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An Effective Strategy of Using Molecular Testing to Screen Mentally Retarded Individuals for Fragile X Syndrome

Tzeng, Ching–Cherng M.D.; Lin, Shio–Jean M.D.; Chen, Yung–Jung M.D.; Kuo, Pao–Lin M.D.; Jong, Yuh–Jyh M.D.; Tsai, Li–Ping M.D.; Chen, Robert M. M.D., PhD

Diagnostic Molecular Pathology: March 2001 - Volume 10 - Issue 1 - pp 34-40
Original Articles

Fragile X syndrome (FXS) is the most common form of familial mental retardation (MR). It is caused by the expansion of the CGG repeat in the FMR1 gene on the X chromosome. To date, FXS is not treatable, but can be prevented by prenatal genetic examination. Identifying women who carry a full mutation or premutation FMR1 gene is thus very important, and can be done by tracing family members of FXS subjects. However, most of the FXS subjects in Taiwan as well as those in many other countries have not been identified. In this study the authors attempt to develop reliable and inexpensive tests suitable for a large-scale screen of subjects with MR for FXS. Together with their previous study, a total of 311 male and 160 female subjects with MR were screened with nonradioactive Southern blot assay using mixed deoxyribonucleic acid from three subjects of the same sex. From these subjects, nine male subjects and one female FXS subject were diagnosed. All male subjects were also screened with nonradioactive polymerase chain reaction (PCR). These nine male FXS subjects were also detected on the basis of PCR amplification failure. No false-negative results were discerned. The PCR procedure was simplified further by combining it with an analysis of a blood spot on filter paper, which is a much simpler and cheaper method for sample collection and DNA preparation. This method was then used to screen 104 boys with MR. Two of them were suspected, and later confirmed with Southern blot assay, as subjects with FXS. This study suggests that simple PCR combined with blood spot analysis could be a reliable, inexpensive test that is feasible for a large-scale screening of male subjects with MR for FXS. However, Southern blot assay with mixed deoxyribonucleic acid is appropriate for screening female subjects. Based on this strategy, most FXS subjects could be identified easily for further management.

From the Division of Cytogenetics and Molecular Genetics, Department of Pathology (C.C.T., R.M.C.), Pediatrics (S.J.L., Y.J.C.), and Obstetrics and Gynecology (P.L.K.), National Cheng Kung University Medical College, Tainan; the Departments of Pediatrics and Clinical Laboratory (Y.J.J.) , Kaohsiung Medical University; and the Department of Pediatrics (L.P.T.), Taipei Municipal Women and Children's Hospital, Taipei, Taiwan, Republic of China.

Supported by grant 87/88-03 from the National Cheng Kung University Medical College and Hospital, and by grant NSC-88-2314-B-006-052 from the National Science Council, Taiwan, Republic of China.

Address correspondence and reprint requests to Dr. Ching–Cherng Tzeng, Department of Pathology, Chi-Mei Foundation Hospital, 901, Chung-Hwa Road, Yung-Kang City, Tainan 710, Taiwan, ROC; Email:

Mental retardation (MR) accounts for 1.2 to 8.4% of the population in different countries. Two thirds are mild cases of MR (IQ, 50–70) and the other one third is severe (IQ, >50). Fragile X syndrome (FXS) is, next to Down syndrome, the second most common cause and the most common familial form of MR (8,18). FXS is caused by a CGG-repeat expansion located in the 5` end of the untranslated region of the FMR1 gene on the X chromosome, which results in hypermethylation and transcriptional silence of the gene. Specific treatment for FXS is not yet feasible. However, this disorder can be prevented through a prenatal genetic examination (27).

It is known that the mutant FMR1 genes in all FXS subjects are derived maternally. The mothers may be carriers of a premutation (PM) or full-mutation (FM) gene. Identification of carrier women is thus very important for the prevention of FXS (27). This has been well established in some developed countries by screening pregnant women who have a family history of MR. However, this history may not be evident or could have been ignored by many people. Negative family history of MR cannot preclude women from being an FXS carrier (21,24). Screening all pregnant women will hopefully avoid any misses, but it is not currently regarded as a cost-effective procedure (2). Identifying carriers by tracing affected subjects is therefore a practical approach.

Recent studies based on molecular testing indicate that the prevalence of FXS is lower than previously estimated by cytogenetic studies and it occurs in approximately 1 in 5,000 men (1,7,27). Several studies suggest that the prevalence of FXS in Taiwan is similar to other populations, although most of the populations were not identified (12,14,24). To increase the efficacy of identifying FXS subjects, it is necessary to preselect certain subjects with MR using a clinical checklist and then to perform the expensive molecular confirmation test on suspect subjects (1,10). Obviously, the more specific the checklist, the higher the positive identification rate. Concomitantly, the false-negative rate will increase along with an increase in specificity. Thus, a simple and reliable test of a broader base with less specific checking criteria would be a more practical approach to identifying FXS subjects.

Recently, we reported that instead of performing Southern blot testing on individual subjects, FXS could be detected invariably with deoxyribonucleic acid (DNA) mixed from three subjects of the same sex (24). In addition, all male subjects were screened with simple, nonradioactive polymerase chain reaction (PCR). We found that all male FXS subjects diagnosed in our previous study (24) failed PCR amplification consistently because of marked expansion of the CGG triplet. This result, and those of others (26), indicate that PCR is a reliable test for detecting male FXS subjects, who could then be reevaluated with standard Southern blot assay.

In the current study we attempted to improve further the efficacy of screening for FXS and to identify reliably the PM carriers. Basically, all female subjects were screened with a nonradioactive Southern blot assay on mixed DNA. The majority of subjects were male, and they were screened reliably with PCR using a blood spot collected on filter paper, which is much simpler and less expensive with regard to DNA preparation (4,6,11). Those subjects suspected of carrying mutant FMR1 genes were then reconfirmed individually with standard tests, including Southern blot assay and PCR sequencing gel electrophoresis for measuring the CGG triplet number.

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Subject Selection and Screening Strategy

In addition to 206 male and 115 female subjects with MR reported recently (24), an additional 45 female and 105 male subjects were screened in the current study with a modified molecular testing strategy for FXS. The majority of the subjects were younger than 12 years of age. All female subjects were screened using Southern blot assay with mixed DNA from three subjects. Before mixing with two other samples, the quality and quantity of digested DNA from each subject was checked, as described later. All male subjects were checked by both Southern blot assay with mixed DNA and a simple, nonradioactive PCR assay.

We also tried to simplify the PCR procedure for screening male subjects by using dried blood on filter paper. For the 105 male subjects tested in the current study, blood spots were made on filter paper on receiving the blood sample. The PCR results with DNA isolated from the blood spot were compared with the DNA prepared from whole blood using a commercial kit (Puregene, Minneapolis, MN, USA). After confirming the reliability of DNA preparation from the blood spot for PCR, we offered this simple test to a total of 104 boys with MR. This greatly enhanced the willingness of undertaking the test by the parents, apparently for economic reasons. However, they were clearly informed of the rare possibility of missing detection of some variant in FXS male subjects, including those with a mosaic with a normal allele and others not associated with the typical form of FM.

Those subjects suspected of carrying the mutant FMR1 gene were reconfirmed by standard tests including Southern blot assay and PCR sequencing gel electrophoresis for measuring the CGG triplet number (23). For the confirmed FXS subjects, we provided genetic counseling and administered genetic tests to their family members.

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Nonradioactive Southern Blot Hybridization

Southern blot analysis of the FMR1 gene based on mixed DNA (1.25 μg from each of three subjects of the same sex) has been described previously (24). DNA was doubly digested with Eco RI and Nru I. Unlike male samples, each female sample was digested individually. Before mixing with two other samples, the quality and quantity of each digested sample was ensured by running it on agarose gel and staining it with ethidium bromide. DNA mixtures were then separated by running them on 0.8% agarose gel at 2 V/cm for 26 hours, followed by transfer to a nylon membrane. After hybridizing with digoxigenin-labeled probe StB12.3 overnight and stringent washes, the blots were then detected using a chemoluminescent kit (Roche Boehringer Mannheim, Mannheim, Germany). A clear result was generally obtained after exposure of the blot to radiographic films at room temperature for 2 hours, and an overexposure for overnight was routinely backed up to avoid missing cases with weak and long smearing signals of FM.

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Nonradioactive PCR

To visualize easily the PCR product in gel with ethidium bromide, we have previously included 0.5 M betaine to replace 7`-deaza-2`-dGTP in the PCR mixture (24). In the current study we modified the protocol further and used a longer primer to amplify a larger fragment flanking the CGG triplet of the FMR1 gene to obtain a better PCR yield (13). A 30-μL PCR mixture contained 5 μl DNA suspension prepared from bloodstained filter paper (or 12.5 ng DNA prepared from whole blood), 7.25 mM Tris HCl (pH, 9.0), 20 mM (NH4)2SO4, 1.5 mM MgCl2, 0.01% (weight per volume) Tween-20, 50 μM dNTP, 0.5 M betaine (Sigma, St. Louis, MO, USA), 10% dimethyl sulfoxide (DMSO), 10% glycerol, both primers at a concentration of 50 nM, and 0.3 U Super-Therm DNA polymerase (Laboratory Product International, Kent, UK). PCR was performed in the Perkin–Elmer thermocycler 9700 (PE Applied Biosystems, Foster City, CA, USA). The cycling profile was as follows: Initial denaturation at 95°C for 5 minutes, 35 cycles at 95°C for 30 seconds, 65°C for 1 minute, 72°C for 2 minutes, and a final extension at 72°C for 10 minutes. In each test, a sample with 98-CGG and a suspension prepared from blank filter paper were included as a positive and negative control respectively. Amplified product was then resolved on a 5% polyacrylamide gel and visualized with ethidium bromide stain and ultraviolet illumination.

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DNA Preparation From Dried Blood on Filter Paper

To prepare high-quality DNA from a blood spot useful for PCR, we compared a variety of procedures reported previously (3–6,11,15,20,25). Ideally, the procedure should be simple and inexpensive. The protocol basically had two major steps: 1) removal of most PCR inhibitors and 2) release of DNA from the white cells by boiling. After brief pilot trials, only a few possible combinations were selected for detailed evaluation.

To evaluate the efficacy of the different combinations, each comparison study was undertaken with a blood spot from 30 male samples. For each test, a 6-mm punch of blood spot was transferred to a 1.5-mL microcentrifuge tube. After incubating with 1 mL red blood cell lysis buffer, as described elsewhere (6) (or distilled water) on a slow roller for 20 minutes at room temperature, the reddish supernatant was decanted. The filter paper was then incubated with 100 μL 1× Tris-EDTA (TE) buffer, heating at 95°C for 8 minutes, or 100 μL 5% Chelex-100 (Sigma) in Tris HCl (pH, 8.5) at 56°C for 20 minutes, then at 95°C for 8 minutes (25). After a brief period of vortexing and centrifugation, the supernatant was used for PCR amplification directly.

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Screening of Female Subjects

Based on the screening of DNA mixed from three subjects, any mutant allele within PM and FM range could be detected unequivocally by the nonradioactive Southern blot assay. As summarized in Table 1, one case of FXS was detected in the previous study of 115 girls and none was detected from the current screening of 45 girls. All the remaining non-FXS subjects were not likely a PM carrier.

As depicted in Figure 1, the FXS female subject (lane A) could be detected unequivocally when mixed with two other female carriers in lane X. Two other PM carriers were also discernible via the DNA mixture (lane X), as tested individually in lanes B (78-CGG) and C (122-CGG). They were female relatives of FXS male subjects diagnosed previously, and the exact CGG number of their PM alleles was determined by radioactive PCR sequencing gel electrophoresis (data not shown). The DNA mixture of lane A was applied routinely in each test as a control sample. Actually, an allele as small as 52-CGG at the upper normal range could also be distinguished when mixed with two other normal female samples (lane Y). This implied that detection of any allele within PM and FM range with this screening procedure would not be a problem. Therefore, the CGG triplet of all other samples tested individually in Figure 1 should be accepted as normal.

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Screening of Male Subjects

The presence of FM genes in DNA mixtures of male subjects should be discerned unequivocally by screening with Southern blot assay. As exemplified in the upper portion of Figure 2, the methylated FM gene was clearly present in lanes X, Y, and Z, which were the mixtures of lanes A-B-C, D-E-F, and G-H-I respectively. FXS subjects, such as those in lanes A, D, and G, were then identified specifically by reexamining the original samples in these mixtures individually. With this screening strategy, nine FXS male subjects were diagnosed, including four from a previous study of 216 subjects and five from the current screening of 105 boys (see Table 1).

However, these nine FXS male subjects could also be detected when screened with a much simpler PCR assay. They all repeatedly failed PCR amplification. As illustrated in the lower portion of Figure 2, lanes A-B-C, D-E-F, and G-H-I were the same samples as those in the upper panel. The three FXS subjects (lanes A, D, and G) failed PCR amplification, and the others presented a PCR product with a normal allele with a CGG triplet near 29 repeats or less. The lanes parallel to X in the lower panel contained a sample with the 98-CGG allele, which was included routinely as a positive control. For any male subjects carrying the PM allele with more than 98 CGG-repeats, the PCR yields might have been weaker or undetectable, and need to be evaluated further by Southern blot assay.

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PCR With Dried Blood on Filter Paper

The initial comparison tried to verify an optimal protocol of preparing DNA from a dried blood spot. We found that there was no difference between distilled water and red blood cell lysis buffer used during the initial step to remove PCR inhibitors (data not shown). However, as illustrated in Figure 3, the final treatment at 95°C would influence the PCR yields strikingly. Based on comparison trials of the same blood spots (lanes 1 through 9), soaking them in 5% Chelex-100 chelating resin (see Fig. 3, upper panel) was obviously superior to incubating in 1× TE (see Fig. 3, lower panel). It indicated that Chelex-100 resin would be mandatory in this PCR amplification of a CG-rich template DNA, although it may not be necessary in many other tests. The DNA stored at −20°C was good for PCR for at least 6 months.

In our previous study using DNA prepared routinely from whole blood, we demonstrated that detection of PM genes up to 98-CGG in male subjects was not a problem (24). However, when applied to the DNA isolated from the blood, the PCR yields were much weaker, which increased false-positive results remarkably. This problem could be overcome by using a longer primer to amplify a larger fragment flanking the CGG triplet of the FMR1 gene (data not shown).

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Application of PCR Combined With Blood Spot

Of the 104 boys referred by submitting blood spots for evaluation of FXS, two (Fig. 4A, lanes 4 and 8) failed repeatedly in PCR amplification and one (see Fig. 4A, lane 6) presented a weak result. These samples were reexamined later with Southern blot assay. The boy demonstrating a weak PCR result was not an FXS subject. However, the other two who failed the PCR were identified as FXS subjects with methylated FM, as demonstrated in lane P of Figures 4B and C respectively. They all inherited maternally. One mother (Fig. 4B, lane M) was a PM carrier with 80-CGG (data not shown). The other mother (Fig. 4C, lane M) was phenotypically normal but a carrier of FM mosaic with a hypermethylated allele of 6.2 Kb (approximately 330-CGG) and multiple nonmethylated ones spanning from 3.5 to 3.9 Kb as a smear.

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The predicted FXS prevalence is approximately 1 in 5,000 boys in the general population (1,7,27). Thus the number of FXS subjects in Taiwan is estimated to be 2,000 boys and 1,000 girls. Most of these families should have one or two affected children (24). These data imply that there are at least 1,500 FXS nuclear families in Taiwan. However, more than 90% of these families have not been identified (12,14,24). There should be a notable number of young female carriers unaware of their carrier status and potential genetic consequence. This is apparently the result of insufficient public awareness of this disorder and the inaccessibility of the sophisticated molecular test.

Instead of random screening, preselection of subjects for tests using checklists was cost-effective, and has been indicated to increase the detection rate of FXS as much as threefold (1,10). However, as described, the possibility of a obtaining a false negative using the checklist method cannot be overlooked. An effective screen using the checklist depends on the education level of the general population, the availability of well-trained professionals, and a thorough health care system. Inadequacy of these factors or a defect in coordination can lead to major negligence and make the entire screening effort fruitless. Conversely, wide screening of all subjects with MR, excluding those with known causes, can be feasible and thorough if we can make the molecular test simple and inexpensive.

Blood spots collected on Guthrie card filter paper has been used widely for newborn screening. Recently it was also used for genetic analysis of many diseases by PCR (4,6,11). However, amplification of Guthrie card DNA presents considerable technical obstacles related to high levels of PCR inhibitors in the blood spot (protein, heavy metals, heme, and heme degradation products) and a low copy number of the genomic template. These inhibitors are especially problematic in the PCR amplification of the FMR1 CGG-repeat region because of the inclusion of 10% DMSO to overcome a high CG component, which would inhibit nearly 50% of polymerase activity (9).

In the current study we found that thermal treatment of the punched filter paper in 5% Chelex-100 suspension is a very simple and efficient method to liberate DNA from bloodstained filter paper. Chelex-100 is a chelating resin that has a high affinity for polyvalent metal ions. The Chelex resin is composed of styrene divinylbenzene copolymers containing paired iminodiacetate ions, which act as chelating groups. The presence of Chelex during boiling prevents the degradation of DNA by chelating metal ions that may act as catalysts for the breakdown of DNA at a high temperature with low ionic strength. It is also possible that some PCR inhibitors could be bound to the Chelex bead matrix and removed during this treatment (25).

Of the MR population, boys were generally twice more common than girls. However, only a small fraction of boys with MR had FXS (1,7,12,24,27). In our cumulative studies we screened a total of 321 boys using Southern blot analysis and simple PCR assay. The results of detecting boys with FXS with both assays were concordant. All nine boys with FXS showed consistent failure of PCR amplification. None of them presented a false-negative result (i.e., a PCR product that corresponded to a normal allele).

Except for extremely rare cases reported thus far, the mosaic FXS subjects usually had PM alleles in either very low proportion or quite large in repeat size (16,17,19,22). Most of the mosaic cases demonstrating a normal CGG repeat deleted not only part of the expanded CGG repeat but also part of the flanking sequence, which would also result in PCR amplification failure (16,17). Therefore, a false-negative result appears less likely to be a problem when applying PCR as a primary screening test. Conversely, a false positive would not be a problem either, because all failed cases or those showing an unusually weak PCR result can be reexamined using Southern blot assay. Of a total of 415 boys screened in this and previous studies, as summarized in Table 1, we diagnosed 11 FXS subjects (2.5%). Negative cases are by far the majority and can be excluded by a simple PCR screening test. Thus, we reconfirmed the most suitable value of PCR for this purpose (24,26).

Besides the 12 FXS probands (as shown in Table 1) and their family members, no other subjects with the PM gene was detected in this and previous studies. Based on PCR alone, any male subject carrying the mutant allele could be detected by showing either a band within the PM range or amplification failure. Female subjects carrying not only FM but also PM of all ranges could be identified unequivocally by screening mixed DNA with Southern blot assay. The exact CGG triplet size of the PM allele could be then measured with standard PCR sequencing gel electrophoresis. For most women with an allelic triplet in the normal range, it is not necessary to measure the triplet size exactly because of its lack of clinical significance. We think that this improved Southern blot assay should be less expensive and would improve markedly the cost-effectiveness of screening pregnant women for the mutant FMR1 gene.

In conclusion, developing a simple, inexpensive, and yet reliable molecular test for widely screening the MR population is important. This can be followed by a confirmation test to diagnose FXS. This approach would clarify the long-term enigma of etiology in the family, as well as provide important risk information to other family members. We demonstrated that PCR of a blood spot sample is a simple and effective method of screening boys with MR for FXS. Southern blot assay using mixed DNA is suitable for screening female subjects. Only a very small fraction of these subjects, who were suspected of having FXS by these screening tests, needed to be reexamined by Southern blot individually. This strategy is economical and feasible for wide screening of mentally or developmentally retarded children for FXS.

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Fragile X syndrome; FMR1; Nonradioactive; PCR; Southern blot analysis; Blood spot

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