Incidence: 1/3500 males; 1/6000 females; all ethnic groups
Phenotype: Abnormal facies, macroorchidism, MR
Karyotype: FRAXA at Xq27.3 in 50% of mitoses
INHERITANCE PATTERN: X-linked...BUT
1. 20% of obligatory carrier males are phenotypically and chromosomally normal (Normal
2. 1/3 of heterozygous females are clinicaly affected; MR only if mutation inherited from a
carrier mother, not from the father.
3. Penetrance (risk of MR) is a function of position in the pedigree, and appears to increase in successive generations (anticipation),------the SHERMAN PARADOX
Polymorphic CGG repeat in first exon
Normal: 6 to 52; mode 30; transcriptionally active
Premutation: 50 to 230; transcriptionally active expands when transmitted by the mother; prob. proportional to size
Full mutation: >230; hypermethylated; transcriptionally off
Warren ST, Nelson DL: Advances in Molecular Analysis of Fragile X Syndrome. JAMA 271: 536-542 (16 Feb. 1994).
ST: Trinucleotide repeat instability: the example of fragile X syndrome.
Amer J Human Genetics, in press.
Policy Statement: American College of Medical Genetics
Fragile X Syndrome: Diagnostic and Carrier Testing
Fragile X syndrome is the most common cause of inherited mental retardation, seen in approximately one in 1,200 males and one in 2,500 females. Males with fragile X syndrome usually have mental retardation and often exhibit characteristic physical features and behavior [Hagerman and Silverman, 1991; Warren and Nelson, 1994]. Affected females exhibit a similar, but usually less severe phenotype.
The diagnosis of fragile X syndrome was originally based on the expression of a folate-sensitive fragile site at Xq27.3 (FRAXA) induced in cell culture under conditions of folate deprivation. Cytogenetic analysis of metaphase spreads demonstrates the presence of the fragile site in less than 60% of cells in most affected individuals. The cytogenetic test has limitations, especially in testing for carrier status, and it exhibits a high degree of variability between individuals and laboratories. Also, interpretation of the cytogenetic test for fragile X syndrome is complicated by the presence of other fragile sites in the same region of the X chromosome (FRAXD, FRAXE, and FRAXF).
In 1991, the fragile X gene (FMR1) was characterized and found to contain a tandemly repeated trinucleotide sequence (COG) near its 5' end. The mutation responsible for fragile X syndrome involves expansion of this repeat segment. The number of CGG repeats in the FMR1 genes of the normal population varies from six to approximately 50. There are two main categories of mutation, premutations of approximately 50 to 200 repeats and full mutations of more than approximately 200 repeats. There is no clear boundary between the upper limit of normal and the lower limit of the premutation range. For this reason, alleles with approximately 45-55 copies of the repeat are said to be in the "grey zone." Some alleles in this size range are unstable and expand from generation to generation, while others are stably inherited. A premutation is susceptible to expansion after passage through a female meiosis. The larger the size of a woman's premutation, the more the risk of expansion to a full mutation in her offspring.
Males and females carrying a premutation are unaffected. Male carriers are referred to as "normal transmitting" males, and they pass on the mutation, relatively unchanged in size, to all of their daughters. These daughters are unaffected, but are at risk of having affected offspring. Variable clinical severity is observed in both sexes. Most, but not all, males with a full mutation are mentally retarded and show typical physical ant behavioral features. Of females with a full mutation, approximately one-third are of normal intelligence, one-third are of borderline intelligence, and one-third are mentally retarded.
Due to the novel nature of the fragile X mutation, inheritance is less straightforward than in classic Mendelian traits. While passage through a female meiosis is necessary for significant trinucleotide repeat expansion, the expansion most likely occurs during early embryonic development. Since expansion occurs in a multicellular embryo and the extent of expansion may vary from cell to cell, individuals often display somatic heterogeneity in allele size. Some affected individuals, termed mosaics, exhibit both a premutation and a full mutation in blood.
Expansion of the trinucleotide repeat to more than 200 repeats (full mutation) is almost always associated with methylation of the promoter region of the gene and correlated gene inactivation. Gene inactivation is an important event in the pathogenesis of the syndrome. Although it is clear that methylation status plays a role in phenotype, its effect on clinical severity is somewhat unpredictable, especially in females.
DNA studies have improved the accuracy of testing for fragile X syndrome. By looking at the size of the trinucleotide repeat segment, as well as the methylation status of the FMR1 gene, the genotype can be determined for both affected individuals and suspected carriers. Two main approaches are used, polymerase chain reaction (PCR) and Southern blot analysis. PCR analysis utilizes flanking primers to amplify a fragment of DNA spanning the repeat region. Thus, the sizes of the PCR products are indicative of the approximate number of repeats present in each allele of the individual being tested. The efficiency of the PCR reaction is inversely related to the number of CGG repeats, so large mutations are more difficult to analyze and may fail to yield a detectable product in the PCR assay. This, and the fact that no information is afforded about FMR1 methylation, are limitations of the PCR approach. On the other hand, PCR analysis permits accurate sizing of alleles in the normal, the premutation, or the "grey zone" size ranges.
FMR1 analysis by Southern blotting allows both size of the repeat segment and methylation status to be assayed simultaneously. A methylation-sensitive restriction enzyme that fails to cleave methylated sites is used to distinguish between methylated and unmethylated alleles. Southern blot analysis is more labor intensive than PCR and requires larger quantities of genomic DNA. Southern blot analysis accurately detects alleles in all size ranges, but precise sizing is not possible. Many labs have both methods available, and perform the type of analysis that is most appropriate to the circumstances.
In a small number of fragile X patients, mechanisms other than trinucleotide expansion, such as deletion or point mutation, are responsible for the syndrome. In these cases, linkage studies and/or studies for rare mutations may be useful for relatives.
Recommendations for Diagnostic Testing
The purpose of these recommendations is to provide general guidelines to aid clinicians in making referrals for fragile X syndrome testing.
Individuals for Whom Testing Should Be Considered
Population carrier screening is not recommended at this time except as part of a well-defined clinical research protocol. The DNA test is very accurate, but it is important to ensure that effective means are in place to adequately inform tested populations of the meaning and implications of results. The nature of the FMR1 mutation and its inheritance are complex, and testing necessitates appropriate follow-up counseling.
Approaches to Testing
Hagerman RJ, Silverman AC (1991): "Fragile X syndrome: Diagnosis, Treatment, and Research." Baltimore: Johns Hopkins University Press.
Warren ST, Nelson DL (1994): Advances in molecular analysis of fragile X syndrome. JAMA 271:536-542.
Working Group of the Genetic Screening Subcommittee of the Clinical Practice Committee:
Approved by the Board of Directors on July 26, 1994.
Received for publication July 27, 1994; revision received July 28,