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Screening for Expanded Alleles of the FMR1 Gene in Blood Spots from Newborn Males in a Spanish Population

2009; Elsevier BV; Volume: 11; Issue: 4 Linguagem: Inglês

10.2353/jmoldx.2009.080173

1943-7811

Isabel Fernández-Carvajal, Paulina Walichiewicz, Xie Xiaosen, Ruiqin Pan, Paul J. Hagerman, Flora Tassone,

Genomic variations and chromosomal abnormalities

Fragile X syndrome, which is caused by expanded CGG repeats of the FMR1 gene, is associated with a broad spectrum of clinical involvement and is the most common inherited form of intellectual disability. Early diagnosis and intervention are likely to lead to improved outcome for children with fragile X syndrome, but such strategies require better estimates of the frequencies of expanded alleles of the FMR1 gene. In this study, we report the results of a newborn screening study of 5267 male blood spots collected from the Northwest region of Spain as part of the national newborn screening program. The blood spots were screened using a rapid polymerase chain reaction-based method that is capable of identifying the presence of all expanded alleles for both males and females. The screened samples included 199 gray zone alleles, 21 premutation alleles, and two full mutation alleles (1 in 2633). The frequency of premutation alleles was three times higher (1 in 251) than the quoted value of 1 in 813 from a Canadian population and is fully consistent with the results of large-scale Israeli screening studies. Our results demonstrate that newborn screening for the presence of expanded FMR1 alleles is an effective means for defining the distribution of expanded FMR1 alleles in newborn populations; as such, this method is suitable for large-scale newborn screening. Fragile X syndrome, which is caused by expanded CGG repeats of the FMR1 gene, is associated with a broad spectrum of clinical involvement and is the most common inherited form of intellectual disability. Early diagnosis and intervention are likely to lead to improved outcome for children with fragile X syndrome, but such strategies require better estimates of the frequencies of expanded alleles of the FMR1 gene. In this study, we report the results of a newborn screening study of 5267 male blood spots collected from the Northwest region of Spain as part of the national newborn screening program. The blood spots were screened using a rapid polymerase chain reaction-based method that is capable of identifying the presence of all expanded alleles for both males and females. The screened samples included 199 gray zone alleles, 21 premutation alleles, and two full mutation alleles (1 in 2633). The frequency of premutation alleles was three times higher (1 in 251) than the quoted value of 1 in 813 from a Canadian population and is fully consistent with the results of large-scale Israeli screening studies. Our results demonstrate that newborn screening for the presence of expanded FMR1 alleles is an effective means for defining the distribution of expanded FMR1 alleles in newborn populations; as such, this method is suitable for large-scale newborn screening. Although fragile X syndrome (FXS) is the most common inherited form of intellectual disability, prior estimates of the frequencies of expanded CGG-repeat alleles have varied widely, ranging from ∼1/2000 to 1/8000, depending on the nature of ascertainment.1Song FJ Barton P Sleightholme V Yao GL Fry-Smith A Screening for fragile X syndrome: a literature review and modelling study.Health Technol Assess. 2003; 7: 1-106Google Scholar Estimates of fragile X syndrome disease prevalence, or FMR1 full mutation (>200 CGG repeats) allele frequency, derived from screening of special education needs populations will likely miss individuals with mild learning disabilities, particularly in females with favorable X-activation ratios.2Hagerman RJ Hagerman PJ Fragile X Syndrome: Diagnosis, Treatment, and Research. 3rd ed. The Johns Hopkins University Press, Baltimore2002Google Scholar Indeed, a higher full mutation allele frequency (1 in ∼2500 females) was reported by Pesso et al,3Pesso R Berkenstadt M Cuckle H Gak E Peleg L Frydman M Barkai G Screening for fragile X syndrome in women of reproductive age.Prenat Diagn. 2000; 20: 611-614Crossref PubMed Scopus (108) Google Scholar who screened a large number of Israeli women in the general population. For premutation alleles (range, 55–200 CGG repeats), allele frequencies are more often estimated through general population screening, where the most solid estimates are for females.1Song FJ Barton P Sleightholme V Yao GL Fry-Smith A Screening for fragile X syndrome: a literature review and modelling study.Health Technol Assess. 2003; 7: 1-106Google Scholar However, there remains some uncertainty regarding the premutation allele frequencies1Song FJ Barton P Sleightholme V Yao GL Fry-Smith A Screening for fragile X syndrome: a literature review and modelling study.Health Technol Assess. 2003; 7: 1-106Google Scholar,3Pesso R Berkenstadt M Cuckle H Gak E Peleg L Frydman M Barkai G Screening for fragile X syndrome in women of reproductive age.Prenat Diagn. 2000; 20: 611-614Crossref PubMed Scopus (108) Google Scholar4Rousseau F Rouillard P Morel ML Khandjian EW Morgan K Prevalence of carriers of premutation-size alleles of the FMR1 gene–and implications for the population genetics of the fragile X syndrome.Am J Hum Genet. 1995; 57: 1006-1018PubMed Google Scholar5Crawford DC Meadows KL Newman JL Taft LF Scott E Leslie M Shubek L Holmgreen P Yeargin-Allsopp M Boyle C Sherman SL Prevalence of the fragile X syndrome in African-Americans.Am J Med Genet. 2002; 110: 226-233Crossref PubMed Scopus (125) Google Scholar6Dombrowski C Lévesque S Morel ML Rouillard P Morgan K Rousseau F Premutation and intermediate-size FMR1 alleles in 10572 males from the general population: loss of an AGG interruption is a late event in the generation of fragile X syndrome alleles.Hum Mol Genet. 2002; 11: 371-378Crossref PubMed Scopus (266) Google Scholar7Toledano-Alhadef H Basel-Vanagaite L Magal N Davidov B Ehrlich S Drasinover V Taub E Halpern G Ginott N Shohat M Fragile-X carrier screening and the prevalence of premutation and full-mutation carriers in Israel.Am J Hum Genet. 2001; 69: 351-360Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar due in part to lingering issues of ascertainment bias,8Hagerman PJ The fragile X prevalence paradox.J Med Genet. 2008; 45: 498-499Crossref PubMed Scopus (274) Google Scholar but also to real frequency differences across ethnic and regional populations. For example, in the screens of males and females in Eastern Canada, allele frequencies were estimated to be ∼1/800 males and ∼1/260 females.4Rousseau F Rouillard P Morel ML Khandjian EW Morgan K Prevalence of carriers of premutation-size alleles of the FMR1 gene–and implications for the population genetics of the fragile X syndrome.Am J Hum Genet. 1995; 57: 1006-1018PubMed Google Scholar,6Dombrowski C Lévesque S Morel ML Rouillard P Morgan K Rousseau F Premutation and intermediate-size FMR1 alleles in 10572 males from the general population: loss of an AGG interruption is a late event in the generation of fragile X syndrome alleles.Hum Mol Genet. 2002; 11: 371-378Crossref PubMed Scopus (266) Google Scholar However, in the Israeli studies,3Pesso R Berkenstadt M Cuckle H Gak E Peleg L Frydman M Barkai G Screening for fragile X syndrome in women of reproductive age.Prenat Diagn. 2000; 20: 611-614Crossref PubMed Scopus (108) Google Scholar,7Toledano-Alhadef H Basel-Vanagaite L Magal N Davidov B Ehrlich S Drasinover V Taub E Halpern G Ginott N Shohat M Fragile-X carrier screening and the prevalence of premutation and full-mutation carriers in Israel.Am J Hum Genet. 2001; 69: 351-360Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar the frequency of premutation alleles in women is closer to ∼1/130.7Toledano-Alhadef H Basel-Vanagaite L Magal N Davidov B Ehrlich S Drasinover V Taub E Halpern G Ginott N Shohat M Fragile-X carrier screening and the prevalence of premutation and full-mutation carriers in Israel.Am J Hum Genet. 2001; 69: 351-360Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar,9Hagerman RJ Hagerman PJ The fragile X premutation: into the phenotypic fold.Curr Opin Genet Dev. 2002; 12: 278-283Crossref PubMed Scopus (213) Google Scholar By contrast, in an Asian (Taiwanese) population, the frequency of premutation alleles in males was reported to be much lower (∼1/1670).10Tzeng C Tsai L Hwu W Lin S Chao M Jong Y Chu S Chao W Lu C Prevalence of the FMR1 mutation in Taiwan assessed by large-scale screening of newborn boys and analysis of DXS548-FRAXAC1 haplotype.Am J Med Genet A. 2005; 133: 37-43Crossref Scopus (55) Google Scholar Within the past decade, there has been increasing recognition of the breadth of phenotypes associated with expanded FMR1 alleles, especially in the premutation range.11Hagerman PJ Hagerman RJ The fragile-X premutation: a maturing perspective.Am J Hum Genet. 2004; 74: 805-816Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar In particular, two disorders specific to the premutation range have been described: primary ovarian insufficiency (formerly premature ovarian failure), which occurs in approximately 20% of females with the premutation, as compared with 1% of the general population12Allingham-Hawkins DJ Babul-Hirji R Chitayat D Holden JA Yang KT Lee C Hudson R Gorwill H Nolin SL Glicksman A Jenkins EC Brown WT Howard-Peebles PN Becchi C Cummings E Fallon L Seitz S Black SH Vianna-Morgante AM Costa SS Otto PA Mingroni-Netto RC Murray A Webb J MacSwinney F Dennis N Jacobs PA Syrrou M Georgiou I Patsalis PC Giovannucci Uzielli ML Guarducci S Lapi E Cecconi A Ricci U Ricotti G Biondi C Scarselli B Vieri F Fragile X premutation is a significant risk factor for premature ovarian failure: the international collaborative POF in fragile X study—preliminary data.Am J Med Genet. 1999; 83: 322-325Crossref PubMed Scopus (411) Google Scholar; and the late-adult-onset neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome.13Amiri K Hagerman RJ Hagerman PJ Fragile X-associated tremor/ataxia syndrome: an aging face of the fragile X gene.Arch Neurol. 2008; 65: 19-25Crossref PubMed Scopus (62) Google Scholar14Hagerman PJ Hagerman RJ Fragile X-associated tremor/ataxia syndrome–an older face of the fragile X gene.Nat Clin Pract Neurol. 2007; 3: 107-112Crossref PubMed Scopus (52) Google Scholar15Berry-Kravis E Potanos K Weinberg D Zhou L Goetz CG Fragile X-associated tremor/ataxia syndrome: clinical features, genetics, and testing guidelines.Mov Disord. 2007; 22: 2018-2030Crossref PubMed Scopus (264) Google Scholar16Willemsen R Mientjes E Oostra BA FXTAS: a progressive neurologic syndrome associated with Fragile X premutation.Curr Neurol Neurosci Rep. 2005; 5: 405-410Crossref PubMed Scopus (23) Google Scholar17Hagerman RJ Leehey M Heinrichs W Tassone F Wilson R Hills J Grigsby J Gage B Hagerman PJ Intention tremor, parkinsonism, and generalized brain atrophy in male carriers of fragile X.Neurology. 2001; 57: 127-130Crossref PubMed Scopus (784) Google Scholar Therefore, it is critical for understanding the broader societal impact of the fragile X family of disorders to obtain an accurate estimate of allele frequencies for both premutation and full mutation alleles, in diverse ethnic and geographical populations. Previously, large-scale screening of the newborn populations has been hampered by the lack of a rapid, inexpensive screening test that is capable of using blood spots to register all expanded (premutation and full mutation) FMR1 alleles, for both males and females. However, with the recent development of such a test,18Tassone F Pan R Amiri K Taylor AK Hagerman PJ A rapid polymerase chain reaction-based screening method for identification of all expanded alleles of the fragile X (FMR1) gene in newborn and high-risk populations.J Mol Diagn. 2008; 10: 43-49Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar screening of blood spots to detect all expanded alleles is now feasible, which in turn permits large-scale screening of newborn populations. Here we report the results of an anonymous blood spot screening of 5267 newborns (males) collected across the region of Castilla y León, Spain. The screened samples included 199 gray zone alleles (1 in 26; 95% confidence interval, 1/23–1/30), 21 premutation alleles (1 in 251; 95% confidence interval, 1/164–1/385), and two full mutation alleles (1 in 2633; 95% confidence interval, 1/714–1/10,000), which is in line with estimates based on the Israeli population screens.8Hagerman PJ The fragile X prevalence paradox.J Med Genet. 2008; 45: 498-499Crossref PubMed Scopus (274) Google Scholar Blood spots used in the current study were obtained from an existing archive of bloodspots representing routine newborn screening at designated hospitals of the region of Castilla y León (nine different provinces), with bloodspots routinely collected between the third and fifth day of life. Samples were collected on 903 specimen collection paper (Whatman, Inc., NJ). The dried blood spots were received at the Metabolic Diseases Laboratory of the Institute of Biology and Molecular Genetics of the University of Valladolid, reference laboratory for the newborn screening of metabolic and genetic diseases (hypothyroidism, phenylketonuria, and cystic fibrosis) in the Castilla y León region of Spain. For the current study, a total of 5267 samples (labeled as male), representing consecutive samples received during the first 6 months of 2007, were used. The samples were stripped of all identifiers, patient codes, and/or accession numbers at Institute of Biology and Molecular Genetics, preserving only stated sex and ethnicity of the donor, to ensure that the samples were not traceable to the donors; thus, only completely de-identified samples were sent to the M.I.N.D. Institute molecular laboratory at the University of California, Davis, for genotyping. On reaching the University of California, Davis, each sample was assigned a local accession number. The majority of the subjects were Caucasian and Spanish from the catchments region. Based on regional census figures, approximately 4% of the population was found to be foreign to the catchment region. All protocols involving human subjects were performed under an existing Institutional Review Board for anonymous screening. A disk 1.2 mm in diameter was removed by punch from each dried blood spot and was placed in a clean 0.5-μl polymerase chain reaction (PCR) tube. Two hundred microliters of Qiagen RBC lysis solution (Qiagen, Valencia, CA) was added to the tube followed by incubation for 5 minutes at room temperature. The supernatant was removed, including any excess liquid adhering to the disk, and the disk was left to dry for several minutes before the PCR master mix (FastStart PCR kit; Roche Diagnostics, Indianapolis, IN) was added to each sample. Master mix containing primers c and f19Fu YH Kuhl DPA Pizzuti A Pieretti M Sutcliffe JS Richards S Verkert AJMH Holden JJA Fenwick Jr, RG Warren ST Oostra BA Nelson DL Caskey CT Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox.Cell. 1991; 67: 1047-1058Abstract Full Text PDF PubMed Scopus (1770) Google Scholar was prepared and used according to the manufacturer's instructions; primers c and f yield amplicons of 221 + 3 × CGG repeat number bp. Using the applied Biosystems 9700 thermocycler, the PCR conditions were: 10 minutes initial denaturation at 95°C, 10 cycles of 95°C for 35 seconds, 64°C for 35 seconds, 68°C for 4 minutes; followed by 25 cycles of 95°C for 35 seconds, 64°C for 35 seconds, 68°C for 4 minutes (with 20 seconds increase each cycle); followed by a final extension of 10 minutes at 68°C. The PCR products were stored at 4°C until analysis or were immediately analyzed using the Qiaxcel genetic analyzer (Qiagen), which utilizes a preassembled cartridge (cartridge type Qiaxcel DNA high-resolution cartridge, injection time 10 seconds, Qiaxcel DNA size marker 100 bp, 3 kb) to simultaneously run samples and collect data. Using conditions as recommended by the manufacturer, Figure 1 shows PCR products derived from 14 bloodspots using the Qiaxcel capillary system. Data were analyzed on a PC running BioCalculator software, which saves the data collected by the unit and allows CGG repeat size analysis after collection. Using DNA size marker as indicated in Figure 1, alleles were classified as normal ( 200 CGG repeats). Samples that did not yield a band after the first round PCR with primers c and f were subjected to a secondary CGG-primer-based PCR screening as previously described.18Tassone F Pan R Amiri K Taylor AK Hagerman PJ A rapid polymerase chain reaction-based screening method for identification of all expanded alleles of the fragile X (FMR1) gene in newborn and high-risk populations.J Mol Diagn. 2008; 10: 43-49Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar Figure 2 shows PCR products obtained from secondary screening of blood spots using the chimeric CGG-targeted primer for the detection of large CGG repeat expansions run on a 2% agarose gel. An extensive smear is produced with the chimeric primer when an expanded allele is present, as shown in lanes 1 and 4 for the two full mutation samples, while no smear is visible in the presence of a normal allele as shown in lanes 2 and 3. Blood spots that underwent the CGG primer-based PCR screening were washed two times for 15 minutes in 1 ml of ddH2O and used immediately in a PCR reaction.18Tassone F Pan R Amiri K Taylor AK Hagerman PJ A rapid polymerase chain reaction-based screening method for identification of all expanded alleles of the fragile X (FMR1) gene in newborn and high-risk populations.J Mol Diagn. 2008; 10: 43-49Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar PCR products were run on a 2% agarose gel. Isolation of DNA from blood spots was performed on the two full mutation samples to rule out the possibility that the two samples were indeed large premutation alleles (see Results). DNA was obtained by using a 1 × 3 mm punch bloodspot directly into a 500-μl Eppendorf tube containing 60 μl of cell lysis solution (Qiagen) and 3 μl of 20 mg/ml proteinase K (Roche Diagnostics, Indianapolis, IN). Spots were incubated at 55°C overnight and then treated with 2.5 μl of RNase A (5 mg/ml) at 37°C for 15 minutes. Proteins were precipitated by adding 200 μl of protein precipitation solution (Qiagen). The solution was spun down at 12,000 rpm for 5 minutes and the DNA was precipitated from the supernatant with 1 volume of isopropanol and 1 μl of glycogen solution (20 mg/ml). The DNA pellet was washed with 70% ethanol, dissolved in DNA hydration solution (Qiagen), and stored at −20°C until use.Figure 2Detection of large CGG repeat expansions using a CGG-targeting PCR primer. Lanes 1 and 4, the two full-mutation alleles identified in this study; lanes 2 and 3, normal alleles identified in this study; lanes 5 and 6, normal and full mutation controls; lane 7, negative control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Of the 5267 total male blood spots screened, 32 (0.6%) showed two main bands using two different sets of primers and were not analyzed further. Although some of those samples may have been mislabeled with respect to the sex of the infant, one would expect that 5 to 10 samples would have come from Klinefelter subjects. Given the anonymous nature of the sample, no follow-up was possible. It should be noted that all bands in those 32 samples were in the normal range (data not shown). Some of the blood spots were run twice if they failed to amplify the first time. Of the remaining 5235 alleles, 5013 had a CGG repeat number within the normal range; 199 were gray zone alleles (1 in 26; 95% confidence interval, 1/23–1/30), 21 were premutation alleles (1 in 251; 95% confidence interval, 1/164–1/385), and two were presumptive full mutation alleles (1 in 2633; 95% confidence interval, 1/714–1/10,000) (Table 1, Figure 3). With the CGG primer approach, a smear is detectable also in the presence of a large premutation allele. Therefore, to rule out the possibility that the two samples harbored alleles in the upper premutation range, which we failed to amplify using the first PCR step (with primer c and f), we mixed the DNA isolated from the two blood spots separately with the same amount of DNA from a known premutation carrier harboring premutation alleles of 144 and 185 CGG repeats. After PCR with primer c and f, only the two premutation bands corresponding to the known premutation carrier were detected and visualized on the agarose gel (data not shown). These findings reinforce our position that two full mutation alleles were detected.Table 1Allele Frequencies Within the Screened SampleAllele class (range)Number of samplesFrequency*Frequencies are based on the total sample size, which includes the 32 samples ejected for sizing.95% confidence intervalNormal (6-44 CGG)5013Intermediate (45-54 CGG)1991/261/23–1/30Premutation (55-200 CGG)211/2511/164–1/385Full mutation (>200 CGG)21/26331/714–1/10,000Samples with two bands32Total5267* Frequencies are based on the total sample size, which includes the 32 samples ejected for sizing. Open table in a new tab Whereas the frequency of gray zone alleles (1/26) is not significantly different from literature values for the Canadian cohorts,6Dombrowski C Lévesque S Morel ML Rouillard P Morgan K Rousseau F Premutation and intermediate-size FMR1 alleles in 10572 males from the general population: loss of an AGG interruption is a late event in the generation of fragile X syndrome alleles.Hum Mol Genet. 2002; 11: 371-378Crossref PubMed Scopus (266) Google Scholar,20Dawson AJ Chodirker BN Chudley AE Frequency of FMR1 premutations in a consecutive newborn population by PCR screening of Guthrie blood spots.Biochem Mol Med. 1995; 56: 63-69Crossref PubMed Scopus (47) Google Scholar the value of 1/251 for premutation alleles is three times larger than the value of 1/813 previously reported in males by Dombrowski et al,6Dombrowski C Lévesque S Morel ML Rouillard P Morgan K Rousseau F Premutation and intermediate-size FMR1 alleles in 10572 males from the general population: loss of an AGG interruption is a late event in the generation of fragile X syndrome alleles.Hum Mol Genet. 2002; 11: 371-378Crossref PubMed Scopus (266) Google Scholar and is in close agreement with the expectation derived from the female Israeli cohorts.8Hagerman PJ The fragile X prevalence paradox.J Med Genet. 2008; 45: 498-499Crossref PubMed Scopus (274) Google Scholar The premutation alleles ranged in size from 55 to 77 CGG repeats; thus, all were toward the small end of the premutation range, again consistent with previous observations.21Jacquemont S Leehey MA Hagerman RJ Beckett LA Hagerman PJ Size bias of fragile X premutation alleles in late-onset movement disorders.J Med Genet. 2006; 43: 804-809Crossref PubMed Scopus (105) Google Scholar Using the current PCR screening method, we could not establish whether any of the 21 identified premutation subjects were actually size and/or methylation mosaic individuals; that is, that they were also carrying a full mutation allele that we did not detect. Were any one of these 21 cases actually an undetected mosaic, the current study would have underestimated the true frequency of full mutation alleles, which would then yield 1/1756 for the frequency of full mutation alleles (ie, for three full mutations, not two), which is much higher than other estimates for full mutation frequency. The presence of mosaics and full mutation alleles can be established by Southern blot analysis; however, given the limited amount of DNA that can be extracted from the filters, such an analysis is not possible. Methylation analysis (ie, using long PCR in combination with bisulfite modification) of the promoter region of the FMR1 region could also be used to identify methylated (and hence full mutation) alleles; again, the method requires much larger quantities of DNA than can be obtained from the filters. We are currently attempting to improve the sensitivity of the current PCR approach. In this work we have used a newly developed methodology to answer a number of questions regarding FMR1 allele frequency, size distribution, and feasibility of newborn screening on a large scale. Previous studies, aimed at establishing allele frequencies in different populations, have yielded dissimilar results mainly due to screening selection bias. Moreover, no studies have demonstrated the practicability of large-scale population screening for all expanded FMR1 alleles (for both males and females) from newborn blood spots. Our current findings underscore the feasibility of large population screening, as for example newborn screening. The availability of an easy, rapid, and inexpensive test may facilitate the introduction of newborn screening for FMR1 mutations into the established public health infrastructure for existing newborn screening programs, and thus early childhood developmental intervention strategies could be enhanced for children who are diagnosed with FMR1 mutations. One important outcome from this study is that the frequency of premutation alleles (1/251) in an unbiased sample of male newborns in Spain appears to be three times higher than the frequency most often quoted in the literature (1/813).6Dombrowski C Lévesque S Morel ML Rouillard P Morgan K Rousseau F Premutation and intermediate-size FMR1 alleles in 10572 males from the general population: loss of an AGG interruption is a late event in the generation of fragile X syndrome alleles.Hum Mol Genet. 2002; 11: 371-378Crossref PubMed Scopus (266) Google Scholar This higher frequency of premutation alleles has important implications for the prevalence in Spain of developmental and behavioral problems (eg, attention deficit hyperactivity disorder and autism spectrum disorders22Farzin F Perry H Hessl D Loesch D Cohen J Bacalman S Gane L Tassone F Hagerman P Hagerman R Autism spectrum disorders and attention-deficit/hyperactivity disorder in boys with the fragile X premutation.J Dev Behav Pediatr. 2006; 27: S137-S144Crossref PubMed Scopus (280) Google Scholar,23Goodlin-Jones BL Tassone F Gane LW Hagerman RJ Autistic spectrum disorder and the fragile X premutation.J Dev Behav Pediatr. 2004; 25: 392-398Crossref PubMed Scopus (115) Google Scholar) that are frequently observed in children who carry premutation alleles, and for fragile X-associated neuroendocrine (fragile X primary ovarian insufficiency) and neurodegenerative (fragile X-associated tremor/ataxia syndrome) disorders among adult carriers. However, the current results speak more broadly of the potential for significant differences in allele frequencies across different populations. It should be kept in mind that founder effects could contribute to the discrepancies in allele frequencies observed in different population groups. The estimated frequency of full mutation alleles in this Spanish cohort of 5267 males, 1/2633, is in line with results from recent population screening studies (see1Song FJ Barton P Sleightholme V Yao GL Fry-Smith A Screening for fragile X syndrome: a literature review and modelling study.Health Technol Assess. 2003; 7: 1-106Google Scholar,8Hagerman PJ The fragile X prevalence paradox.J Med Genet. 2008; 45: 498-499Crossref PubMed Scopus (274) Google Scholar); however, since this estimate was based on only two samples, the confidence interval is quite large. Assuming that the observed frequency is correct, a sample size of approaching 50,000 would be needed to reduce the upper limit of the confidence interval to within about 25% of the mean value. Previously reported frequencies for full mutation alleles (∼1 in 2500 to 1 in 8000)3Pesso R Berkenstadt M Cuckle H Gak E Peleg L Frydman M Barkai G Screening for fragile X syndrome in women of reproductive age.Prenat Diagn. 2000; 20: 611-614Crossref PubMed Scopus (108) Google Scholar,5Crawford DC Meadows KL Newman JL Taft LF Scott E Leslie M Shubek L Holmgreen P Yeargin-Allsopp M Boyle C Sherman SL Prevalence of the fragile X syndrome in African-Americans.Am J Med Genet. 2002; 110: 226-233Crossref PubMed Scopus (125) Google Scholar,24Morton JE Bundey S Webb TP MacDonald F Rindl PM Bullock S Fragile X syndrome is less common than previously estimated.J Med Genet. 1997; 34: 1-5Crossref PubMed Scopus (76) Google Scholar25Turner G Webb T Wake S Robinson H Prevalence of fragile X syndrome.Am J Med Genet. 1996; 64: 196-197Crossref PubMed Scopus (599) Google Scholar26de Vries BB van den Ouweland AM Mohkamsing S Duivenvoorden HJ Mol E Gelsema K van Rijn M Halley DJ Sandkuijl LA Oostra BA Tibben A Niermeijer MF Screening and diagnosis for the fragile X syndrome among the mentally retarded: an epidemiological and psychological survey: Collaborative Fragile X Study Group.Am J Hum Genet. 1997; 61: 660-667Abstract Full Text PDF PubMed Scopus (184) Google Scholar have generally been based on screening of target populations with significant developmental problems extrapolated to the general population. Such projections tend to underestimate the disease prevalence, and hence allele frequencies, since individuals with only mild or no apparent learning difficulties would be excluded. Interestingly, a recent report8Hagerman PJ The fragile X prevalence paradox.J Med Genet. 2008; 45: 498-499Crossref PubMed Scopus (274) Google Scholar that used an average of the known frequency for premutation females (1/126)3Pesso R Berkenstadt M Cuckle H Gak E Peleg L Frydman M Barkai G Screening for fragile X syndrome in women of reproductive age.Prenat Diagn. 2000; 20: 611-614Crossref PubMed Scopus (108) Google Scholar,7Toledano-Alhadef H Basel-Vanagaite L Magal N Davidov B Ehrlich S Drasinover V Taub E Halpern G Ginott N Shohat M Fragile-X carrier screening and the prevalence of premutation and full-mutation carriers in Israel.Am J Hum Genet. 2001; 69: 351-360Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar to estimate the expected frequencies of full mutation and (male) premutation alleles yielded frequencies of 1/2355 (males and females) for full mutation alleles and 1/282 for premutation alleles (male); remarkably close to the observations of the current study. The current distribution of FMR1 alleles indicates that the most common alleles in this population are 29 and 30 CGG repeats (Figure 3). Previous studies have indicated that the most common alleles vary of few repeats in different populations including 28 CGG repeats,20Dawson AJ Chodirker BN Chudley AE Frequency of FMR1 premutations in a consecutive newborn population by PCR screening of Guthrie blood spots.Biochem Mol Med. 1995; 56: 63-69Crossref PubMed Scopus (47) Google Scholar 29 CGG repeats in Taiwan,27Wang YC Li C Lin ML Lin WH Li SY Molecular diagnosis of fragile X syndrome and distribution of CGG repeats in the FMR-1 gene in Taiwanese.J Formos Med Assoc. 2000; 99: 402-407PubMed Google Scholar 30 CGG repeats,28Snow K Doud LK Hagerman R Pergolizzi RG Erster SH Thibodeau SN Analysis of a CGG sequence at the FMR-1 locus in fragile X families and in the general population.Am J Hum Genet. 1993; 53: 1217-1228PubMed Google Scholar 29 and 30 CGG repeats in Spain,29Milà M Kruyer H Glover G Sánchez A Carbonell P Castellví-Bel S Volpini V Rosell J Gabarrón J López I Villa M Ballesta F Estivill X Molecular analysis of the (CGG)n expansion in the FMR-1 gene in 59 Spanish fragile X syndrome families.Hum Genet. 1994; 94: 395-400Crossref PubMed Scopus (22) Google Scholar and 29 CGG repeats in a Chinese population.30Chen TA Lu XF Che PK Ho WK Variation of the CGG repeat in FMR-1 gene in normal and fragile X Chinese subjects.Ann Clin Biochem. 1997; 34: 517-520Crossref PubMed Scopus (16) Google Scholar However, it should be noted that small differences among studies (1–2 CGG repeats) may be a consequence of experimental errors in various labs in the absence of single repeat precision and sequenced CGG standards. In a previous screen for expanded FMR1 alleles using blood spots, Rife et al31Rife M Badenas C Mallolas J Jiménez L Cervera R Maya A Glover G Rivera F Milà M Incidence of fragile X in 5,000 consecutive newborn males.Genet Test. 2003; 7: 339-343Crossref PubMed Scopus (50) Google Scholar presented a general population screening of 4937 newborn blood spots from males collected throughout the Catalonia region in Spain. The screening yielded a frequency of 1/2466 full mutation males and 1/1233 premutation carriers. Whereas the frequency of full mutation alleles observed in the Rife et al study is in agreement with previous studies,32Crawford DC Acuna JM Sherman SL FMR1 and the fragile X syndrome: human genome epidemiology review.Genet Med. 2001; 3: 359-371Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar,33Patsalis PC Sismani C Hettinger JA Boumba I Georgiou I Stylianidou G Anastasiadou V Koukoulli R Pagoulatos G Syrrou M Molecular screening of fragile X (FRAXA) and FRAXE mental retardation syndromes in the Hellenic population of Greece and Cyprus: incidence, genetic variation, and stability.Am J Med Genet. 1999; 84: 184-190Crossref PubMed Scopus (45) Google Scholar their frequency of premutation alleles was lower than the value of 1/813 reported in the Canadian study6Dombrowski C Lévesque S Morel ML Rouillard P Morgan K Rousseau F Premutation and intermediate-size FMR1 alleles in 10572 males from the general population: loss of an AGG interruption is a late event in the generation of fragile X syndrome alleles.Hum Mol Genet. 2002; 11: 371-378Crossref PubMed Scopus (266) Google Scholar; a paradoxical result both in terms of the expectation from the genetic model for transmission,1Song FJ Barton P Sleightholme V Yao GL Fry-Smith A Screening for fragile X syndrome: a literature review and modelling study.Health Technol Assess. 2003; 7: 1-106Google Scholar,8Hagerman PJ The fragile X prevalence paradox.J Med Genet. 2008; 45: 498-499Crossref PubMed Scopus (274) Google Scholar and given the fact that both the Rife et al study and the current investigation used newborn samples from Spain, albeit from different regions. This discrepancy could be explained by the genotype methodology used by this group. Two additional studies20Dawson AJ Chodirker BN Chudley AE Frequency of FMR1 premutations in a consecutive newborn population by PCR screening of Guthrie blood spots.Biochem Mol Med. 1995; 56: 63-69Crossref PubMed Scopus (47) Google Scholar,34Holden JJ Chalifoux M Wing M Julien-Inalsingh C White BN A rapid, reliable, and inexpensive method for detection of di- and trinucleotide repeat markers and disease loci from dried blood spots.Am J Med Genet. 1996; 64: 313-318Crossref PubMed Scopus (16) Google Scholar reported screens of newborns for fragile X using blood spots. Dawson et al20Dawson AJ Chodirker BN Chudley AE Frequency of FMR1 premutations in a consecutive newborn population by PCR screening of Guthrie blood spots.Biochem Mol Med. 1995; 56: 63-69Crossref PubMed Scopus (47) Google Scholar analyzed cohorts of 1000 males and 1000 females; however, the number of samples successfully analyzed in that study was quite small, owing to a high (∼25%) failure rate on PCR. In the second study, Holden et al34Holden JJ Chalifoux M Wing M Julien-Inalsingh C White BN A rapid, reliable, and inexpensive method for detection of di- and trinucleotide repeat markers and disease loci from dried blood spots.Am J Med Genet. 1996; 64: 313-318Crossref PubMed Scopus (16) Google Scholar reported blood spot screening results for 2050 newborn males; however, their study also suffered from difficulty with PCR amplification for alleles larger than ∼75 CGG repeats. Finally, a recent study by Saul et al35Saul RA Friez M Eaves K Stapleton GA Collins JS Schwartz CE Stevenson RE Fragile X syndrome detection in newborns-pilot study.Genet Med. 2008; 10: 714-719Abstract Full Text Full Text PDF PubMed Google Scholar reported an equal frequency of both premutation and full mutation (1:730), which most likely reflect the low number of subject screened (1459 newborn blood spots). The current pilot screening study for expanded FMR1 alleles, using newborn blood spots, demonstrates the applicability of our methodology to large-scale newborn screening. The methodological approach satisfies the principal requirements for a screening tool: that it reliably detect expanded alleles at least through the upper portion of the premutation range; that it be both rapid and cost effective (ie, less than $3 for reagents per test); and that it works effectively with the small amount of DNA typically yielded from a portion of a single blood spot, the principal resource for newborn screening. It should be noted that although the CGG-primer-based screening method is capable of registering large expansions in the full mutation range, it is not designed to determine the actual sizes of full mutation alleles. Rather, full mutation alleles flagged by the screening method would be sized by Southern blot analysis as part of a newborn follow-up assessment and early intervention program. In this context, newborn screening would provide parents the opportunity to learn about their child's fragile X status and their own reproductive risk, in addition to other likely benefits provided by accessing early intervention programs, which have been shown to positively influence child development and provide support to families of children with fragile X syndrome.36Bailey Jr, DB Newborn screening for fragile X syndrome.Ment Retard Dev Disabil Res Rev. 2004; 10: 3-10Crossref PubMed Scopus (58) Google Scholar37Bailey Jr, DB Skinner D Warren SF Newborn screening for developmental disabilities: reframing presumptive benefit.Am J Public Health. 2005; 95: 1889-1893Crossref PubMed Scopus (69) Google Scholar38Bailey Jr, DB Beskow LM Davis AM Skinner D Changing perspectives on the benefits of newborn screening.Ment Retard Dev Disabil Res Rev. 2006; 12: 270-279Crossref PubMed Scopus (48) Google Scholar This work is dedicated to the memory of Matteo.