This offering: 0.1 CEU or 1 Contact Hour
Upon completion of this peer-reviewed article, the participant will be able to:
- Identify the four allelic forms of the FMR1 gene that are used for classification of the fragile X syndrome.
- Define the terms penetrence and anticipation.
- Describe the function of the FRM1 gene in the fragile X syndrome.
- Evaluate the use of Southern blot technique and PCR amplification in the diagnosis of the fragile X syndrome.
- Summarize the use of genetic screening in the general population for the fragile X site.
Fragile X syndrome (FXS), also termed Martin-Bell syndrome or marker X syndrome, is the most common cause of familial-inherited mental retardation. This disease is caused by an amplification of CGG triplet repeats located in the 5'untranslated region of the fragile X mental retardation-1 (FMR1) gene. Patients with FXS have expanded CGG trinucleotides, which are hypermethylated, causing the expression of FRMP to cease.
This in turn leads to the absence of the FMR1 protein (FMRP), resulting in mental retardation. The FXS name comes from the observation of the X chromosome Xq27.3 fragile site. This site reveals a slightly constricted portion near the tip of the long arm of the X chromosome, hence the "fragile" name. In some karyotypic pictures, the X actually has broken at this site, releasing a small piece containing the end of the chromosome (Fig. 1)
The recognition of an excess of mentally retarded males first was noted in the late 1800s. It was speculated that the males were more aggressive and expectations were higher, therefore, this male majority resulted from bias assessment. Genetic explanation for this inequity was never considered. In 1943, Martin and Bell were able to link cognitive disorders to a family with multiple male relatives exhibiting mental retardation. They speculated that the mode of transmission, while unidentifiable at the time, appeared to be X-linked inheritance. During a 1969 doctorial dissertation, Lehreke argued for the first time that an increase in male retardation was due to X-linked genes. In 1971, Turner suggested that X-linked mental retardation (XLMR) with no other clinical signs should belong to a unique diagnostic category.
The discovery of excessive genetic material by Lubs in 1969 showed that this material extended beyond the long arm on the X chromosome in affected males and in their unaffected female relatives. During this decade, it was realized that nonspecific XLMR was not uncommon and that this cytogenetic marker might be useful for diagnostic testing. Confirmation of this finding depended on Sutherland's culturing technique using folic acid- or thymidine-deficient cell culture medium to stimulate expression. The original work of Martin and Bell went unnoticed until 1982, when Richards and Web re-evaluated the original pedigree showing both macroorchidism and the cytogenetic marker on the X chromosome. In recognition of the first published pedigree, XLMR associated with this X chromosome cytogenetic marker was named the Martin-Bell syndrome. The FXS is the term currently used to refer to this unique genetic mutation, but the term Martin-Bell phenotype is used to refer to the facial characteristics associated with FXS.
Wide variability in the clinical presentation of the disorder is one reason the diagnosis is missed. Cognitive behavior and neuropsychological difficulties summarize the impairment. Adults with FXS exhibit several physical anomalies, including an unusually large head; long, narrow face; large, prominent ears and large testicles (macroochidism) in males.
Approximately 80 percent of patients with FXS will have one or more of these features, but their presence varies with age. Problems can include autistic-like behavior, hand flapping and avoidance of eye contact. Other features include aggressive tendencies, deficiency in abstract thinking and decreasing IQ with increasing age. Females exhibit similar but less severe phenotype because the additional X chromosome can produce the FMRP. FXS has been found in men and women of all races and ethnic backgrounds. The molecular basis for this phenotypic variability in both males and females is believed to be linked to the variable number of neurons in the brain that express FMRP. Penetrance is defined as the probability that a particular gene mutation will produce the disease.
In 1991, several labs independently isolated the gene responsible for FXS and identified its location. The molecular basis of FXS was revealed by cloning and shown to be associated with explosive expansion of trinucleotide repeats within the gene FMR1. FXS occurs because of the massive expansion in the promoter region of this gene. The function of the band Xq27.3, which is termed the FMR1 gene, consists of 17 exons and is roughly 38 kb in size. FMR1 is a highly-conserved gene and, within the 4.4 kb of the FMR1 transcript, a CGG triplet repeat is located at the 5'-untranslated region (5'UTR). Among normal individuals, this CGG repeat is highly polymorphic in length and content, often punctuated by AGG interruptions. The normal repeat size ranges from 7 to 60, with 30 repeats found on the most common allele (Table 1).
The understanding of the trinucleotide repeat expansions, described in 1991 by Fu and Warren, has helped with the understanding of the genetic cause of many neurological diseases, such as FXS. The repeat sequence CGG is stably inherited from one generation to another. The expansion of the repeat beyond the normal range results in abnormal levels of gene product and, ultimately, the disease state. In this disease, the trinucleotide repeat demonstrates instability when transmitted from parents to offspring.
Repeat instability is related to size. Therefore, the longer the repeat, the greater probability of expansion. Stability of the repeats appears to be related to the primary sequence. In FXS, the sequences are not perfect sequences, with interruptions where AGG is interspersed rather than CGG. Absence of these interruption sequences seems to render the resulting repeat subject to greater instability with minimal expansion. However, when a repeat reaches a critical size threshold, the repeat becomes very unstable. A repeat size of 100 or greater in FXS almost always leads to a full mutation.
With this trinucleotide repeat disorder, a correlation exists between increasing repeat size and disease severity. The FMR1 gene can be categorized in at least four forms, based on the size of the repeat: full mutation (>200-230 repeats); premutation (61-200 repeats); intermediate (41-60 repeats) and normal (6-40 repeats). The most extreme cases of mentally-challenged FXS patients have tremendous repeat expansions, but those with smaller expansions have a milder form of the disease.
There also is a "premutation" range considered to be intermediate between normal repeat and "full mutation," usually causing minimal phenotypic abnormalities. It should be noted that the instability of the premutation often leads to expansion and phenotypic expression in generations to follow. The term anticipation often is used to describe this phenomenon, which means that greater numbers of affected individuals are observed in later generations than in earlier ones. Evidence shows that parental bias occurs with respect to expansion in subsequent generations. The untranslated CGG repeats of FXS tend to have maternal bias of transmission. The expansion from premutation to full mutation occurs through a female. The mechanism responsible for this transmission bias has not been determined. CGG repeat expansion in the FMR1 gene is associated with decreased mRNA and protein levels. These decreases are often accompanied by increased DNA methylation of an adjacent CpG island. Increased methylation itself may cause decreased transcription of the FMR1 gene.
Unaffected individuals have 5-55 CGG repeats in the first exon at the 5' end of band Xq27.3. A span of 65-230 repeats is known as a premutation, whereas more than 230 repeats is a full mutation. Full mutation results in hypermethylation of the cysteine bases and restricts protein binding, leading to gene inactivation. The number of repeats is unstable from generation to generation, making the pattern of inheritance difficult to predict.
Methylation of the FMR1 Gene
The mechanism by which the CGG repeat causes the molecular pathology of fragile X phenotype has not been elucidated, but hypermethylation appears to play a part. The repeat sequence is located in the noncoding portion of the FMR1 gene. Once more than 230 repeats occurs, the DNA surrounding the repeat becomes hypermethylated. Methylation is the addition of methyl groups (CH3) to the C (cytosine) bases in CpG sequences in the DNA molecule.
Hypermethylation causes the inactivation of the FMR1 gene containing the triplet-repeat sequence, and it no longer can be transcripted. Lack of FMR1 gene product is the presumptive cause of the abnormal phenotype of FXS. Methylation plays a role, but it is not the only process influencing the function of the FMR1. In the absence of methylation, the gene is transcribed, but the mRNA is inefficiently translated and the degree of inefficiency increases in proportion to repeat copy number.
The trinucleotide repeat mutation that underlies FXS has unpredictable transmission features. Alleles with the full-mutation are preceded by premutation alleles that carry an intermediate repeat expansion of greater than 50 but less than 200. Premutation alleles do not generate FXS symptoms in most carriers, but they show significant instability and, thus, forecast the risk of genetic disease in a carrier's progeny. The greater the number of repeats in a premutation allele, the higher the risk of disease in that person's children. A woman with a 60 repeat expansion versus one with 90 triplet repeat has only a 17 percent chance of having FXS instead of 50 percent, respectively.
The change that produces a premutation allele increases the likelihood that the FMR1 gene will have more mutations. Consequently this leads to a larger original number of additional CGG repeats, which escalates a larger number of subsequent additions.
The expansion of the FMR1 premutation alleles has an explained relation to the parental origin of repeats. Most male carriers transmit their FMR1 allele with only a small change in the number of repeats, whereas women with premutation alleles bear children with excessive expansions of 4,000 CGG repeats in their FMR1 gene. Speculation is that the conditions that generate fragile X mutations occur during oogenesis. Due to the novel nature of the fragile X mutation, inheritance is less straight forward than in classic Mendelian traits. While passage through a female meiosis is necessary for significant triplet repeat expansion, the expansion most likely occurs during early embryonic development (Table 2).
At this time, estimates of the prevalence of FXS are 1 in 4,000 males and 1 in 8,000 females, with the full mutation manifesting intellectual disability. Limited data exists on the prevalence of females of normal intelligence who carry premutations and, thus, are at risk of having children with either a full mutation or a larger premutation.
The prevalence of premutation carrier males who are at risk of having FXS grandchildren is estimated at 1 in 5,000. As we move from identifying FXS with cytogenetics to molecular genetics, the accuracy of diagnosis will increase the reliability of the risk factor.
Before the cloning of FMR1, the inducible fragile site was developed as a cytogenetic marker and proved valuable in diagnosing this nonspecific form of the X-linked mental retardation. The fragile sites on the X chromosome are not spontaneously expressed using the general cytogenetics methods. An induction method is used in culture, leading to a deficiency of either thymidine or deoxycytidine at the time of DNA synthesis. Either condition will induce fragile site expression of the fragile X chromosome. The limitation of the cytogenetic test to determine the prevalence of FXS was recognized when studies of Turner and Webb were re-analyzed by testing the fragile X-positive males using the DNA diagnostic test for FXS. They found a high false-positive rate that they attributed to the expression of other fragile sites.
Since the discovery of the FMR1 gene, the first line of laboratory testing is molecular analysis. DNA studies have improved the accuracy of testing for FXS. DNA-based testing that determines the size of the fragile X CGG repeat are considered diagnostic and are in the magnitude of 99 percent sensitive and specific. This test method can provide a prenatal diagnosis when amniotic fluid and chorionic villus is used as the sample source. However, as with any DNA-based test, each has interpretation rules.
New DNA diagnostic tests have been based on the use of the polymerase chain reaction (PCR). Many different PCR protocols have been developed for the fragile X CGG repeat, with differing degrees of amplification abilities and sizing accuracies. PCR uses flanking primers to amplify a fragment of DNA spanning the repeat region. 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. PCR analysis is recommended for accurate sizing of alleles in the normal, intermediate and premutation range. The biggest disadvantage of this method is the test is not straightforward. The amplification of large repeats is difficult, especially when run with a second sample of smaller repeats. Large repeat samples often are not even amplified with many PCR testing methods. This is a problem for females and for persons with mosaicism that could appear to have a single, normal repeat size. Negative testing is often followed by the Southern blot method for any sample that fails to amplify and for females that appear homozygous. Even when one takes into account the variations in testing, the PCR tool is inexpensive, automated and fast. Another advantage of PCR is that sample size can be very small, collection is painless and live cells are not necessary.
Currently, the most popular and accepted method for DNA-based testing for the expanded CGG repeat is the Southern blot. Many different restriction enzymes can be used in combination to determine both expansion and methylation status. The restriction enzymes for expansion are EcoRI, PstI, BglII, HindIII and BclI. Methylation determinations use SacII, BssHII, EagI or BstZI restriction enzymes. Methylation status is particularly useful for distinguishing between borderline premutation and full-mutation alleles (200-230 repeats). Methylation-sensitive enzymes also can describe the degree of methylation of full mutation allele for both males and females. Southern blot analysis is more labor-intensive than PCR and requires larger quantities of genomic DNA.
The availability of antibodies directed against FMRP stimulated the development of a new diagnostic tool to identify fragile X patients. This test is based on the presence of FMRP in cells from unaffected individuals and the absence of FMRP in cells from the fragile X patients. To determine affection status for FXS, repeat expansion must be determined, as well as the subsequent lack of FMRP as well. In 1995, Willensen developed an antibody test that detects the presence of FMRP in blood smears. This protein-based assay measures the percentage of FMRP detected in lymphocytes from blood smears. Fragile X patients can be identified by the absence of FMRP or, in the case of female fragile X patients, by the diminished percentage of cells that express FMRP. This test also can identify patients who do not produce FMR1 protein due to a small deletion in the FMR1 gene. Males with the fragile X syndrome demonstrate fewer than 40 percent of their lymphocytes with this protein.
For males, the diagnostic power of the test is high because there is no overlap between the values of unaffected individuals and of affected males with the full mutation. The cutoff point of the diagnostic test for males has been determined to be 42 percent. This noninvasive test can be used for screening large groups of mentally challenged patients for fragile X syndrome, but is less specific in identifying females with the full mutation.
Plucked hairs containing the inner root sheath, a large part of the outer root sheath and the upper part of the bulb express high levels of FMRP. A low percentage of FMRP-stained hair roots are diagnostic of fragile X syndrome. For males, the diagnostic power of this new noninvasive test is high, and can be used to identify male patients. Willensen found that females with the full mutation could also be diagnostic. The power of this test has identified four fragile X patients that could not be diagnosed with DNA analysis.
In 2004, Zhou's lab reported a technique using methylation-specific triple PCR. This modified PCR assay attempts to address the deficiencies of PCR-based methods. This alternative molecular diagnostic test for FXS accurately differentiated between normal, premutation and full mutation of both males and females. Methylated and notmethylated FMR1 alleles are separated using sodium bisulphate, producing distinct difference in nucleotide sequence. Amplification and sizing of the non-methylated FMR1 repeat is accomplished with primers designated as non-Met-F and non-Met-R. This reaction detects all nonmethylated normal and premutation FMR1 repeats. Two different PCR reactions are used to detect the methylated allele. The first reaction uses Met-F and Met-R. This reaction determines all methylated allele sizes, as well as full mutation alleles up to 350 repeats. The second methylated-allele PCR reaction, called triplet primed PCR "mTP-PCR," uses the primers mTP-F and mTP-R to determine the methylated premutation or full-mutation allele of 300 bp repeats and higher. The potential for this assay to be used in the clinical laboratory offers the advantage of a stand-alone test versus the current use of both PCR and Southern blot analysis.
It should be noted that a small number of fragile X patients have mechanisms other than trinucleotide expansion. These would include deletion or point mutation. In these cases, linkage studies or rare mutation studies are needed.
A Steady Role
With the ability to diagnosis FXS accurately and economically, the question of whom should receive testing still is not clear. The American College of Medical Genetics has set the guidelines for patient recommendation for FXS testing.
Individuals of either sex with mental retardation, developmental delay or autism, who show physical or behavior characteristics of FXS, should be offered testing. A family history of FXS or relatives with undiagnosed mental retardation warrants testing, as well as fetuses of known carrier mothers.
New rapid test kits are on the horizon. General population screening soon will be easy to accomplish. However, ethical issues surface when identifying asymptomatic carriers or new cases of FXS.
Information is powerful, but with it comes inherent problems. These include a lack of a cure or effective treatment available for this disorder. While diagnostic tests can identify the FXS, no current test exists to determine the degree of mental retardation in either male of females. Whatever the future brings, clinical laboratory scientists will be providing the information.
- Gardner RJM, Sutherland GR. Chromosome Abnormalities and Genetic Counseling. Third edition. Oxford University Press, 2004:218-232.
- Jin P, Warren ST. Understanding the molecular basis of fragile X syndrome. Hum Mol Genet 2000;9(6):901-908.
- Bell MV, Hirst MC, Nakahori Y, et al. Physical mapping across the fragile X: Hypermethylation and clinical expression of the fragile X syndrome. Cell 1991;64(4):861-866.
- Fu YH, Kuhl DP, Pizzuti A, et al. Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox. Cell 1991;67(6):1047-1058.
- Weinhausel A, Haas OA. Evaluation of the fragile X (FRAXA) syndrome with methylation-sensitive PCR. Hum Genet 2001;108(6):450-8.
- Crawford DC, Acuna JM, Sherman SL. FMR1 and the fragile X syndrome: human genome epidemiology review. Genet Med 2001;3(5):359-371.
- Willemsen, R, Smits, A. Severjnen, LA, et. al. Predictive Testing for Cognitive functioning in female carries of the fragile X syndrome using hair root analysis. J Med Genet 2003:40:377-379.
- Zhou Y, Law HY, Boehm CD, et al. Robust fragile X (CGG)n genotype classification using a methylation specific triple PCR assay. J Med Genet 2004 Apr;41(4):e45.
- Taylor A. Fragile X DNA Testing: Guide for Physicians and Families. The National Fragile X Foundation. Available at: www.fragilex.org. Last accessed June 13, 2005.
- Policy Statement: American College of Medical Genetic. Fragile X Syndrome: Diagnostic and Carrier Testing. Available at: http://genetics.faseb.org/genetics/acmg/pol-16.htm. Last accessed June 13, 2005.
Roxanna Alter is assistant professor, school of allied health, division of clinical laboratory science, University of Nebraska Medical Center.
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