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.

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.

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.

Identification of miRNA changes in Alzheimer's disease brain and CSF yields putative biomarkers and insights into disease pathways.

MicroRNAs have essential functional roles in brain development and neuronal specification but their roles in neurodegenerative diseases such as Alzheimer's disease (AD) is unknown. Using a sensitive qRT-PCR platform we identified regional and stage-specific deregulation of miRNA expression in AD patient brains. We used experimental validation in addition to literature to reveal how the deregulated brain microRNAs are biomarkers for known and novel pathways in AD pathogenesis related to amyloid processing, neurogenesis, insulin resistance, and innate immunity. We additionally recovered miRNAs from cerebrospinal fluid and discovered AD-specific miRNA changes consistent with their role as potential biomarkers of disease.

FNA needle rinses preserved in Cytolyt are acceptable specimen type for mutation testing of thyroid nodules.

INTRODUCTION: This study investigated the application of molecular testing to residual thyroid fine-needle aspiration material from needle rinses collected in Cytolyt. MATERIALS AND METHODS: Two thyroid needle rinses from 135 patients were collected in Cytolyt during routine diagnostic workup in our institution. Molecular testing was performed to detect 14 genetic alterations in BRAF, K-, H-, N-RAS genes as well as RET/PTC1, RET/PTC3, and PAX8/PPARγ and verified by next generation sequencing and correlated with cytologic diagnoses. RESULTS: Molecular testing revealed a total of 17 mutations across specimens with benign nodule (n = 5; HRAS, NRAS), Hürthle cell neoplasm (n = 2; BRAF, HRAS) and Papillary thyroid carcinoma (n = 10, 9 BRAF, 1 KRAS) cytology. No RNA gene rearrangements were detected. CONCLUSIONS: Mutations and translocations associated with thyroid cancer can be detected in thyroid fine-needle aspiration needle rinses preserved in Cytolyt specimens collected during routine patient management, which are typically discarded when a diagnosis is attained.

Response-surface modeling of the effect of 5alpha-dihydrotestosterone and androgen receptor levels on the response to the androgen antagonist vinclozolin.

Androgens secreted by the testes bind the androgen receptor in developing target tissues to induce the expression of genes required for male sexual differentiation and development. Androgen concentration and androgen receptor levels vary in male reproductive target tissues during development. Exposure to environmental androgen antagonists during critical windows of fetal and postnatal development can inhibit male sexual development by blocking transcription of androgen-dependent genes. As the sensitivity to androgen antagonists under conditions of varying androgen concentrations and varying androgen receptor levels is unknown, we used a luciferase reporter assay to investigate the transcriptional effects of a known androgen antagonist (the vinclozolin metabolite M2) at different androgen concentrations and different androgen receptor levels. The ability of M2 to inhibit transcription was greater at lower concentrations of androgen (5alpha-dihydrotestosterone) and androgen receptor. The data were modeled to determine the dose-response surface of M2 and androgen receptor concentrations at different 5alpha-dihydrotestosterone levels and the relationship of the 3 components to the response. The model and hypothesis testing results suggest that, at 0.01 and 0.1 nM 5alpha-dihydrotestosterone concentrations within the expected in vivo range of free androgen levels during development, the response-surface shapes were similar and the interaction of the androgen receptor and M2 concentrations to the response were similarly antagonistic. Thus, two components of the developmental stage, androgen and androgen receptor concentrations, are critical for sensitivity to the inhibitory effects of an androgen antagonist.

Functional DNA quantification guides accurate next-generation sequencing mutation detection in formalin-fixed, paraffin-embedded tumor biopsies.

The formalin-fixed, paraffin-embedded (FFPE) biopsy is a challenging sample for molecular assays such as targeted next-generation sequencing (NGS). We compared three methods for FFPE DNA quantification, including a novel PCR assay ('QFI-PCR') that measures the absolute copy number of amplifiable DNA, across 165 residual clinical specimens. The results reveal the limitations of commonly used approaches, and demonstrate the value of an integrated workflow using QFI-PCR to improve the accuracy of NGS mutation detection and guide changes in input that can rescue low quality FFPE DNA. These findings address a growing need for improved quality measures in NGS-based patient testing.

miRNA biomarkers in cyst fluid augment the diagnosis and management of pancreatic cysts.

PURPOSE: The diagnosis of pancreatic cystic lesions has increased dramatically. Most are benign, whereas some, such as intraductal papillary mucinous neoplasms (IPMN), represent precursors of pancreatic adenocarcinoma. Therapeutic stratification of IPMNs is challenging without precise information on dysplasia grade and presence of invasion. We assessed the diagnostic benefit of using miRNAs as biomarkers in pancreatic cyst fluid, focusing on IPMNs because of their frequency and malignant potential. EXPERIMENTAL DESIGN: RNA was extracted from 55 microdissected formalin-fixed, paraffin-embedded (FFPE) IPMN specimens, and 65 cyst fluid specimens aspirated following surgical resection. Expression of 750 miRNAs was evaluated with TaqMan miRNA Arrays using 22 FFPE and 15 cyst fluid specimens. Differential expression of selected miRNA candidates was validated in 33 FFPE and 50 cyst fluid specimens using TaqMan miRNA Assays. RESULTS: We identified 26 and 37 candidate miRNAs that distinguish low-grade from high-grade IPMNs using FFPE and cyst fluid specimens, respectively. A subset of 18 miRNAs, selected from FFPE and cyst fluid data, separated high-grade IPMNs from low-grade IPMNs, serous cystadenomas (SCA) and uncommon cysts, such as solid pseudopapillary neoplasms (SPN) and cystic pancreatic neuroendocrine tumors (PanNET). A logistic regression model using nine miRNAs allowed prediction of cyst pathology implying resection (high-grade IPMNs, PanNETs, and SPNs) versus conservative management (low-grade IPMNs, SCAs), with a sensitivity of 89%, a specificity of 100%, and area under the curve of 1. CONCLUSIONS: We found candidate miRNAs that helped identify patients with high-grade IPMN and exclude nonmucinous cysts. These classifiers will require validation in a prospective setting to ultimately confirm their clinical usefulness.