Features Of Fraxa And Fraxe

Clinical Phenotype, Genetics, and Biochemistry

The fragile X syndrome affects males to a greater extent than females: in males, learning and social deficits are moderate to severe, with social impairments usually preventing them from fathering offspring although sperm production is thought to be unaffected. In adult males, visible signs of the condition often include a large head, long face, large ears, and macroorchidism; however, in children, the phenotype is likely to be restricted to developmental delay with perhaps some autistic-like features such as gaze avoidance or hand-flapping.[2] Females with fragile X syndrome vary from apparently normal phenotypes to moderate learning disability, often with heightened anxiety, social withdrawal, or depression.1-3-1 The appearance of fragile X phenotypes in women may be simply a consequence of nonrandom X inactiva-tion, although this has not been proven.

In 1991, the gene responsible for fragile X syndrome was isolated by several independent research groups[4,5] and designated FMR1 (fragile X mental retardation 1). Most surprising was the discovery of the novel mutational mechanism—the progressive expansion of a tract of trinucleotide (CGG) repeats in the 5' untranslated region of the gene—culminating in the shutdown of transcription and hypermethylation of the promoter for repeat numbers in excess of around 200. Repeat numbers between around 60 and 200 show pronounced instability at meiosis, with a heavy bias toward expansion; therefore, these behave as ''premutations.'' This process of progressive repeat expansion has been termed dynamic mutation[6] and provides an explanation for the unusual inheritance pattern of fragile X syndrome with many nonpenetrant ''transmitting'' males observed in pedigrees. A far greater instability is observed during maternal transmission; although minor instability of premutations may be observed in paternal meiosis, a paternally transmitted premutation rarely, if ever, expands into a full mutation. Rare cases of fragile X syndrome are found to have a point mutation or deletion in FMR1 instead of a trinucleotide expansion, confirming the loss of gene product as a cause of fragile X pathology.

In addition, in 1991, the existence of the distinct condition of FRAXE was established when it became clear that several ''fragile X'' families (i.e., showing expressed site fragility in Xq27-28 and learning disability) did not have expansions in the CGG repeat tract in the FMR1 gene. Shortly afterward, these families were found to be segregating an expansion mutation in a GCC repeat tract in exon 1 of another gene, designated FMR2.[7] Pedigrees of FRAXE families differ notably from those of FRAXA families in that affected males frequently have children, with the degree of intellectual and behavioral impairment being much less pronounced than for FRAXA. Males with FRAXE mutations do not show the syndromic features typical of FRAXA, and FRAXE is now considered as a nonspecific X-linked mental retardation condition with a much lower incidence than FRAXA. The FRAXE repeat shows a dynamic mutation behavior similar to that of FRAXA, with unstable premutations

Fig. 1 Fragile X chromosome. (Courtesy of Prof. Pat Jacobs.)

expanding progressively to become full mutations. However, rather surprisingly, there is no obvious homology between FMR1 and FMR2 apart from the repeat tract.

The FMR1 gene responsible for fragile X syndrome comprises 17 exons, is alternatively spliced, and codes for a protein-designated FMRP,[8] expressed in various tissues but especially strongly in neurones and spermatogo-nia. Although not yet fully understood, the function of FMRP is becoming rapidly elucidated in recent years. It is an RNA-binding protein whose domains also include a nuclear localization signal and a nuclear export signal. It shows affinity for a number of partner proteins in the cytoplasm, where it associates closely with ribosomes. FMRP appears to shuttle in and out of the nucleus, binding a variety of mRNA including that of FMR1 itself, forming a ribonuclear particle that is transported along neurones to dendrites, where some local protein synthesis appears to take place. One of the key processes influenced by FMRP seems to be the maturation of dendritic spines; these fine protuberances are the principal determinants of synaptic plasticity, which in turn affects learning, memory, and cognition. Abnormal or immature dendritic spines have been demonstrated in both genetically and environmentally induced mental retardation syndromes. Knockout mice deficient in FMRP have been shown to exhibit learning deficits and immature and overabundant dendritic

Population Genetics

The dynamic mutation process creates a situation where new mutations are rarely observed, with all cases of premutations and full mutations in the population having been derived from parents with smaller expansion mutations. It is presumed that the smallest premutations arise from within the size range of 6-59 repeats, which exhibits a stable polymorphism within the general population. In the upper part of this ''normal'' size range, from about 45 repeats upward, the probability of unstable meiotic transmission becomes slightly elevated, but this tendency seems to be greater in some families than others, which creates a broad region of overlap in size between the ''normal'' and ''premutation'' allelic states.[10] This range of sizes, approximately 45-60 repeats for FRAXA and possibly lower for FRAXE, has been termed the ''intermediate range'' or ''grey zone.'' There are substantial difficulties in the genetic counseling of patients found to carry an intermediate-sized allele, especially where the genetic family history is incomplete. The risk of expansion to a full mutation increases progressively with the size of premutation in the mother; the smallest premutation, to date, found to expand to a full mutation in one generation has been 59 repeats.

The main factor believed to differentiate stable intermediate alleles from unstable potential premutations is the presence of interspersed AGG motifs at regular intervals, typically every 9th or 10th trinucleotide, within the CGG repeat tract.[11] These are thought to anchor the repeat sequence and prevent major unequal exchanges leading to expansion. Most normal, stably transmitted alleles are found to contain at least two interspersed AGGs, whereas almost all unstable premutations have pure CGG tracts or a single AGG. Where an AGG is retained, it is almost invariably the most proximal one in the sequence, implying that loss of interspersions and consequent expansion are effected at the distal (3') end. The evidence is circumstantial because the loss of an AGG leading to acquired instability within a pedigree has yet to be reported. However, the GCC repeat tract at the FRAXE locus appears to be devoid of interspersions even among normal alleles; hence, the presence of interspersed motifs is clearly not the sole mechanism for restricting the mutability of trinucleotide repeat sequences.

Diagnostic Testing

Full mutations at both the FRAXA and FRAXE loci may be readily identified by a Southern blot of a genomic DNA digest followed by hybridization with a specific labeled probe (Fig. 2). Use of a double digest employing one restriction enzyme sensitive to methylation enables the observation of hypermethylation associated with full mutations, thereby providing a more sensitive resolution of full mutations and premutations based on both size and methylation status.[12] Occasionally, mosaics for a full mutation and a premutation are observed; these constitute up to 20% of FRAXA full-mutation patients and may be less severely affected with fragile X syndrome. Care should be taken when interpreting the FRAXE blots using

Fmr1 Southern Blot

Fig. 2 Southern blot of FMR1 gene. Digest with BstZUEcoRI hybridized to probe StB12.3. (From Ref. [12].) Lane 1: Size marker XPstI digest; lane 2: normal male; lane 3: full mutation/ premutation mosaic male; lane 4: normal female (note methylated upper fragment corresponding to inactive X chromosome); lane 5: male with intermediate allele; lane 6: premutation male; lane 7: normal female; lane 8: premutation female.

Fig. 2 Southern blot of FMR1 gene. Digest with BstZUEcoRI hybridized to probe StB12.3. (From Ref. [12].) Lane 1: Size marker XPstI digest; lane 2: normal male; lane 3: full mutation/ premutation mosaic male; lane 4: normal female (note methylated upper fragment corresponding to inactive X chromosome); lane 5: male with intermediate allele; lane 6: premutation male; lane 7: normal female; lane 8: premutation female.

HindlH digests because there is a restriction site polymorphism that can mimic full mutations;[13] if this is suspected, a different enzyme digest should be used to confirm the diagnosis. Southern blot analysis is generally performed on samples of peripheral blood but may also be carried out on chorionic villus samples, facilitating reliable prenatal diagnosis of fragile X syndrome.

The high demand for fragile X testing creates a need for a high-throughput exclusion technique, and polymerase chain reaction (PCR) analysis of the repeat sequence using either a radioactive nucleotide incorporation (Fig. 3A) or a fluorescently labeled primer (Fig. 3B) provides rapid and convenient detection of males with a normal-sized allele and of females with two normal-sized alleles.[14] This technique also detects the smaller-sized premutations up to approximately 70 repeats, and enables fairly accurate estimates of repeat size to be made. However, it will not detect the larger premutations or full mutations due to the sharp reduction in efficiency of PCR for long CG-rich repeat sequences. Hence, males who show no PCR product, or females who show just a single allele require further analysis by Southern blot to determine their genotype. It should also be noted that the exclusion of fragile X by this method depends on the assumption that mosaicism for a normal allele and a full mutation is absent or very rare. If there is not enough DNA for conventional Southern blot analysis, it is possible to detect large premutations or full mutations by electrophoresing PCR products, transferring them to a membrane by electroblotting and hybridizing to a (CGG)n oligonucleotide probe.[15]

An alternative way of diagnosing fragile X syndrome is by in situ FMRP detection using an anti-FMRP antibody, which is detected as a red stain in the presence of FMRP.[16] The technique may be employed using blood cells, but has met with most success using hair root tissues. Clearly, as it is the absence of staining due to lack of FMRP that is indicative of fragile X syndrome, this diagnostic strategy is highly reliant on the efficiency of staining and must, in any case, be followed up by genetic testing of any suspected patients. Furthermore, it will not identify premutation carriers. However, it does have the advantage of detecting the minority of fragile X patients with a null mutation in the coding sequence of the gene rather than a trinucleotide repeat expansion.

Screening and Prevalence

Estimates of the prevalence of fragile X mutations have shown wide variation. Early screening studies employing cytogenetic fragile-site expression as a diagnostic criterion were subject to errors resulting from nonpenetrance of cytogenetic expression, low-level ''background'' expression leading to false positives, and the pooling of FRAXA and FRAXE site expression—these errors created probable overestimates of the prevalence of FMR1 mutations. More recent screening studies using molecular testing to identify mutations have arrived at a consensus of 1/4000 to 1/6000 as a prevalence for the FRAXA full muta-


although lower than previous estimates, this would still make fragile X syndrome the single most common inherited cause of intellectual disability. There is some evidence of ethnic variation: a few isolated populations have a significantly higher prevalence of FRAXA

mutations.[19] The only recorded estimate of FRAXE full mutation prevalence is 1/23,000,[18] but this needs to be corroborated by further studies.

Other FMR1 Phenotypes

Until recently, it was thought that premutations had no adverse phenotypic consequences. However, two conditions have now been found to be associated with FMR1 premutation alleles: premature ovarian failure (POF) and fragile X-associated tremor and ataxia syndrome (FXTAS).

POF, defined as the onset of menopause before the age of 40 years, affects some 20% of female FMR1 premutation carriers.[20] As there is no evidence of POF among full-mutation carriers, it seems likely that this is a causal effect of the premutation rather than any mutation

in an adjacent gene in linkage disequilibrium with FMR1. The pathological mechanism by which FMR1 premutations cause POF is unknown, but a possible biochemical basis for this and other premutation phenotypes has been established by the discovery of elevated FMR1 mRNA levels and reduced FMRP levels in premutation carriers.1-21-1

FXTAS is a more recently discovered condition than POF and its properties are still being characterized.1-22-1 However, preliminary studies suggest that it may have a penetrance similar (20-30% of male premutation carriers) to POF, and again no comparable condition has been found in full mutation carriers. It appears that FXTAS may be restricted to male carriers, although studies continue to look for possible equivalent effects in female carriers.

The discovery of the FMR1 gene and its novel mutational mechanism provided the catalyst for the identification of an abundant class of genes harboring trinucleotide repeats, including those responsible for common genetic conditions such as Huntington disease, myotonic dystrophy, and Friedreich ataxia. Although the first trinucleotide repeat disease to be discovered, FRAXA has not proved to be typical of the genre of such loci: peculiar features include association with fragile sites and loss of gene function due to methylation, extreme parent-of-origin bias in repeat expansion, and pleiotropic phenotypes attributed to premutations. Fragile X syndrome, despite a wealth of intensive study and many significant advances, continues to rank among the most intriguing and enigmatic of all human genetic diseases.

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