and droplet digital PCR, and were 100% concordant with Sanger sequencing for the three variants. Furthermore, copy number values were 98.6% (SMN1) and 97.1% (SMN2) concordant to each laboratory's own reference results.

A Novel Triplet-Primed PCR Assay to Detect the Full Range of Trinucleotide CAG Repeats in the Huntingtin Gene (HTT).

The expanded CAG repeat number in HTT gene causes Huntington disease (HD), which is a severe, dominant neurodegenerative illness. The accurate determination of the expanded allele size is crucial to confirm the genetic status in symptomatic and presymptomatic at-risk subjects and avoid genetic polymorphism-related false-negative diagnoses. Precise CAG repeat number determination is critical to discriminate the cutoff between unexpanded and intermediate mutable alleles (IAs, 27-35 CAG) as well as between IAs and pathological, low-penetrance alleles (i.e., 36-39 CAG repeats), and it is also critical to detect large repeat expansions causing pediatric HD variants. We analyzed the HTT-CAG repeat number of 14 DNA reference materials and of a DNA collection of 43 additional samples carrying unexpanded, IAs, low and complete penetrance alleles, including large (>60 repeats) and very large (>100 repeats) expansions using a novel triplet-primed PCR-based assay, the AmplideX PCR/CE HTT Kit. The results demonstrate that the method accurately genotypes both normal and expanded HTT-CAG repeat numbers and reveals previously undisclosed and very large CAG expansions >200 repeats. We also show that this technique can improve genetic test reliability and accuracy by detecting CAG expansions in samples with sequence variations within or adjacent to the repeat tract that cause allele drop-outs or inaccuracies using other PCR methods.

A Genotype-Phenotype Study of High-Resolution FMR1 Nucleic Acid and Protein Analyses in Fragile X Patients with Neurobehavioral Assessments.

Fragile X syndrome (FXS) is caused by silencing of the FMR1 gene, which encodes a protein with a critical role in synaptic plasticity. The molecular abnormality underlying FMR1 silencing, CGG repeat expansion, is well characterized; however, delineation of the pathway from DNA to RNA to protein using biosamples from well characterized patients with FXS is limited. Since FXS is a common and prototypical genetic disorder associated with intellectual disability (ID) and autism spectrum disorder (ASD), a comprehensive assessment of the FMR1 DNA-RNA-protein pathway and its correlations with the neurobehavioral phenotype is a priority. We applied nine sensitive and quantitative assays evaluating FMR1 DNA, RNA, and FMRP parameters to a reference set of cell lines representing the range of FMR1 expansions. We then used the most informative of these assays on blood and buccal specimens from cohorts of patients with different FMR1 expansions, with emphasis on those with FXS (N = 42 total, N = 31 with FMRP measurements). The group with FMRP data was also evaluated comprehensively in terms of its neurobehavioral profile, which allowed molecular-neurobehavioral correlations. FMR1 CGG repeat expansions, methylation levels, and FMRP levels, in both cell lines and blood samples, were consistent with findings of previous FMR1 genomic and protein studies. They also demonstrated a high level of agreement between blood and buccal specimens. These assays further corroborated previous reports of the relatively high prevalence of methylation mosaicism (slightly over 50% of the samples). Molecular-neurobehavioral correlations confirmed the inverse relationship between overall severity of the FXS phenotype and decrease in FMRP levels (N = 26 males, mean 4.2 ± 3.3 pg FMRP/ng genomic DNA). Other intriguing findings included a significant relationship between the diagnosis of FXS with ASD and two-fold lower levels of FMRP (mean 2.8 ± 1.3 pg FMRP/ng genomic DNA, p = 0.04), in particular observed in younger age- and IQ-adjusted males (mean age 6.9 ± 0.9 years with mean 3.2 ± 1.2 pg FMRP/ng genomic DNA, 57% with severe ASD), compared to FXS without ASD. Those with severe ID had even lower FMRP levels independent of ASD status in the male-only subset. The results underscore the link between FMR1 expansion, gene methylation, and FMRP deficit. The association between FMRP deficiency and overall severity of the neurobehavioral phenotype invites follow up studies in larger patient cohorts. They would be valuable to confirm and potentially extend our initial findings of the relationship between ASD and other neurobehavioral features and the magnitude of FMRP deficit. Molecular profiling of individuals with FXS may have important implications in research and clinical practice.

Correction: Novel Insight into Mutational Landscape of Head and Neck Squamous Cell Carcinoma.

[This corrects the article DOI: 10.1371/journal.pone.0093102.].

Validation of Fragile X Screening in the Newborn Population Using a Fit-for-Purpose FMR1 PCR Assay System.

Newborn screening is designed for presymptomatic identification of serious conditions with effective early interventions. Clinical laboratories must perform prospective pilot studies to ensure that the analytical performance and workflow for a given screening test are appropriate. We assessed the potential to screen newborns for fragile X syndrome, a monogenic neurodevelopmental disorder, by establishing a customized, high-throughput PCR and analysis software system designed to detect fragile X mental retardation 1 gene repeat expansions from dried blood spots (DBSs). Assay precision, accuracy, sensitivity, and specificity were characterized across the categorical range of repeat expansions. The assay consistently resolved genotypes within three CGG repeats of reference values up to at least 137 repeats and within six repeats for larger expansions up to 200 repeats. Accuracy testing results were concordant with reference results. Full and premutation alleles were detected from subnanogram DNA inputs eluted from DBSs and from mixtures with down to 1% relative abundance of the respective expansion. Analysis of 963 deidentified newborn DBS samples identified 957 normal and 6 premutation specimens, consistent with previously published prevalence estimates. These studies demonstrate that the assay system can support high-throughput newborn screening programs.

Expansions and contractions of the FMR1 CGG repeat in 5,508 transmissions of normal, intermediate, and premutation alleles.

Instability of the FMR1 repeat, commonly observed in transmissions of premutation alleles (55-200 repeats), is influenced by the size of the repeat, its internal structure and the sex of the transmitting parent. We assessed these three factors in unstable transmissions of 14/3,335 normal (~5 to 44 repeats), 54/293 intermediate (45-54 repeats), and 1561/1,880 premutation alleles. While most unstable transmissions led to expansions, contractions to smaller repeats were observed in all size classes. For normal alleles, instability was more frequent in paternal transmissions and in alleles with long 3' uninterrupted repeat lengths. For premutation alleles, contractions also occurred more often in paternal than maternal transmissions and the frequency of paternal contractions increased linearly with repeat size. All paternal premutation allele contractions were transmitted as premutation alleles, but maternal premutation allele contractions were transmitted as premutation, intermediate, or normal alleles. The eight losses of AGG interruptions in the FMR1 repeat occurred exclusively in contractions of maternal premutation alleles. We propose a refined model of FMR1 repeat progression from normal to premutation size and suggest that most normal alleles without AGG interruptions are derived from contractions of maternal premutation alleles.

An Integrated Next-Generation Sequencing System for Analyzing DNA Mutations, Gene Fusions, and RNA Expression in Lung Cancer.

We developed and characterized a next-generation sequencing (NGS) technology for streamlined analysis of DNA and RNA using low-input, low-quality cancer specimens. A single-workflow, targeted NGS panel for non-small cell lung cancer (NSCLC) was designed covering 135 RNA and 55 DNA disease-relevant targets. This multiomic panel was used to assess 219 formalin-fixed paraffin-embedded NSCLC surgical resections and core needle biopsies. Mutations and expression phenotypes were identified consistent with previous large-scale genomic studies, including mutually exclusive DNA and RNA oncogenic driver events. Evaluation of a second cohort of low cell count fine-needle aspirate smears from the BATTLE-2 trial yielded 97% agreement with an independent, validated NGS panel that was used with matched surgical specimens. Collectively, our data indicate that broad, clinically actionable insights that previously required independent assays, workflows, and analyses to assess both DNA and RNA can be conjoined in a first-tier, highly multiplexed NGS test, thereby providing faster, simpler, and more economical results.

Design, Optimization, and Multisite Evaluation of a Targeted Next-Generation Sequencing Assay System for Chimeric RNAs from Gene Fusions and Exon-Skipping Events in Non-Small Cell Lung Cancer.

Lung cancer accounts for approximately 14% of all newly diagnosed cancers and is the leading cause of cancer-related deaths. Chimeric RNA resulting from gene fusions (RNA fusions) and other RNA splicing errors are driver events and clinically addressable targets for non-small cell lung cancer (NSCLC). The reliable assessment of these RNA markers by next-generation sequencing requires integrated reagents, protocols, and interpretive software that can harmonize procedures and ensure consistent results across laboratories. We describe the development and verification of a system for targeted RNA sequencing for the analysis of challenging, low-input solid tumor biopsies that includes reagents for nucleic acid quantification and library preparation, run controls, and companion bioinformatics software. Assay development reconciled sequence discrepancies in public databases, created predictive formalin-fixed, paraffin-embedded RNA qualification metrics, and eliminated read misidentification attributable to index hopping events on the next-generation sequencing flow cell. The optimized and standardized system was analytically verified internally and in a multiphase study conducted at five independent laboratories. The results show accurate, reproducible, and sensitive detection of RNA fusions, alternative splicing events, and other expression markers of NSCLC. This comprehensive approach, combining sample quantification, quality control, library preparation, and interpretive bioinformatics software, may accelerate the routine implementation of targeted RNA sequencing of formalin-fixed, paraffin-embedded samples relevant to NSCLC.

Comprehensive genotyping of the C9orf72 hexanucleotide repeat region in 2095 ALS samples from the NINDS collection using a two-mode, long-read PCR assay.

OBJECTIVE: Expansion of the G4C2 repeat tract in the C9orf72 gene is linked to frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Here, we provide comprehensive genotyping of the C9orf72 repeat region for the National Institute of Neurological Disorders and Stroke (NINDS) ALS collection (n = 2095), using a novel bimodal PCR assay capable of amplifying nearly 100% GC-rich sequences. METHODS: A single-tube 3-primer PCR assay mode, resolved using capillary electrophoresis, was used for sizing up to 145 repeats with single-repeat accuracy, for detecting expansions irrespective of their overall size, and for flagging confounding 3' sequence variations (SVs). A modified two-primer PCR mode, resolved via agarose gel electrophoresis, provided further size information for hyper-expanded samples (>145 repeats) up to ∼5.8 kb amplicons (∼950 G4C2 repeats). RESULTS: Within the evaluated cohort, 177 (8.4%) samples were expanded, with 175 (99%) samples being hyper-expanded. 3'-SVs were identified in 64 (3.1%) samples, and were most common in expanded alleles. Genotypes of all 606 (29%) homozygous samples were confirmed using an orthogonal PCR assay. CONCLUSION: This study and PCR method may improve and standardize molecular characterization of the C9orf72 locus, and have the potential to inform phenotype-genotype correlations and therapeutic development in ALS/FTD.

Use of human-derived stem cells to create a novel, in vitro model designed to explore FMR1 CGG repeat instability amongst female premutation carriers.

OBJECTIVE: Create a model, using reprogrammed cells, to provide a platform to identify the mechanisms of CGG repeat instability amongst female fragile X mental retardation 1 gene (FMR1) premutation (PM) carriers. METHODS: Female PM carriers (with and without POI) and healthy controls were enrolled from June 2013 to April 2014. Patient-derived fibroblasts (FB) were reprogrammed to induced pluripotent stem cells (iPSC) using viral vectors, encoding KLF4, OCT4, SOX2, and MYC. FMR1 CGG repeat-primed PCR was used to assess the triplet repeat structure of the FMR1 gene. FMR1 promoter methylation (%) was determined using FMR1 methylation PCR (mPCR). Quantification of FMR1 transcripts by RT-qPCR was used to evaluate the effect of reprogramming on gene transcription, as well as to correlate patient phenotype with FMR1 expression. Production of FMR1 protein (FMRP) was determined using a liquid bead array-based immunoassay. RESULTS: Upon induction to pluripotency, all control clones exhibited maintenance of progenitor cell CGG repeat number, whereas 10 of 12 clones derived from PM carriers maintained their input CGG repeat number, one of which expanded and one contracted. As compared to parent FB, iPSC clones exhibited a skewed methylation pattern; however, downstream transcription and translation appeared unaffected. Further, the PM carriers, regardless of phenotype, exhibited similar FMR1 transcription and translation to the controls. CONCLUSIONS: This is the first study to establish a stem cell model aimed to understand FMR1 CGG repeat instability amongst female PM carriers. Our preliminary data indicate that CGG repeat number, transcription, and translation are conserved upon induction to pluripotency.

Absence of AGG Interruptions Is a Risk Factor for Full Mutation Expansion Among Israeli FMR1 Premutation Carriers.

Introduction: Fragile X syndrome (FXS) is a common form of X-linked intellectual and developmental disability with a prevalence of 1/4000-5000 in males and 1/6000-8000 in females. Most cases of the syndrome result from expansion of a premutation (55-200 CGGs) to a full mutation (>200 CGGs) repeat located in the 5' untranslated region of the fragile X mental retardation (FMR1) gene. The risk for full mutation expansions increases dramatically with increasing numbers of CGG repeats. Recent studies, however, revealed AGG interruptions within the repeat area function as a "protective factor" decreasing the risk of intergenerational expansion. Materials and Methods: This study was conducted to validate the relevance of AGG analysis for the ethnically diverse Israeli population. To increase the accuracy of our results, we combined results from Israel with those from the New York State Institute for Basic Research in Developmental Disabilities (IBR). To the best of our knowledge this is the largest cohort of different ethnicities to examine risks of unstable transmissions and full mutation expansions among FMR1 premutation carriers. Results: The combined data included 1471 transmissions of maternal premutation alleles: 369 (25.1%) stable and 1,102 (74.9%) unstable transmissions. Full mutation expansions were identified in 20.6% (303/1471) of transmissions. A total of 97.4% (388/397) of transmissions from alleles with no AGGs were unstable, 79.6% (513/644) in alleles with 1 AGG and 46.7% (201/430) in alleles with 2 or more AGGs. The same trend was seen with full mutation expansions where 40% (159/397) of alleles with no AGGs expanded to a full mutation, 20.2% (130/644) for alleles with 1 AGG and only 3.2% (14/430) in alleles with 2 AGGs or more. None of the alleles with 3 or more AGGs expanded to full mutations. Conclusion: We recommend that risk estimates for FMR1 premutation carriers be based on AGG interruptions as well as repeat size in Israel and worldwide.

Association of skewed X-chromosome inactivation with FMR1 CGG repeat length and anti-Mullerian hormone levels: a cohort study.

BACKGROUND: Premutation range CGGn repeats of the FMR1 gene denote risk toward primary ovarian insufficiency (POI), also called premature ovarian failure (POF). This prospective cohort study was undertaken to determine if X-chromosome inactivation skew (sXCI) is associated with variations in FMR1 CGG repeat length and, if so, is also associated with age adjusted antimüllerian hormone (AMH) levels as an indicator of functional ovarian reserve (FOR). METHODS: DNA samples of 58 women were analyzed for methylation status and confirmation of CGGn repeat length. Based on previously described FMR1 genotypes, there were 18 women with norm FMR1 (both alleles in range of CGG n=26-34), and 40 women who had at least one allele at CGGn<26 or CGG>34 ( not-norm FMR1). As part of a routine evaluation of ovarian reserve, patients at our fertility center have their serum AMH assessed at first visit. Regression models were used to test the association of ovarian reserve, as indicated by serum AMH, with sXCI. RESULTS: sXCI was significantly lower among infertility patients with norm FMR1 (6.5 ± 11.1, median and IQR) compared to those with not-norm FMR1 (12.0 ± 14.6, P = 0.005), though among young oocyte donors the opposite effect was observed. Women age >30 to 38 years old demonstrated greater ovarian reserve in the presence of lower sXCI as evidenced by significantly higher AMH levels (GLM sXCI_10%, f = 11.27; P = 0.004). CONCLUSIONS: Together these findings suggest that FMR1 CGG repeat length may have a role in determining X-chromosome inactivation which could represent a possible mechanism for previously observed association of low age adjusted ovarian reserve with FMR1 variations in repeat length. Further, larger, investigations will be required to test this hypothesis.

A methylation PCR method determines FMR1 activation ratios and differentiates premutation allele mosaicism in carrier siblings.

BACKGROUND: Epigenetic modifications of the fragile X mental retardation 1 (FMR1) gene locus may impact the risk for reproductive and neurological disorders associated with expanded trinucleotide repeats and methylation status in the 5' untranslated region. FMR1 methylation is commonly assessed by Southern blot (SB) analysis, yet this method suffers a cumbersome workflow and relatively poor sizing resolution especially for premutation allele characteristic for fragile X-associated disorders. In this study, a methylation PCR (mPCR) assay was used to evaluate correlations among genotype, epitype, and phenotype in fragile X premutation (PM) carrier women in order to advance the understanding of the association between molecular determinants and the presence of fragile X-associated tremor and ataxia (FXTAS). RESULTS: Activation ratios (ARs) in 39 PM women were determined by mPCR and compared with SB analysis. ARs were distributed across a range of values including five samples with <20% AR and six with >80% AR. The two methods were correlated (R2 of 0.87 and F test of <0.001), indicating that mPCR can measure AR in agreement with established assays. However, mPCR was unique in identifying novel and distinct patterns of methylation mosaicism in premutation carrier women, including seven sibling pairs that were assessed using FXTAS clinical rating scales. Of note, four of six pairs with defined age of onset for neurological signs showed ARs consistent with skewed activation of the pathogenic expanded allele. One subject with severe FXTAS had a mosaic full mutation allele identified using mPCR but not detected by SB analysis. CONCLUSIONS: We utilized a repeatable and streamlined methodology to characterize FMR1 inactivation in premutation carrier women. The method was concordant with SB analysis and provided higher resolution information on allele and methylation mosaicism. This approach can facilitate the characterization of epigenetic factors influencing fragile X phenotypes in larger cohort studies that can advance understanding and treatment of fragile X-associated disorders.

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies.

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench ("wet bench") and data analyses conducted using bioinformatics pipelines ("dry bench"). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible "tag" PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

Fragile X syndrome screening in Chinese children with unknown intellectual developmental disorder.

BACKGROUND: Fragile X syndrome is the most common genetic disorder of intellectual developmental disorder/mental retardation (IDD/MR). The prevalence of FXS in a Chinese IDD children seeking diagnosis/treatment in mainland China is unknown. METHODS: Patients with unknown moderate to severe IDD were recruited from two children's hospitals. Informed consent was obtained from the children's parents. The size of the CGG repeat was identified using a commercial TP-PCR assay. The influence of AGG interruptions on the CGG expansion during maternal transmission was analyzed in 24 mother-son pairs (10 pairs with 1 AGG and 14 pairs with 2 AGGs). RESULTS: 553 unrelated patients between six months and eighteen years of age were recruited. Specimens from 540 patients (male:female = 5.2:1) produced high-quality TP-PCR data, resulting in the determination of the FMR1 CGG repeat number for each. The most common repeat numbers were 29 and 30, and the most frequent interruption pattern was 2 or 3 AGGs. Five full mutations were identified (1 familial and 4 sporadic IDD patients), and size mosaicism was apparent in 4 of these FXS patients (4/5 = 80%). The overall yield of FXS in the IDD cohort was 0.93% (5/540). Neither the mean size of CGG expansion (0.20 vs. 0.79, p > 0.05) nor the frequency of CGG expansion (2/10 vs. 9/14, p > 0.05) was significantly different between the 1 and 2 AGG groups following maternal transmission. CONCLUSIONS: The FMR1 TP-PCR assay generates reliable and sensitive results across a large number of patient specimens, and is suitable for clinical genetic diagnosis. Using this assay, the prevalence of FXS was 0.93% in Chinese children with unknown IDD.

Fragile X full mutation expansions are inhibited by one or more AGG interruptions in premutation carriers.

PURPOSE: Fragile X CGG repeat alleles often contain one or more AGG interruptions that influence allele stability and risk of a full mutation transmission from parent to child. We have examined transmissions of maternal and paternal alleles with 45-90 repeats to quantify the effect of AGG interruptions on fragile X repeat instability. METHODS: A novel FMR1 polymerase chain reaction assay was used to determine CGG repeat length and AGG interruptions for 1,040 alleles from 705 families. RESULTS: We grouped transmissions into nine categories of five repeats by parental size and found that in every size category, alleles with no AGGs had the greatest risk for instability. For maternal alleles <75 repeats, 89% (24/27) that expanded to a full mutation had no AGGs. Two contractions in maternal transmission were accompanied by loss of AGGs, suggesting a mechanism for generating alleles that lack AGG interruptions. Maternal age was examined as a factor in full mutation expansions using prenatal samples to minimize ascertainment bias, and a possible effect was observed though it was not statistically significant (P = 0.06). CONCLUSION: These results strengthen the association of AGG repeats with CGG repeat stability and provide more accurate risk estimates of full mutation expansions for women with 45-90 repeat alleles.