Early Detection of Fragile X Syndrome: Applications of a Novel Approach for Improved Quantitative Methylation Analysis in Venous Blood and Newborn Blood Spots

Abstract BACKGROUND Standard fragile X syndrome (FXS) diagnostic tests that target methylation of the fragile X mental retardation 1 (FMR1) CpG island 5′ of the CGG expansion can be used to predict severity of the disease in males from birth, but not in females. METHODS We describe methylation specific–quantitative melt analysis (MS-QMA) that targets 10 CpG sites, with 9 within FMR1 intron 1, to screen for FXS from birth in both sexes. The novel method combines the qualitative strengths of high-resolution melt and the high-throughput, quantitative real-time PCR standard curve to provide accurate quantification of DNA methylation in a single assay. Its performance was assessed in 312 control (CGG <40), 143 premutation (PM) (CGG 56–170), 197 full mutation (FM) (CGG 200–2000), and 33 CGG size and methylation mosaic samples. RESULTS In male and female newborn blood spots, MS-QMA differentiated FM from control alleles, with sensitivity, specificity, and positive and negative predictive values between 92% and 100%. In venous blood of FM females between 6 and 35 years of age, MS-QMA correlated most strongly with verbal IQ impairment (P = 0.002). In the larger cohort of males and females, MS-QMA correlated with reference methods Southern blot and MALDI-TOF mass spectrometry (P < 0.05), but was not significantly correlated with age. Unmethylated alleles in high-functioning FM and PM males determined by both reference methods were also unmethylated by MS-QMA. CONCLUSIONS MS-QMA has an immediate application in FXS diagnostics, with a potential use of its quantitative methylation output for prognosis in both sexes.

Download Full-text

Learning-disabled Males With a Fragile X CGG Expansion in the Upper Premutation Size Range

PEDIATRICS ◽

10.1542/peds.97.1.122 ◽

1996 ◽

Vol 97 (1) ◽

pp. 122-126

Author(s):

Randi J. Hagerman ◽

Louise W. Staley ◽

Rebecca O'Conner ◽

Kellie Lugenbeel ◽

David Nelson ◽

...

Keyword(s):

Mental Retardation ◽

Fragile X Syndrome ◽

Size Range ◽

Cpg Island ◽

Fragile X ◽

Learning Disabled ◽

Full Mutation ◽

Cgg Repeat ◽

Responsible Gene ◽

Repeat Segment

There is a broad spectrum of clinical involvement in both boys and girls affected by fragile X syndrome. Although this disorder is best known as the most common inherited cause of mental retardation, it also can manifest as learning disabilities in individuals with IQs in the broad range of normal. Boys are usually retarded, and girls are usually learning disabled with fragile X syndrome.1 The responsible gene, fragile X mental retardation 1 (FMR1), was isolated in 1991, and the mutation was found to involve expansion of a trinucleotide (CGG) repeat segment. Individuals with fragile X syndrome have a CGG expansion of more than 200 repeats associated with hypermethylation of both the expansion and an adjacent CpG island (full mutation).2,3

Download Full-text

Blood spots screening for identification of Fragile X Syndrome among intellectual disability students in Flores Island, INDONESIA

International Journal of Pediatric Endocrinology ◽

10.1186/1687-9856-2013-s1-p204 ◽

2013 ◽

Vol 2013 (S1) ◽

Author(s):

Agustini Utari ◽

Joyce Lo ◽

Tzuhan Tong ◽

Tri Indah Winarni ◽

Sultana MH Faradz ◽

...

Keyword(s):

Intellectual Disability ◽

Fragile X Syndrome ◽

Fragile X ◽

Blood Spots

Download Full-text

DNA Methylation at Birth Predicts Intellectual Functioning and Autism Features in Children with Fragile X Syndrome

International Journal of Molecular Sciences ◽

10.3390/ijms21207735 ◽

2020 ◽

Vol 21 (20) ◽

pp. 7735

Author(s):

Claudine M Kraan ◽

Emma K Baker ◽

Marta Arpone ◽

Minh Bui ◽

Ling Ling ◽

...

Keyword(s):

Dna Methylation ◽

Fragile X Syndrome ◽

Fragile X ◽

Single Gene ◽

Intellectual Functioning ◽

Dried Blood Spots ◽

Reference Ranges ◽

Mrna Levels ◽

Blood Spots ◽

Fmr1 Mrna

Fragile X syndrome (FXS) is a leading single-gene cause of intellectual disability (ID) with autism features. This study analysed diagnostic and prognostic utility of the Fragile X-Related Epigenetic Element 2 DNA methylation (FREE2m) assessed by Methylation Specific-Quantitative Melt Analysis and the EpiTYPER system, in retrospectively retrieved newborn blood spots (NBS) and newly created dried blood spots (DBS) from 65 children with FXS (~2–17 years). A further 168 NBS from infants from the general population were used to establish control reference ranges, in both sexes. FREE2m analysis showed sensitivity and specificity approaching 100%. In FXS males, NBS FREE2m strongly correlated with intellectual functioning and autism features, however associations were not as strong for FXS females. Fragile X mental retardation 1 gene (FMR1) mRNA levels in blood were correlated with FREE2m in both NBS and DBS, for both sexes. In females, DNAm was significantly increased at birth with a decrease in childhood. The findings support the use of FREE2m analysis in newborns for screening, diagnostic and prognostic testing in FXS.

Download Full-text

Detection of skewed X-chromosome inactivation in Fragile X syndrome and X chromosome aneuploidy using quantitative melt analysis

Expert Reviews in Molecular Medicine ◽

10.1017/erm.2015.11 ◽

2015 ◽

Vol 17 ◽

Cited By ~ 9

Author(s):

David E. Godler ◽

Yoshimi Inaba ◽

Charles E. Schwartz ◽

Quang M. Bui ◽

Elva Z. Shi ◽

...

Keyword(s):

Fragile X Syndrome ◽

X Chromosome ◽

Sex Chromosome ◽

Fragile X ◽

Venous Blood ◽

Reference Method ◽

X Chromosome Inactivation ◽

Chromosome Inactivation ◽

X Chromosomes ◽

Skewed X Chromosome Inactivation

Methylation of the fragile X mental retardation 1 (FMR1) exon 1/intron 1 boundary positioned fragile X related epigenetic element 2 (FREE2), reveals skewed X-chromosome inactivation (XCI) in fragile X syndrome full mutation (FM: CGG > 200) females. XCI skewing has been also linked to abnormal X-linked gene expression with the broader clinical impact for sex chromosome aneuploidies (SCAs). In this study, 10 FREE2 CpG sites were targeted using methylation specific quantitative melt analysis (MS-QMA), including 3 sites that could not be analysed with previously used EpiTYPER system. The method was applied for detection of skewed XCI in FM females and in different types of SCA. We tested venous blood and saliva DNA collected from 107 controls (CGG < 40), and 148 FM and 90 SCA individuals. MS-QMA identified: (i) most SCAs if combined with a Y chromosome test; (ii) locus-specific XCI skewing towards the hypomethylated state in FM females; and (iii) skewed XCI towards the hypermethylated state in SCA with 3 or more X chromosomes, and in 5% of the 47,XXY individuals. MS-QMA output also showed significant correlation with the EpiTYPER reference method in FM males and females (P < 0.0001) and SCAs (P < 0.05). In conclusion, we demonstrate use of MS-QMA to quantify skewed XCI in two applications with diagnostic utility.

Download Full-text

The fragile X syndrome: 13 years of experience

Proceedings of the Latvian Academy of Sciences Section B Natural Exact and Applied Sciences ◽

10.2478/v10046-011-0020-6 ◽

2011 ◽

Vol 65 (3-4) ◽

pp. 67-72

Author(s):

Zanda Daneberga ◽

Zita Krūmiņa ◽

Baiba Lāce ◽

Daiga Bauze ◽

Rita Lugovska

Keyword(s):

Fragile X Syndrome ◽

Dna Analysis ◽

Clinical Symptoms ◽

Cpg Island ◽

Fragile X ◽

Psychomotor Development ◽

Molecular Diagnostic ◽

Years Of Experience ◽

Male Population ◽

Cgg Repeats

The fragile X syndrome: 13 years of experience Fragile X syndrome (FXS; MIM #300624; FRAXA, Xq27.3) is well known and a common cause of X-linked mental retardation. The syndrome is caused by dynamic mutation of FMR1 gene CpG island CGG repeats. Clinically FXS patients demonstrate delayed developmental milestones, particularly speech, attention-deficit/hyperactivity disorder, autistic features, and psychomotor development delay. Dysmorphic face and macroorchidism are important features in the postpubertal age. We present our 13-year experience with FXS patients who were confirmed by molecular diagnostic. Phenotype-genotype evaluation was made for 12 male FXS patients. Genotype-phenotype analysis did not reveal significant correlation between clinical symptoms observed in FXS patients and genotypes obtained from leucocytes DNA analysis. The prevalence of the fragile X syndrome in the Latvian male population was estimated to be 1/6428 (95% CI 5538-7552) or 15.55/100 000 males (95% CI 13.24 - 18.05). The prevalence of the fragile X syndrome among mentally retarded male patients was estimated to be 2.67%. The low number of diagnosed patients with fragile X syndrome demonstrated in our study led to the conclusion that fragile X syndrome is generally clinically unrecognised.

Download Full-text

Diagnosis of the fragile X syndrome in males using methylation-specific PCR of the FMRI locus

Genetics and Molecular Biology ◽

10.1590/s1415-47571999000200005 ◽

1999 ◽

Vol 22 (2) ◽

pp. 169-172 ◽

Cited By ~ 3

Author(s):

Sérgio D.J. Pena ◽

Rosane Sturzeneker

Keyword(s):

Fragile X Syndrome ◽

Internal Control ◽

High Efficiency ◽

Cpg Island ◽

Fragile X ◽

Pcr Amplification ◽

Sodium Bisulfite ◽

Chromosome 15 ◽

Methylation Specific Pcr ◽

Specific Pcr

We have developed a non-isotopic technique based on methylation-specific PCR (MSP) of the FMR1 promoter for the identification of fragile X full mutations among men. DNA samples were first treated with sodium bisulfite to convert unmethylated, but not methylated, cytosines to uracil, followed by PCR amplification with oligonucleotide primers specific for methylated versus unmethylated DNA. We designed two primer pairs: one produced a 142-bp fragment from the bisulfite-treated methylated CpG island, while the other generated an 84-bp product from the treated non-methylated promoter. In normal males only the 84-bp fragment was seen, while the diagnosis of FRAXA was doubly indicated by the appearance of a 142-bp product together with absence or weak visualization of the 84-bp band. As an indispensable internal control for the efficiency of the sodium bisulfite treatment, we used a primer pair specific for the imprinted maternal methylated version of the CpG island of the SNRPN gene on human chromosome 15. Using the methylation-specific PCR we identified with 100% sensitivity and accuracy eight previously diagnosed FRAXA male patients mixed with 42 normal controls. Because of its simplicity and high efficiency, methylation-specific PCR may become the method of choice for the diagnosis of the fragile X syndrome in mentally retarded males.

Download Full-text

The Accuracy of Dried Blood Spots Compared to Plasma or Serum Retinol for the Diagnosis of Vitamin A Deficiency: A DTA Systematic Review and Meta-Analysis

Current Developments in Nutrition ◽

10.1093/cdn/nzaa041_010 ◽

2020 ◽

Vol 4 (Supplement_2) ◽

pp. 106-106

Author(s):

Bryan Gannon ◽

Rachel Herrmann ◽

Anju Sinha ◽

Kate Ghezzi-Kopel ◽

Lisa Rogers ◽

...

Keyword(s):

Systematic Review ◽

Vitamin A ◽

Sensitivity And Specificity ◽

Meta Analysis ◽

Venous Blood ◽

Dried Blood Spots ◽

Cold Chain ◽

Serum Retinol ◽

Blood Spots ◽

Reference Methods

Abstract Objectives Dried blood spots (DBS) do not require a cold chain, making them easier to store and transport compared to serum or plasma samples in resource-limited settings. Our objective was to determine the accuracy of DBS compared to serum or plasma retinol for assessing vitamin A status in individuals and populations. Methods A systematic review of diagnostic test accuracy (DTA) was performed using standard Cochrane Methods for Screening and Diagnostic Tests. All human studies were included if they determined retinol in DBS (index test) and compared results to retinol determined in either plasma or serum samples (reference standard). Index and reference methods could use either capillary or venous blood with retinol quantified by HPLC. We used the WHO threshold of 0.70 μmol/L to define low retinol concentrations. Meta-analysis determined summary estimates of sensitivity and specificity using the bivariate model, which models logit transformed sensitivities and specificities as a bivariate normal distribution including a parameter for the negative correlation between sensitivity and specificity. Rstudio (Version 1.1.456, RStudio, Inc. Boston, MA) was used for analysis. Results Our search (conducted April 10, 2019) identified 9021 records, and 285 studies were assessed in full-text for inclusion; 6 publications reported 8 studies comprising 397 participants, 50 of which had low serum retinol by the reference method. The use of DBS to determine a low retinol level had summary estimates (95% CIs) for sensitivity of 86.7% (71.4 to 94.5) and specificity of 99.5% (89.7 to 100.0). Conclusions Use of DBS to determine low retinol has been shown to have good sensitivity and high specificity compared to serum or plasma. Use of DBS can reduce the need for centrifugation and cold chain for storage and transport. The mixed use of capillary and venous blood in both index and reference methods merits further investigation for the determination of retinol in DBS. Funding Sources None declared.

Download Full-text

A Homogeneous Assay for Analysis of FMR1 Promoter Methylation in Patients with Fragile X Syndrome

Clinical Chemistry ◽

10.1373/clinchem.2006.080762 ◽

2007 ◽

Vol 53 (4) ◽

pp. 790-793 ◽

Cited By ~ 26

Author(s):

Christina Dahl ◽

Karen Grønskov ◽

Lars A Larsen ◽

Per Guldberg ◽

Karen Brøndum-Nielsen

Keyword(s):

Fragile X Syndrome ◽

Southern Blot ◽

Cpg Island ◽

Fragile X ◽

Methylation Status ◽

Melting Peak ◽

Melting Curve Analysis ◽

Full Mutation ◽

Cgg Repeat ◽

Male Patients

Abstract Background: Fragile X syndrome is caused by the expansion of a CGG trinucleotide repeat at the 5′ untranslated region of the fragile X mental retardation 1 gene (FMR1). When expanded to >200 repeats (full mutation), the repeat region and the adjacent promoter CpG island become hypermethylated, rendering FMR1 transcriptionally inactive. Conventional molecular diagnosis of fragile X syndrome involves determination of the CGG repeat number by Southern blot analysis. Methods: A homogeneous methylation-specific melting curve analysis (MS-MCA) assay for methylation status of the FMR1 promoter region was developed on the LightCycler platform. Genomic DNA was treated with sodium bisulfite, and a region containing 8 CpG sites was amplified in the presence of SYBR Green I, using primers that do not differentiate between methylated and unmethylated FMR1 molecules. After amplification, the samples were melted at 0.05 °C/s, and fluorescence melting curves were recorded. We studied samples, previously characterized by Southern blot analyses, from 10 female and 10 male donors with normal numbers of CGG trinucleotide repeats, 9 male donors who were premutation carriers, 4 male donors who carried both a premutation and a full mutation, and 25 patients with fragile X syndrome. Results: Samples from all 20 male patients with fragile X syndrome showed a high melting peak corresponding to fully methylated FMR1, whereas samples from healthy males showed a single low melting peak corresponding to unmethylated FMR1. Of 24 samples from affected males, 9 (38%) showed 2 melting peaks, suggesting that cellular methylation mosaicism is common in fragile X syndrome. Conclusions: MS-MCA allows rapid and reliable identification of fragile X syndrome in male patients.

Download Full-text