Rapid Fragile X Carrier Screening and Prenatal Diagnosis Using a Nonradioactive PCR Test

Brown, W. Ted; Houck, George E.; Jeziorowska, Anna; Levinson, F.; Ding, Xiaohua; Dobkin, Carl; Zhong, Nan; Henderson, J.; Brooks, Susan Sklower; Jenkins, Edmund C.

doi:10.1001/jama.1993.03510130075034

Cited by 218 publications

(38 citation statements)

References 47 publications

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“…Blood was obtained from 175 females ( (Meadows et al 1996). For male samples that did not amplify or females with only one allele, a second PCR protocol was used (Brown et al 1993). The PCRs for FRAXA consisted of 1× PCR Buffer (Gibco/BRL), 10% dimethyl sulfoxide (DMSO), 370 µM deazaG, 500 µM d(ACT), 0.3 µM each primer, 15 ng T4 gene 32, and 1.05 U Roche Expand Long Taq.…”

Section: Samplesmentioning

confidence: 99%

A study of the distributional characteristics of FMR1 transcript levels in 238 individuals

et al. 2004

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Fragile X syndrome, the most common form of inherited mental retardation, is caused by hyperexpansion and hypermethylation of a CGG repeat tract in the 5' untranslated region of the FMR1 gene. This methylation causes the gene to be transcriptionally silenced. In addition to the common allele form with less than 41 repeats, there are two other allelic forms of the FMR1 gene that are unmethylated: premutation (61-200 CGG repeats) and intermediate (41-60 CGG repeats). Recently, premutation-specific phenotypes not related to fragile X syndrome have been reported: a 20-fold increased risk for premature ovarian failure (POF) among female carriers and an increased risk for a tremor ataxia syndrome (TAS) primarily among older male carriers. At the molecular level, increased levels of FMR1 transcript have been observed among premutation carriers. Increased levels of transcript may be causally related to the POF or TAS phenotypes or may be a surrogate of some other allelic property. In this report, we have examined the distributional properties of transcript levels by repeat size and gender among 238 individuals. We have confirmed a significant linear relationship between transcript level and repeat size in males and females. The evidence for the linear effect is primarily within the premutation size alleles.

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Section: Samplesmentioning

confidence: 99%

A study of the distributional characteristics of FMR1 transcript levels in 238 individuals

et al. 2004

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“…The discrete differences observed between the mode and mean/median values point to the apparent skewness of the FMR2 allele frequency distribution in our population. Using the g 1 Chiurazzi et al, 1996Fu et al, 1991 United States 2,500 12-59 42 30 -79.9 Brown et al, 1996Brown et al, 1993 1,069 11-52 35 29 -NR Brown et al, 1997Brown et al, 1993 Fu et al, 1991 (Caucasian) a Calculated by authors; only for those reports in which frequency distributions were available. Methods used for CGG repeat analysis: Fu et al, 1991, radioactive PCR/PAGE with primers c and f;Fu et al, 1991, radioactive PCR/PAGE with primers a and f; Pergolizzi et al, 1992, agarose gel/Southern using primers 1-21, and 203-181;Brown et al, 1993, PAGE/Southern using primers 1 and 3; Haddad et al, 1996, PAGE using primers Eag-U and Eag-L. NR, not reported.…”

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confidence: 99%

Expansion mutation frequency and CGG/GCC repeat polymorphism inFMR1 andFMR2 genes in an Indian population

2000

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Based on molecular screening, we estimated the frequencies of fragile X syndrome and FRAXE syndrome in an institutionalized population (n = 130) in New Delhi, India. Eligibility criteria for inclusion of subjects in the study were mild/moderate mental retardation, with/without family history, and the fragile X clinical phenotype. Screening by Southern hybridization revealed an overall frequency of 0.077 of the syndrome in the sample population. The disorder was observed with a high frequency (0.1) among males as compared to females (0.027). No expansions of FMR2 allele were observed in the same study sample. CGG/GCC allelic polymorphism of FMR1 and FMR2 were established from a total of 392 X chromosomes, using the radioactive polymerase chain reaction–polyacrylamide gel electrophoresis method. Distinct repeat sizes, repeat ranges, and repeat modes characterised the FMR1 and FMR2 alleles. In the X chromosomes of both MR individuals and unaffected controls, unimodal values of 29 and 15 repeats in FMR1 and FMR2 genes, respectively, were observed. Allele frequency distribution was symmetrical at the FMR1 locus whereas a significant positive skew was observed for the FMR2 alleles. The observed heterozygosity of the FMR1 gene was 0.772 compared to 0.839 of FMR2. Correlation of CGG/GCC repeats of FMR1 and FMR2 did not show any association of repeat sizes at these two loci (correlation coefficient, ρ = 0.09). CGG/GCC repeat variation at FMR1 and FMR2 loci observed in this study sample are different from that reported for the other Caucasian and Asian populations. Genet. Epidemiol. 20:129–144, 2001. © 2001 Wiley‐Liss, Inc.

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“…Currently available procedures for the diagnostic evaluation of the FRAXA syndrome include cytogenetic, Southern-blot, polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), and immunohistochemical analyses (Rousseau et al 1991a;Brown et al 1993;Cao et al 1994;Pai et al 1994;El-Aleem et al 1995;Wang et al 1995;Carrel and Willard 1996;Haddad et al 1996;Holden et al 1996;Spence et al 1996;Willemsen and Oostra 2000). The most commonly applied molecular geneticbased techniques depend on either the detection of the expanded repeat, the de novo methylation of the affected gene regions, or both (Brown et al 1993;Cao et al 1994;Pai et al 1994;El-Aleem et al 1995;Wang et al 1995;Carrel and Willard 1996;Haddad et al 1996;Holden et al 1996;Spence et al 1996;Hecimovic et al 1997;Panagopoulos et al 1999;Strelnikov et al 1999).…”

Section: Andreas Weinhäusel · Oskar a Haasmentioning

confidence: 99%

“…The most commonly applied molecular geneticbased techniques depend on either the detection of the expanded repeat, the de novo methylation of the affected gene regions, or both (Brown et al 1993;Cao et al 1994;Pai et al 1994;El-Aleem et al 1995;Wang et al 1995;Carrel and Willard 1996;Haddad et al 1996;Holden et al 1996;Spence et al 1996;Hecimovic et al 1997;Panagopoulos et al 1999;Strelnikov et al 1999). Only radioactive or non-radioactive Southern-blotting of DNA doubledigested with methyl-sensitive and methyl-insensitive restriction enzymes is able reliably to cover the whole spectrum of premutations, full mutations, and mosaicism that may be encountered in the context of this syndrome (Brown et al 1993;Kolehmainen and Karant 1994;Kirchgessner et al 1995;Allingham-Hawkins et al 1996;Carrel and Willard 1996;de Vries et al 1998). PCR is mainly applied to determine the length of the triplet repeat in the normal and premutation range.…”

Section: Andreas Weinhäusel · Oskar a Haasmentioning

confidence: 99%

“…The high CG content of the repeat region renders a direct and reliable amplification of full mutations difficult. It is only reliable within the lower repeat unit range, particularly of male patients, either following 7-deaza-2'-dGTP incorporation (Brown et al 1993) or by applying a commercial test system (PE Biosystems).…”

Section: Andreas Weinhäusel · Oskar a Haasmentioning

confidence: 99%

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Evaluation of the fragile X (FRAXA) syndrome with methylation-sensitive PCR

Weinhäusel

Haas

2001

Hum Genet

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The fragile X (FRAXA) syndrome is the most common form of inherited mental retardation in males. Its peculiar pattern of inheritance results from the parent of origin-specific expansion of a CGG-repeat within the FMR1 gene on the X chromosome. In patients, gene function is abolished by hypermethylation of the promoter and the massively expanded repeat. We have developed a methylation-sensitive polymerase chain reaction (MS-PCR) strategy that combines repeat-length and methylation analysis of the CGG-repeat and the promoters of the FMR1 and XIST genes. The allelic methylation of the latter opposes that of the FMR promoter and serves as an internal control and standard for semiquantitative analyses. This system enables the delineation of 11 distinct patterns encountered in nonaffected, carrier, and affected males and females. We have evaluated our system on well-defined samples with different FMR1 mutations and have used it for the diagnostic evaluation of 253 male and 80 female probands. In the male group, we have identified five full mutations, and three gray-zone and premutation alleles with 54, 55, and 62 repeats, respectively. The female group consists of 33 normal homozygote and 41 heterozygote individuals, two of whom harbor a gray-zone allele with 47 repeats, none with a premutation, and six with a full mutation. Our MS-PCR approach allows the currently most comprehensive diagnostic evaluation of the FRAXA syndrome in a cost- and time-efficient fashion. In addition, it is a valuable tool for the analysis of clonality and skewing phenomena in females.

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Rapid Fragile X Carrier Screening and Prenatal Diagnosis Using a Nonradioactive PCR Test

Cited by 218 publications

References 47 publications

A study of the distributional characteristics of FMR1 transcript levels in 238 individuals

A study of the distributional characteristics of FMR1 transcript levels in 238 individuals

Expansion mutation frequency and CGG/GCC repeat polymorphism inFMR1 andFMR2 genes in an Indian population

Evaluation of the fragile X (FRAXA) syndrome with methylation-sensitive PCR

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