Fragile X carrier detection before or at early pregnancy through a wide screening program may not only confer a risk of having offspring with Fragile X syndrome (FXS), but may also confer a risk for Fragile X-associated primary ovarian insufficiency and Fragile X-associated tremor/ataxia syndrome. However, prior to the implementation of such a program, the carrier prevalence in a population and the availability of effective screening test should be evaluated. The aim of our study was to determine the prevalence of premutation carriers and to evaluate the feasibility of screening test.
The blood samples were obtained from 8,641 pregnant women with no family history of mental retardation. We performed a three-primer CGG repeat primed (RP) PCR using the AmplideX™
Among the 8,641 women, we found 8 premutation carriers (1:1,090, 0.09%) and 46 women with an intermediate allele (1:190, 0.53%). No woman was found to carry the fully mutated allele. All the detected alleles were within the CGG repeat range of 8-117. Among the 8,641 samples, 29 and 30 CGG repeats represent 66.6% of all cases. The CGG RP PCR method provides robust detection of expanded alleles and resolves allele zygosity, thus minimizing the number of samples that require Southern blot analysis.
This is the first study that has focused on the prevalence of FXS premutation carriers and
Fragile X syndrome (FXS, OMIM 300624) is caused by the expansion of the CGG repeat in the 5’ untranslated region (UTR) of the
Until recently, however, the technical difficulties of performing population-based carrier detection precluded offering screening to the general population. As large expansions are refractory to PCR amplification, strategies for prenatal population-based carrier screening for pregnant women required both a PCR and Southern blot analysis. Combined, these are costly and laborintensive, have prolonged turnaround times, and ultimately have limited utility for population-based screening. Recent studies have described a method, CGG repeat primed PCR (CGG RP PCR) coupled with capillary electrophoresis, that overcomes the technical hurdles to implementing carrier screening and that has the potential to be used for population-based screening.4) In this study, using CGG RP PCR by reducing the number of Southern blot analyses, we determined the
A total of 8,641 pregnant women applied for testing on their own initiative or on the advice of their physicians, on a self-pay basis, from various medical sites in Korea. Institutional review board (IRB) approval was obtained for the study from our IRB committee. They were given printed information on fragile X and each completed a questionnaire to ascertain any family history of mental retardation, and all women with such a family history were excluded from the study. All the women who were found to be carriers of premutation alleles were offered genetic counseling. They were provided with information about prenatal diagnosis based on amniotic fluid analysis or chorionic villus sampling (CVS). Genomic DNA was isolated from peripheral blood leukocytes (5 mL of whole blood) using a standard method (QIAamp DNA Blood Mini kit; Qiagen, Hilden, Germany).
We performed the CGG RP PCR using an AmplideX™
Among 8,641 pregnant women screened for
A total of 44 different CGG repeat alleles ranging in number from 8-117 were present. As seen in Fig. 1, the most common allele was 29 repeats (37.1%), followed by 30 repeats (29.5%). Among the 8,641 samples, 29 and 30 CGG repeats represent 66.6% of all cases. Besides this major peak, there was a second peak with 35 and 36 repeats (Fig. 1). The overall normal allele prevalence was 99.5% (8,598/8,641). Intermediate size was defined as 45-54 repeats; a total of 46 women were found to be within this range, with an intermediate allele prevalence of 1:190 (0.53%).
Despite recent interest in widespread carrier screening for
Although mutation frequencies were reported in all studies, it is difficult to make comparisons between studies as different repeat length cutoffs, sample sizes, and sampling methods have been used. Consistency in repeat length cutoffs and large studies with a wide range of population groups are needed.14) While the lack of any premutation alleles has been reported in other Asian studies in Japan and Taiwan, as well as in a previous Korean study,15-17) the number of collected samples in those studies was much fewer than the number of samples collected in this study. This is the first report to conclusively demonstrate the premutation carrier frequency in a general population in Korea. According to a report by Musci et al., a prenatal Fragile X carrier screening program that aims to screen all pregnant women regardless of family history or specific risk factors, would be cost effective based on a reasonably wide range of assumptions.3) Considering the very low premutation frequency in our data, we doubt such a wide screening program in Korea would be cost-effective or appropriate. However, arguments in favor of introducing population- based screening for FXS center on the severity of the condition and its impact on individuals, families, and society.3, 14)
The intermediate alleles have been shown to be slightly unstable upon transmission. The American College of Medical Genetics has recommended that intermediate alleles be considered a possible risk factor for repeat expansion.6) Here, we estimated the intermediate allele prevalence was 1:190 females. This frequency was lower than the intermediate allele frequencies reported in previous reports for Caucasian populations.14)
In previous studies on the variation of the CGG repeat in the
PCR amplification of the CGG repeats region in
In conclusion, we demonstrated that the premutation carrier frequency of FXS in the normal pregnant Korean women is 1:1,090, which is much lower than in Western countries. It is now assumed that the prevalence of FXPOI is low in Korea. Several limitations are noted in our present study, mainly due to its subject enrollment. Regardless of these limitations, however, our data could offer groundwork for a proper prenatal population-based Fragile X carrier screening program for reproductive women in Korea.
Allele frequencies for CGG repeats in
Number of Premutation Carriers for Each Range of the Number of CGG Repeats
|CGG repeat range||No. of women (n=8)|
Carrier Frequency of
|Populations||Samples screened||Premutation identified||Carrier Frequency (%)||References|
|Israel-1||8,426||58||1:143 (0.70)||Pesso et al.7)|
|Israel-2||14,334||127||1:113 (0.88)||Toledano-Alhadef et al.8)|
|Israel-3||36,483||231||1:158 (0.63)||Berkenstadt et al.9)|
|USA-1||474||3||1:158 (0.63)||Spence et al.10)|
|USA-2||2,299||6||1:382 (0.26)||Cronister et al.11)|
|Finland-1||1,477||6||1:246 (0.41)||Ryynanen et al.12)|
|Finland-2||220||1||1:220 (0.45)||Kallinen et al.13)|
|Korea||8,641||8||1:1,090 (0.09)||Present study|
Pregnant or non-pregnant women without a family history of mental retardation
Pregnant or non-pregnant women with no information about a family history of mental retardation