scholarly journals Methyl-CpG-Binding PCR of Bloodspots for Confirmation of Fragile X Syndrome in Males

2009 ◽  
Vol 2009 ◽  
pp. 1-8 ◽  
Author(s):  
Ching-Cherng Tzeng ◽  
Chiou-Ping Liou ◽  
Chien-Feng Li ◽  
Ming-Chi Lai ◽  
Li-Ping Tsai ◽  
...  
Keyword(s):  
Fragile X Syndrome ◽  
Promoter Methylation ◽  
Fragile X ◽  
Blot Hybridization ◽  
Methylation Status ◽  
Southern Blot Hybridization ◽  
Detection Sensitivity ◽  
Simple Method ◽  
X Chromosomes ◽  
Blinded Manner

This study demonstrates that methyl-CpG-binding PCR (MB-PCR) is a rapid and simple method for detecting fragile X syndrome (FXS) in males, which is performed by verifying the methylation status of theFMR1promoter in bloodspots. Proteins containing methyl-CpG-binding (MB) domains can be freeze-stored and used as stocks, and the entire test requires only a few hours. The minimum amount of DNA required for the test is 0.5 ng. At this amount, detection sensitivity is not hampered, even mixing with excess unmethylated alleles up to 320 folds. We examined bloodspots from 100 males, including 24 with FXS, in a blinded manner. The results revealed that the ability of MB-PCR to detectFMR1promoter methylation was the same as that of Southern blot hybridization. Since individuals with 2 or more X chromosomes generally have methylatedFMR1alleles, MB-PCR cannot be used to detect FXS in females.

Microisolation of the chicken Z chromosome and construction off microclone libraries

Genome ◽  
1997 ◽  
Vol 40 (6) ◽  
pp. 865-872 ◽  
Author(s):  
Régis Zimmer ◽  
Alon Haberfeld ◽  
Ann M. Verrinder Gibbins
Keyword(s):  
Light Microscope ◽  
Genomic Dna ◽  
Size Range ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Z Chromosome ◽  
Mitotic Metaphase ◽  
Simple Method ◽  
Dna Library ◽  
Male Chicken

A simple method was used to adapt a standard light microscope for the collection of chicken Z chromosomes from mitotic-metaphase spreads. The DNA of the collected chromosomes was enzymatically amplified using a partially degenerate primer. The resulting sequences, within a size range of 200–800 bp, were cloned to produce a Z chromosome DNA library, using blunt-end ligation into a SmaI-digested pUC18 plasmid (the SureClone system; Pharmacia, U.S.A.). The microcloning experiments produced 1250 clones; the size range of the cloned inserts was 250–800 bp, with an average of 480 bp (176 clones examined). Using male chicken genomic DNA as a probe, 10 out of 17 randomly selected clones showed strong positive signals on Southern blots, confirming the origin of the inserts as chicken DNA. In addition, the Z-chromosome origin of a selected microclone was verified in a semiquantitative Southern blot hybridization that showed positive signals with intensities that were approximately twice as strong for male (ZZ) as for female (ZW) chicken genomic DNA when the clone was used as a probe. The value of these libraries in further analysis of the chicken Z chromosome is discussed.Key words: microdissection, microcloning, chicken Z chromosome.

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

2015 ◽  
Vol 17 ◽  
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.

Fragile X Syndrome in Humans and Mice

1996 ◽  
Vol 45 (1-2) ◽  
pp. 93-108 ◽  
Author(s):  
B. A. Oostra
Keyword(s):  
Mental Retardation ◽  
Fragile X Syndrome ◽  
Dendritic Spines ◽  
Pyramidal Neurons ◽  
Fragile Site ◽  
Fragile X ◽  
Methylation Status ◽  
Physical Symptoms ◽  
Post Mortem Brain ◽  
Iq Scores

Fragile X syndrome is the most common cause of interited mental retardation in humans, with a frequency of approximately 1 in 1200 males and 1 in 2500 females [1]. It is second only to Down syndrome as a genetic cause of mental retardation, which has an overall frequency of 1 in 600. These frequency estimates suggest that fragile X syndrome accounts for approximately 3% of mental retardation in males, and perhaps as much as 20% in males with IQs between 30 and 55 [2]. The disease derives its name from the observation of a fragile site at Xq27.3 in cultured lymphocytes, fibroblasts and amniocytes [3].The phenotype of the fragile X syndrome is mental retardation, usually with an IQ in the 4-70 range [4] and a number of dysmorphic features: long face, everted ears and large testicles [for review see ref. 5] (Fig. 1). Not every patient shows all the physical symptoms, which are generally more apparent after childhood. Macroorchidism is a common feature of fragile X syndrome in more than 90% of postpuberal males. Some patients show hyperactivity and attention deficits as well as avoidance behaviour similar to autism. Affected females generally have a less severe clinical presentation, and their IQ scores are generally higher, with typically borderline IQs or mild mental retardation.No gross pathological abnormalities have been described in the brains of fragile X patients. Only a few post-mortem brain studies of fragile X males have been described and the information is very limited, presenting only non-specific findings such as brain atrophy, ventricular dilatation and pyramidal neurons with abnormal dendritic spines. It has been shown that the volume of the hippocampus was enlarged compared to controls [6], while a significantly decreased size of the posterior cerebellar vermis and increased size of the fourth ventricle was found [7]. Using magnetic resonance imaging it was shown that fragile X patients have an increased volume of the caudate nucleus [8]. The caudate volume is correlated with IQ and methylation status of the FMR1 gene.

scholarly journals Deleted X chromosomes in patients with the fragile X syndrome.

1984 ◽  
Vol 21 (5) ◽  
pp. 373-373 ◽  
Author(s):  
M Fitchett ◽  
M Seabright
Keyword(s):  
Fragile X Syndrome ◽  
Fragile X ◽  
X Chromosomes

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

2007 ◽  
Vol 53 (4) ◽  
pp. 790-793 ◽  
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 &gt;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.

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