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Repeat expansion diseases are caused by the aberrant repeat expansions within specific genes. RNAs derived from aberrant repeat sequences form non-canonical secondary structures, contributing to induce cell toxicity. In particular, RNA G-quadruplexes (G4RNAs) formed in guanine-rich repeat expanded RNAs trigger neurodegeneration. We have previously shown that the expanded CGG repeat-derived G4RNAs initiate aggregation of FMRpolyG, a neuropathogenic protein generated by repeat-associated non-AUG (RAN) translation in Fragile X-associated tremor/ataxia syndrome (FXTAS). In this review, we describe the neuropathological mechanism attributed to G4RNAs in guanine-rich repeat expansion diseases, including FXTAS.

G-quadruplex (G4) is a unique nucleic acid structure that formed when a four-stranded structure is produced within a single-stranded guanine-rich sequence. Four guanine molecules form a square planar arrangement, termed G-quartet, which are stacked on top of each other to form the G4 structure in DNA (G4DNA) and in RNA (G4RNA). Recent studies have revealed that G4DNA and G4RNA are folded in cells, which suggested their biological and pharmacological significance in DNA replication, transcription, epigenetic modification, and RNA metabolism. So far, we have reported the following; 1) G4 is a target of cognitive function therapy for ATR-X intellectual disability syndrome, in which mutations are found in a G4 binding protein ATRX. 2) G4 is formed in heterochromatin depending on neuronal development. 3) G4 promotes prionoids in a CGG triplet repeat disease, Fragile X-associated tremor/ataxia syndrome (FXTAS). 4) 5-aminolevulinic acid is a potential candidate drug for treating some neurological diseases through the G4 binding ability. In this review, we summarized the significant roles of G4 in neurological diseases.

We assessed the utilization of diagnostic analyses for fragile X syndrome by a mail-in questionnaire on 1) the number of patients analyzed and diagnosed with the syndrome in the past year, 2) types of diagnostic analyses used, 3) clinical features that made physicians to decide analyses, 4) purpose of analyses, and 5) informed consent for analyses. Facilities for the mentally handicapped, as well as hospitals and physicians specialized in genetics, completed our questionnaire. Among 101 responders, total of 543 cases underwent analyses. Nine cases (including 3 cases over 20 years old) were finally diagnosed in a year. The rate of positive findings was 0.6% for chromosomal analyses, and 8% for gene analyses. Physicians dicided to make analyses based on clinical features such as mental retardation, characteristic face, and autistic features, in order to find the cause (s) of the subjects' condition. For gene analyses, more than a half of physicians obtained a form of informed consent. Specialists should have interest in this syndrome because the analyses identified new adult cases. Establishment of a guideline for diagnosis of this syndrome requires gene analyses based on evidence and informed consent.

We assessed the present status of chromosome and gene analyses utilized in Japan for the diagnosis of fragile X syndrome. Nursery facilities for the mentally handicapped and hospitals completed a mail-in-questionnaire including information on the number of patients diagnosed with the syndrome, the types of diagnostic analyses used, and the need for such analyses. Among 517 responders, ninety-five (18.4%, more frequently in hospitals) reportedly conducted chromosome or gene analyses, which established the diagnosis of fragile X syndrome in 56 patients, accounting for 2.2% of patients who underwent the analyses, and 0.13% of the patients included in this study. Each responder's experience with chromosome or gene analyses were related to his/her demand for them. Some facilities that had not utilized these analyses requested information on them as well as access to them. A larger number of specialists interested in diagnostic screening are essential to establish a diagnosis system of fragile X syndrome.

Fragile X syndrome is the most common heritable form of mental retardation, and affects 1 in 1500(male)-2500(female), with minor dysmorphic manifestations such as long face with large protruding ears and macro-orchidism in mentally retarded male patients. The syndrome is caused by dynamic mutation(trinucleotide repeat expansion) at FRAXA located on the long arm of X chromosome. Molecular diagnosis enables carrier identification as well as prenatal diagnosis, in which the cytogenetic method was not feasible. Premutation in phenotypically normal carriers and full mutation in mentally retarded patients explain the characteristic inheritance of the disease called anticipation. This article describes the recent advancements in molecular cytogenetics of fragile X syndrome.

The folate sensitive fragile site FRAXE is located in Xq28, 600 kb distal to the fragile X syndrome (FRAXA) fragile site. An unstable GCC triplet repeat responsible for FRAXE exists in the 5' untranslated region of FMR2 gene. Normal alleles range from 10-35 repeats and more frequently 15-20 GCCs. An expanded GCC repeat over 200 is methylated, inactivates FMR2, and results in FRAXE mental retardation. Affected individuals show mild mental retardation and/or autistic symptoms FMR2 is a large gene consisting of 21 exons spanning about 600 kb. FMR2 mRNA is about 9.5 kb and is expressed in the brain(especially in the hippocampus and the amygdala) in adults and also in placenta. The FMR2 protein is 1311 amino acids, contains nuclear localization signals, and is a putative transcription factor.

The fragile X syndrome(FRAXA) is the disease characterized by X-linked mental retardation, large ears, behavioral problems and macroorchidism. FMR-1 gene expression and its function in human organs are not fully understood. We studied the age dependent and tissue specific FMR-1 gene expression using human autopsy materials by means of RT-PCR. Total RNAs were extracted from the tissues and RT-PCR were performed to detect FMR-1 gene expression. Semiquantitative analysis were performed and the amount of FMR-1 gene products were compared with PGK gene products. Significant FMR-1 gene expressions were noted in all tissues tested, however cerebrum, cerebellum and testis demonstrated relatively higher FMR-1 transcripts compared with other organs. Relatively high expressions were noticed in all tissues during fetal/infantile periods. Semiquantitative analysis reveals that FMR-1 gene expressed much stronger especially around the perinatal periods. These results suggested that FMR-1 gene is widely expressed in human tissues and may play an important role during human maturation not only in the central nervous systems but also in other organs including liver and kidney.

Dynamic mutation, the mechanism of allelic expansion of the heritable unstable triplet repeat DNA sequences, has been the subject of intense investigation since its discovery in 1991 as the molecular basis for fragile X syndrome. The fundamental distinction between dynamic and other forms of mutation is that dynamic mutation is a process rather than a single event. The process involves a relationship between the copy number of perfect repeat DNA sequence and its instability which accounts for the anticipation seen in families carrying a dynamic mutation disease. Recently, dynamic mutation due to allelic expansion of minisatellite repeats has also been demonstrated as either the fragile sites and a genetic disease. In this minireview, dynamic mutations associated with chromosomal fragile sites and their cytogenetics are overviewed.

Chromosome instability is a characteristic cytogenetic feature of a number of genetically determined human disorders collectively known as chromosome breakage syndromes. Included among the disorders are Bloom's syndrome (BS), Fanconi's anemia (FA), ataxia telangiectasia (AT). In each of the syndromes chromosome instability exists in the form of increased frequencies of breaks and interchanges occurring either spontaneously or following treatment with various DNA-damaging agents. These diseases have in common an autosomal recessive transmission and an increased tendency to develop malignancies. The blood cells of subjects with AT, BS, or FA are significantly more radiosensitive than those of controls, particularly in the occurrence of chromosome aberrations.

The fragile X syndrome is one of the most common forms of genetic mental retardation and is caused by elongation of a small target DNA fragment containing a repeat of the trinucleotide CGG located in a 5' exon of the FMR-1 gene on the X chromosome. The genetic mutations were classified as two main types, premutation and full mutation, according to the size of the elongation. Because clinical findings are varied in patients with the fragile X syndrome, diagnosis of the disease is very difficult when based only on clinical symptoms. Molecular findings in three families with the fragile X syndrome showed that individuals with the full mutation were moderately mentally retarded, but individuals with the premutation had normal intelligence. The intellectual quotient levels in the families related to the sizes of the mutation which were unstable from generation to generation. These findings suggested that molecular studies in patients with mental retardation are very usefull to diagnose the fragile X syndrome, and that genetic counseling should be carried out based on the DNA analyses of the FMR-1 gene in the family members.

Heritable fragile sites are specific points on human chromosomes which appear as nonstaining gaps or breaks under certain physiological conditions and are inherited in a Mendelian codominant fashion. Recent findings of a correlation between fragile sites and breakpoints involved in specific chromosomal rearrangements seen in some neoplasias suggest that fragile sites may be unique sites particularly susceptible to chromosome breakage and rearrangement under in vivo physiological conditions. The majority of known fragile sites, including fragile X, are classified in the folate-sensitive group and are expressed under conditions of thymidylate stress. In thymidine-auxotrophic somatic cell hybrids constructed by cell fusion between fragile X cells and thymidylate synthase-negative mouse mutant cells, we have shown that thymidine deprivation alone can induce the expression of fragile X, suggesting that the primary DNA structure itself is responsible for its expression. Elucidation of the DNA structure of fragile sites may provide a clue to the understanding of genetic instability in the human genome and its possible involvement in cancer-specific chromosomal rearrangements.