Fragile X-associated tremor/ataxia syndrome (FXTAS) is generally considered to be uncommon in older female carriers of premutation alleles (55-200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene; however, neither prevalence, nor the nature of the clinical phenotype, has been well characterized in female carriers. In this study, we evaluated 146 female carriers (mean, 42.3 years; range, 20-75 years) with and without core features of FXTAS (tremor; gait ataxia), and 69 age-matched controls (mean, 45.8 years; range, 21-78 years). Compared with controls, carriers with definite or probable FXTAS had greater medical co-morbidity, with increased prevalence of thyroid disease (P ¼ 0.0096), hypertension (P ¼ 0.0020), seizures (P ¼ 0.0077), peripheral neuropathy (P ¼ 0.0040), and fibromyalgia (P ¼ 0.0097), in addition to the typical symptoms of FXTAS-tremor (P < 0.0001) and ataxia (P < 0.0001). The non-FXTAS premutation group had more complaints of chronic muscle pain (P ¼ 0.0097), persistent paraesthesias in extremities (P < 0.0001), and history of tremor (P < 0.0123) than controls. The spectrum of clinical involvement in female carriers with FXTAS is quite broad, encompassing a number of medical co-morbidities as well as the core movement disorder. The remarkable degree of thyroid dysfunction (17% in the non-FXTAS group and 50% in the FXTAS group) warrants consideration of thyroid function studies in all female premutation carriers, particularly those with core features of FXTAS. ß

Fragile X syndrome , the most common inherited cause of intellectual impairment and the most common single gene associated with autism , generally occurs for fragile X mental retardation 1 (FMR1) alleles that exceed 200 CGG repeats (full-mutation range). Currently, there are no unbiased estimates of the number of full-mutation FMR1 alleles in the general population; a major obstacle is the lack of an effective screening tool for expanded FMR1 alleles in large populations. We have developed a rapid polymerase chain reaction (PCR)-based screening tool for expanded FMR1 alleles. The method utilizes a chimeric PCR primer that targets randomly within the expanded CGG region , such that the presence of a broad distribution of PCR products represents a positive result for an expanded allele. The method is applicable for screening both males and females and for allele sizes throughout the premutation (55 to 200 CGG repeats) and full-mutation ranges. Furthermore , the method is capable of rapid detection of expanded alleles using as little as 1% of the DNA from a single dried blood spot. The methodology presented in this work is suitable for screening large populations of newborn or those at high risk (eg , autism , premature ovarian failure , ataxia , dementia) for expanded FMR1 alleles. The test described herein costs less than $5 per sample for materials; with suitable scale-up and automation , the cost should approach $1 per sample. (J Mol

(CGG) n repeat expansion in the FMR1 gene is associated with fragile X syndrome and other disorders. Current methods for FMR1 molecular testing rely on Southern blot analysis to detect expanded alleles too large to be PCR-amplified and to identify female homozygous alleles that often confound interpretations of PCR data. A novel , single-tube CGG repeat primed FMR1 PCR technology was designed with two genespecific primers that flank the triplet repeat region, as well as a third primer that is complementary to the (CGG) n repeat. This PCR was evaluated with 171 unique DNA samples , including a blinded set of 146 clinical specimens. The method detected all alleles reported by Southern blot analysis , including full mutations in 66 clinical samples and comprised up to 1300 CGG. Furthermore , a blinded cohort of 42 female homozygous and heterozygous specimens, including 21 with full mutation alleles , was resolved with 100% accuracy. Last , AGG interrupter sequences, which may influence the risk of (CGG) n expansion in the children of some carriers , were each correctly identified in 14 male and female clinical samples as referenced to DNA sequencing. As a result , this PCR provides robust detection of expanded alleles and resolves allele zygosity , thus minimizing the number of samples that require Southern blot analysis and producing more comprehensive FMR1 genotyping data than other methods. Expansion of cytosine-guanine-guanine (CGG) triplet repeats in the 5Ј-untranslated region of the fragile X mental retardation 1 (FMR1, NM_002024.4) gene is associated with several disorders, including fragile X syndrome, fragile X-associated tremor/ataxia syndrome, and fragile X-associated primary ovarian insufficiency. [1][2][3][4] Patients with the FMR1 full mutation (Ͼ200 CGG repeats) may be affected by a range of neurological, psychiatric, or emotional challenges, including mental retardation and/or autism.5 Deficits in development and particularly in attention and social communication have also been noted for many children with the FMR1 premutation. Moreover, premutation carriers (55 to 200 CGG repeats) are known to be at risk for fragile X-associated primary ovarian insufficiency and fragile X-associated tremor/ataxia syndrome, and some of these individuals may present additional complications, such as hypothyroidism and fibromyalgia.6 As a result, FMR1 disorders are linked to a range of clinical conditions, necessitating testing patients at different times during their life span. 7Fragile X syndrome molecular diagnosis is usually based on quantification of the (CGG) n repeat elements and the assessment of the methylation state of expanded alleles.5 Although PCR is the preferred approach to determine the (CGG) n repeat length of FMR1 alleles, typically only alleles with less than 100 to 150 CGG have

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.