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.

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.

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.

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.

X-inactivation in the clinical phenotype of fragile X premutation carrier sisters.

OBJECTIVE: The purpose of this study is to describe a case series of 4 sisters with discordant clinical phenotypes associated with fragile X-associated tremor/ataxia syndrome (FXTAS) that may be explained by varying CGG repeat sizes and activation ratios (ARs) (the ratio of cells carrying the normal fragile X mental retardation 1 [FMR1] allele on the active X chromosome). METHODS: Four sisters with premutation size FMR1 gene repeats underwent detailed clinical characterization. CGG repeat length was determined by PCR, and AR was determined using a newly developed commercial methylation PCR assay and was compared with the results from Southern blot with densitometric image analysis. RESULTS: Sister 1 had the largest CGG expansion (82) and the lowest AR (12%), with the most severe clinical presentation. Sister 2 had a lower CGG expansion (70) and an AR of 10% but had a milder clinical presentation.Sister 3 had a similar CGG expansion (79) but a slightly higher AR of 15% and less neurologic involvement. Sister 4 had a similar CGG expansion size of 80 but had the largest AR (40%) and was the only sister not to be affected by FXTAS or have any neurologic signs on examination. CONCLUSIONS: These results suggest that premutation carrier women who have higher ARs may be less likely to show manifestations of FXTAS. If larger studies show similar patterns, AR data could potentially be beneficial to supplement CGG repeat size when counseling premutation carrier women in the clinic.

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.

Evaluation of an integrated clinical workflow for targeted next-generation sequencing of low-quality tumor DNA using a 51-gene enrichment panel.

BACKGROUND: Improvements in both performance and cost for next-generation sequencing (NGS) have spurred its rapid adoption for clinical applications. We designed and optimized a pan-cancer target-enrichment panel for 51 well-established oncogenes and tumor suppressors, in conjunction with a bioinformatic pipeline informed by in-process controls and pre- and post-analytical quality control measures. METHODS: The evaluation of this workflow consisted of sequencing mixtures of intact DNA to establish analytical sensitivity and precision, utilization of heuristics to identify systematic artifacts, titration studies of intact and FFPE samples for input optimization, and incorporation of orthogonal sequencing strategies to increase both positive predictive value and variant detection. We also used 128 FFPE samples to assess clinical accuracy and incorporated the previously described quantitative functional index (QFI) for sample qualification as part of detailing complete system performance. RESULTS: We observed a concordance correlation coefficient of 0.99 between the observed versus expected percent variant at 250 ng input across 4 independent sequencing runs. A subset of the systematic variants were confirmed to be barely detectable on an independent sequencing platform (Wilcox signed-rank test p-value <10(-16)), and the incorporation of orthogonal sequencing strategies increased the harmonic mean of sensitivity and positive predictive value of mutation detection by 41%. In one cohort of FFPE tumor samples, coverage and inter-platform concordance were positively correlated with the QFI, emphasizing the need for pre-analytical sample quality control to reduce the risk of false positives and negatives. In a separate cohort of FFPE samples, the 51-gene panel achieved 78% sensitivity (95% CI = 56.3, 92.5) with 100% PPV (95% CI = 81.5, 100.0) based on known mutations at 7.9% median abundance. By sequencing specimens using an orthogonal NGS technology, sensitivity was improved to 87.0% (95% CI = 66.4,97.2) while maintaining PPV. CONCLUSIONS: The results highlight the value of process integration in a comprehensive targeted NGS system, enabling both discovery and diagnostic applications, particularly when sequencing low-quality cancer specimens.

Recoding aminoacyl-tRNA synthetases for synthetic biology by rational protein-RNA engineering.

We have taken a rational approach to redesigning the amino acid binding and aminoacyl-tRNA pairing specificities of bacterial glutaminyl-tRNA synthetase. The four-stage engineering incorporates generalizable design principles and improves the pairing efficiency of noncognate glutamate with tRNA(Gln) by over 10(5)-fold compared to the wild-type enzyme. Better optimized designs of the protein-RNA complex include substantial reengineering of the globular core region of the tRNA, demonstrating a role for specific tRNA nucleotides in specifying the identity of the genetically encoded amino acid. Principles emerging from this engineering effort open new prospects for combining rational and genetic selection approaches to design novel aminoacyl-tRNA synthetases that ligate noncanonical amino acids onto tRNAs. This will facilitate reconstruction of the cellular translation apparatus for applications in synthetic biology.

AGG interruptions and maternal age affect FMR1 CGG repeat allele stability during transmission.

BACKGROUND: The presence of AGG interruptions in the CGG repeat locus of the fragile X mental retardation 1 (FMR1) gene decreases the instability of the allele during transmission from parent to child, and decreases the risk of expansion of a premutation allele to a full mutation allele (the predominant cause of fragile X syndrome) during maternal transmission. METHODS: To strengthen recent findings on the utility of AGG interruptions in predicting instability or expansion to a full mutation of FMR1 CGG repeat alleles, we assessed the outcomes of 108 intermediate (also named gray zone) and 710 premutation alleles that were transmitted from parent to child, and collected from four international clinical sites. We have used the results to revise our initial model that predicted the risk of a maternal premutation allele expanding to a full mutation during transmission and to test the effect of AGG interruptions on the magnitude of expanded allele instability of intermediate or premutation alleles that did not expand to a full mutation. RESULTS: Consistent with previous studies, the number of AGG triplets that interrupts the CGG repeat locus was found to influence the risk of allele instability, including expansion to a full mutation. The total length of the CGG repeat allele remains the best predictor of instability or expansion to a full mutation, but the number of AGG interruptions and, to a much lesser degree, maternal age are also factors when considering the risk of transmission of the premutation allele to a full mutation. CONCLUSIONS: Our findings demonstrate that a model with total CGG length, number of AGG interruptions, and maternal age is recommended for calculating the risk of expansion to a full mutation during maternal transmission. Taken together, the results of this study provide relevant information for the genetic counseling of female premutation carriers, and improve the current predictive models which calculate risk of expansion to a full mutation using only total CGG repeat length.

Coevolution of specificity determinants in eukaryotic glutamyl- and glutaminyl-tRNA synthetases.

The glutaminyl-tRNA synthetase (GlnRS) enzyme, which pairs glutamine with tRNA(Gln) for protein synthesis, evolved by gene duplication in early eukaryotes from a nondiscriminating glutamyl-tRNA synthetase (GluRS) that aminoacylates both tRNA(Gln) and tRNA(Glu) with glutamate. This ancient GluRS also separately differentiated to exclude tRNA(Gln) as a substrate, and the resulting discriminating GluRS and GlnRS further acquired additional protein domains assisting function in cis (the GlnRS N-terminal Yqey domain) or in trans (the Arc1p protein associating with GluRS). These added domains are absent in contemporary bacterial GlnRS and GluRS. Here, using Saccharomyces cerevisiae enzymes as models, we find that the eukaryote-specific protein domains substantially influence amino acid binding, tRNA binding and aminoacylation efficiency, but they play no role in either specific nucleotide readout or discrimination against noncognate tRNA. Eukaryotic tRNA(Gln) and tRNA(Glu) recognition determinants are found in equivalent positions and are mutually exclusive to a significant degree, with key nucleotides located adjacent to portions of the protein structure that differentiated during the evolution of archaeal nondiscriminating GluRS to GlnRS. These findings provide important corroboration for the evolutionary model and suggest that the added eukaryotic domains arose in response to distinctive selective pressures associated with the greater complexity of the eukaryotic translational apparatus. We also find that the affinity of GluRS for glutamate is significantly increased when Arc1p is not associated with the enzyme. This is consistent with the lower concentration of intracellular glutamate and the dissociation of the Arc1p:GluRS complex upon the diauxic shift to respiratory conditions.

The role of AGG interruptions in fragile X repeat expansions: a twenty-year perspective.

In 1994, it was suggested that AGG interruptions affect the stability of the fragile X triplet repeat. Until recently, however, this hypothesis was not explored on a large scale due primarily to the technical difficulty of determining AGG interruption patterns of the two alleles in females. The recent development of a PCR technology that overcomes this difficulty and accurately identifies the number and position of AGGs has led to several studies that examine their influence on repeat stability. Here, we present a historical perspective of relevant studies published during the last 20 years on AGG interruptions and examine those recent publications that have refined risk estimates for repeat instability and full-mutation expansions.

CGG allele size somatic mosaicism and methylation in FMR1 premutation alleles.

BACKGROUND: Greater than 200 CGG repeats in the 5'UTR of the FMR1 gene lead to epigenetic silencing and lack of the FMR1 protein, causing fragile X Syndrome. Individual carriers of a premutation (PM) allele with 55-200 CGG repeats are typically unmethylated and can present with clinical features defined as FMR1-associated conditions. METHODS: Blood samples from 17 male PM carriers were assessed clinically and molecularly by Southern blot, western blot, PCR and QRT-PCR. Blood and brain tissue from an additional 18 PM males were also similarly examined. Continuous outcomes were modelled using linear regression and binary outcomes were modelled using logistic regression. RESULTS: Methylated alleles were detected in different fractions of blood cells in all PM cases (n=17). CGG repeat numbers correlated with percent of methylation and mRNA levels and, especially in the upper PM range, with greater number of clinical involvements. Inter-tissue/intra-tissue somatic instability and differences in percent methylation were observed between blood and fibroblasts (n=4) and also observed between blood and different brain regions in three of the 18 PM cases examined. CGG repeat lengths in lymphocytes remained unchanged over a period of time ranging from 2 to 6 years, three cases for whom multiple samples were available. CONCLUSIONS: In addition to CGG size instability, individuals with a PM expanded allele can exhibit methylation and display more clinical features likely due to RNA toxicity and/or FMR1 silencing. The observed association between CGG repeat length and percent of methylation with the severity of the clinical phenotypes underscores the potential value of methylation in affected PM to further understand penetrance, inform diagnosis and expand treatment options.

Novel insight into mutational landscape of head and neck squamous cell carcinoma.

Development of head and neck squamous cell carcinoma (HNSCC) is characterized by accumulation of mutations in several oncogenes and tumor suppressor genes. We have formerly described the mutation pattern of HNSCC and described NOTCH signaling pathway alterations. Given the complexity of the HNSCC, here we extend the previous study to understand the overall HNSCC mutation context and to discover additional genetic alterations. We performed high depth targeted exon sequencing of 51 highly actionable cancer-related genes with a high frequency of mutation across many cancer types, including head and neck. DNA from primary tumor tissues and matched normal tissues was analyzed for 37 HNSCC patients. We identified 26 non-synonymous or stop-gained mutations targeting 11 of 51 selected genes. These genes were mutated in 17 out of 37 (46%) studied HNSCC patients. Smokers harbored 3.2-fold more mutations than non-smokers. Importantly, TP53 was mutated in 30%, NOTCH1 in 8% and FGFR3 in 5% of HNSCC. HPV negative patients harbored 4-fold more TP53 mutations than HPV positive patients. These data confirm prior reports of the HNSCC mutational profile. Additionally, we detected mutations in two new genes, CEBPA and FES, which have not been previously reported in HNSCC. These data extend the spectrum of HNSCC mutations and define novel mutation targets in HNSCC carcinogenesis, especially for smokers and HNSCC without HPV infection.

Intermediate CGG repeat length at the FMR1 locus is not associated with hormonal indicators of ovarian age.

OBJECTIVE: Premutation and intermediate CGG repeat length at the fragile X mental retardation 1 (FMR1) locus have been associated with premature ovarian failure. We tested whether intermediate length is associated with indicators of ovarian age in a sample of fertile women. Our primary measures of ovarian age were antimüllerian hormone (AMH) and follicle-stimulating hormone (FSH) levels. METHODS: The cross-sectional sample comprised 258 women with karyotyped spontaneous abortions (140 trisomic spontaneous abortions and 118 chromosomally normal spontaneous abortions or spontaneous abortions with anomalies other than trisomy) and 325 women with recent live births (LBs). We analyzed data from the total sample and data from LBs only. We defined CGG repeat length by the length (both continuous and categorical) on the longer allele. RESULTS: CGG repeat length was not significantly associated with either hormone measure. A repeat length of 35 to 54 CGG, versus the modal category of 30 CGG, was associated with an approximately 7% increase in median AMH level and a 3% increase in median FSH level. Results were unaltered when analyses were limited to LBs. Analyses of hormone levels using cutpoints to define older ovarian age showed no associations with repeat length. Among 10 women with repeat lengths of 35 to 54 CGG analyzed for AGG sequences, the uninterrupted CGG length was not significantly longer among women with hormonal indicators of "old" versus "young" ovarian age. CONCLUSIONS: Our data do not support an association between intermediate CGG repeat length and levels of AMH or FSH among fertile women.

A novel methylation PCR that offers standardized determination of FMR1 methylation and CGG repeat length without southern blot analysis.

Fragile X syndrome and associated disorders are characterized by the number of CGG repeats and methylation status of the FMR1 gene for which Southern blot (SB) historically has been required for analysis. This study describes a simple PCR-only workflow (mPCR) to replace SB analysis, that incorporates novel procedural controls, treatment of the DNA in separate control and methylation-sensitive restriction endonuclease reactions, amplification with labeled primers, and two-color amplicon sizing by capillary electrophoresis. mPCR was evaluated in two independent laboratories with 76 residual clinical samples that represented typical and challenging fragile X alleles in both males and females. mPCR enabled superior size resolution and analytical sensitivity for size and methylation mosaicism compared to SB. Full mutation mosaicism was detected down to 1% in a background of 99% normal allele with 50- to 100-fold less DNA than required for SB. A low level of full mutation mosaicism in one sample was detected using mPCR but not observed using SB. Overall, the sensitivity for detection of full mutation alleles was 100% (95% CI: 89%-100%) with an accuracy of 99% (95% CI: 93%-100%). mPCR analysis of DNA from individuals with Klinefelter and Turner syndromes, and DNA from sperm and blood, were consistent with SB. As such, mPCR enables accurate, sensitive, and standardized methods of FMR1 analysis that can harmonize results across different laboratories.

Fragile X AGG analysis provides new risk predictions for 45-69 repeat alleles.

We investigated the effect of AGG interruptions on fragile X repeat instability upon transmission of fragile X intermediate and small premutation alleles with 45-69 CGG repeats. The FMR1 repeat structure was determined for 375 mothers, 48 fathers, and 538 offspring (457 maternal and 81 paternal transmissions) using a novel PCR assay to determine repeat length and AGG interruptions. The number of AGG interruptions and the length of uninterrupted CGG repeats at the 3' end were correlated with repeat instability on transmission. Maternal alleles with no AGGs conferred the greatest risk for unstable transmissions. All nine full mutation expansions were inherited from maternal alleles with no AGGs. Furthermore, the magnitude of repeat expansion was larger for alleles lacking AGG interruptions. Transmissions from paternal alleles with no AGGs also exhibited greater instability than those with one or more AGGs. Our results demonstrate that characterization of the AGG structure within the FMR1 repeat allows more accurate risk estimates of repeat instability and expansion to full mutations for intermediate and small premutation alleles.