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Fragile X syndrome (FXS) is the most common monogenic form of inherited intellectual disability and autism spectrum disorder (ASD). More than 99% of individuals with FXS are caused by the unstable expansion of CGG repeats located within the 5'-untranslated region of the FMR1 gene. The clinical features of FXS include various degrees of cognitive deficit, physical, behavioral and psychiatric problems. Early treatment and prevention from having further affected children can be guided by molecular genetic testing of the FMR1 gene. The following guideline has combined the relevant research, guidelines and consensus worldwide, and summarized the genetic knowledge and clinical treatment for FXS in order to achieve a standardized diagnosis, treatment and prevention for patients and families affected by this disease.

Objective: To screen for mutations of fragile X mental retardation 1 (FMR1) gene during early and middle pregnancy and provide prenatal diagnosis for those carrying high-risk CGG trinucleotide expansions., Methods: Peripheral blood samples of 2316 pregnant women at 12 to 21(+6) gestational weeks were collected for the extraction of genomic DNA. CGG repeats of the FMR1 gene were detected by fluorescence PCR and capillary electrophoresis. Genetic counseling and prenatal diagnosis were provided for 3 women carrying the premutations., Results: The carrier rate of CGG repeats of the FMR1 gene was 1 in 178 for the intermediate type and 1 in 772 for the premutation types. The highest frequency allele of CGG was 29 repeats, which accounted for 49.29%, followed by 30 repeats (28.56%) and 36 repeats (8.83%). In case 1, the fetus had a karyotype of 45,X, in addition with premutation type of CGG expansion of the FMR1 gene. Following genetic counseling, the couple chose to terminate the pregnancy through induced labor. The numbers of CGG repeats were respectively 70/- and 29/30 for the husband and wife. In case 2, amniocentesis was performed at 20 weeks of gestation. The number of CGG repeats of the FMR1 gene was 29/-. No abnormality was found in the fetal karyotype and chromosomal copy number variations. The couple chose to continue with the pregnancy. Case 3 refused prenatal diagnosis after genetic counseling and gave birth to a girl at full term, who had a birth weight of 2440 g and no obvious abnormality found during follow-up., Conclusion: Pregnant women should be screened for FMR1 gene mutations during early and middle pregnancy, and those with high-risk CGG expansions should undergo prenatal diagnosis, genetic counseling and family study.

Objective: To analyze the (CGG)n repeats of FMR1 gene among patients with unexplained mental retardation., Methods: For 201 patients with unexplained mental retardation, the (CGG)n repeats of the FMR1 gene were analyzed by PCR and FragilEase TM PCR. Prenatal diagnosis was provided to carriers of pre- and full-mutations. The pattern of X chromosome inactivation (XCI) was determined for women with mental retardation and full mutations., Results: For the 201 patients with unexplained mental retardation, 15 were identified with full mutations of the FMR1 gene. The prevalence of fragile X syndrome (FXS) in patients with unexplained mental retardation was determined as 7.5% (15/201). Prenatal diagnosis was provided for 6 pregnant women with pre- or full mutations. Analysis revealed that women with mental retardation and full FMR1 mutations exhibited a skewed XCI pattern with primary expression of the X chromosome carrying the mutant allele., Conclusion: FXS has a high incidence among patients with unexplained mental retardation. Analysis of FMR1 gene (CGG)n repeats in patients with unexplained mental retardation can facilitate genetic counseling and prenatal diagnosis for their families. FMR1 gene (CGG)n repeats screening should be recommended for patients with unexplained mental retardation.

Objective: To explore the correlation between Fragile X mental retardation gene-1 (FMR1) gene CGG repeats with diminished ovarian reserve (DOR)., Methods: For 214 females diagnosed with DOR, DNA was extracted from peripheral blood samples. FMR1 gene CGG repeats were determined by PCR and capillary electrophoresis., Results: Three DOR patients were found to carry FMR1 premutations, and one patient was found to carry gray zone FMR1 repeats. After genetic counseling, one patient and the sister of another patient, both carrying FMR1 permutations, conceived naturally. Prenatal diagnosis showed that both fetuses have carried FMR1 permutations., Conclusion: FMR1 gene permutation may be associated with DOR. Determination of FMR1 gene CGG repeats in DOR patients can provide a basis for genetic counseling and guidance for reproduction.

Objective: To determine the carrier rate of Fragile X mental retardation 1 gene (FMR1) mutants in women with a history of adverse pregnancy or childbirth, and to provide prenatal diagnosis for the carriers., Methods: Peripheral blood samples were collected from women with a history of adverse pregnancy or childbirth, and the FMR1 gene cytosine-guanine-guanine repeat number (CGG)n was determined by triple-repeat primer polymerase chain reaction (TP-PCR) combined with capillary electrophoresis. Prenatal diagnosis was provided for the carriers during pregnancy., Results: Among 819 samples, 9 gray zone repeats carriers and 10 premutation carriers were detected, which gave a prevalence of 1 in 91 and 1 in 82, respectively, with a total prevalence of 1 in 43. Prenatal diagnosis was provided during 7 pregnancies for 6 carriers. A female fetus with premutation (n = 30/57) and an affected male fetus with full mutation (n = 336) were detected., Conclusion: FMR1 gene testing in women with a history of adverse pregnancy or childbirth can facilitate genetic counseling and reproductive guidance for carriers of gray zone repeats and premutations. Prenatal diagnosis for carriers of premutation can facilitate reduction of the birth of children with fragile X syndrome.

Objective: To assess the value of genetic testing for Fragile X syndrome (FXS)., Methods: A domestically made diagnostic kit based Tri-primer-PCR method was used to detect mutations of the FMR1 gene among 6 pedigrees with unexplained intellectual disability. The results were verified by methylation PCR and Southern blotting., Results: Pedigrees 1 and 6 were positive for the screening. In pedigree 1, a full-mutation allele with methylation was identified in the proband and his mother, which was passed on to the fetus. In pedigree 6, the proband was mosaic for a full-mutation allele and a pre-mutation allele. His sister was asymptomatic with a full-mutation. His mother carried pre-mutation allele, while his father and sister's baby were normal. The number of CGG repeats of the pedigrees 2 to 5 were in the normal range., Conclusion: Genetic testing can provide an effective way to prevent FXS caused by FMR1 mutations and enable prenatal diagnosis for families with a high risk for the disease.

Objective: To screen for CGG repeats in the FMR1 gene among patients with fragile X syndrome and carriers of pre-mutations., Methods: Potential full and pre-mutations of the FMR1 gene were detected with a Tri-primer-florescence PCR-Sanger sequencing method. The results were validated with positive and negative controls., Results: All positive and negative controls were confirmed. A male patient was found to have > 200 CGG repeats (full mutation). For a pregnant women who was heterozygous for 35/115 CGG repeats, > 200 CGG repeats were also found with amniotic fluid sample from her fetus who was a male. The result was confirmed by following selective abortion with informed consent., Conclusion: Tri-primer-florescence PCR-Sanger sequencing is a simple, effective and reliable method for routine screening of patients/carriers with full/pre-mutations of the FMR1 gene in the population.

Objective: To study the pattern of CGG repeat instability within germline cells derived from two male fetuses affected with Fragile X syndrome (FXS)., Methods: The length and methylation status of CGG repeats within the testes of a fetus carrying a full FXS mutation and another fetus carrying mosaicism FXS mutation were analyzed with Southern blotting and AmplideX FMR1 PCR. Immunohistochemistry was also applied for the measurement of FMR1 protein (FMRP) expression within the testes., Results: For the fetus carrying the full mutation, Southern blotting analysis of the PCR product has detected an expected band representing the full mutation in its brain and a premutation band of > 160 CGG repeats in its testis. Whereas the pattern of premutation/full mutation in mosaic testis was similar to that in peripheral blood and no sign of contracted fragment was found other than a band of about 160 CGG repeats. Immunohistochemistry assay with a FMRP-specific antibody demonstrated a number of FMRP-positive germ cells, which suggested a contraction from full mutation to premutation alleles., Conclusion: This study has clarified the instability pattern of CGG repeat and expression of FMRP protein within the testes of fetuses affected with FXS, confirming that the CGG repeat can contract progressively within the germline. The FMRP expression in the testis is consistent with spermatogonium proliferation, and thus the contraction from full mutation to unmethylated premutations may occur for the requirement of FMRP expression during spermatogenesis. The better understanding of FMRP function during germ cell proliferation may elucidate the mechanism underlying the contraction of full FXS mutation in male germline.

Objective: To investigate the clinical (including reproductive) manifestations and genetic characteristics of familial fragile X syndrome (FXS)., Methods: We collected the clinical data about a case of familial FXS by inquiry, testicular ultrasonography, semen analysis, determination of sex hormone levels, and examinations of the peripheral blood karyotype and Y chromosome microdeletions. Using Southern blot hybridization, we measured the size of the CGG triple repeat sequence of the fragile X mental retardation-1 (FMR1) gene and determined its mutation type of the pedigree members with a genetic map of the FXS pedigree., Results: Among the 34 members of 4 generations in the pedigree, 3 males and 1 female (11.76%) carried full mutation and 9 females (26.47%) premutation of the FMR1 gene. Two of the males with full FMR1 mutation, including the proband showed a larger testis volume (>30 ml) and a higher sperm concentration (>250 ×10⁶/ml), with a mean sperm motility of 50.5%, a mean morphologically normal sperm rate of 17.5%, an average sperm nuclear DNA fragmentation index (DFI) of 18.5%, a low level of testosterone, normal karyotype in the peripheral blood, and integrity of the azoospermia factor (AZF) region in the Y chromosome. One of the second-generation females carrying FMR1 premutation was diagnosed with premature ovarian failure and another 3 with uterine myoma., Conclusions: Some of the FXS males in the pedigree may present macroorchidism and polyzoospermia but with normal semen parameters. In the intergenerational transmission, premutation might extend to full mutation, with even higher risks of transmission and extension of mutation in males, especially in those with >80 CGG triple repeat sequences. Therefore, it is recommended that the couples wishing for childbearing receive genetic testing, clinical guidance, and genetic counseling before pregnancy and, if necessary, prenatal diagnosis and preimplantation genetic diagnosis.

Objective: To compare the CGG-repeat-length and its methylation status in fetal tissues and to explicate the heterogeneity of CGG repeats., Method: Multiple tissues from a full mutation(August 2013) and a mosaic aborted fetus of 23-week gestation(May 2012) were collected and genomic DNA from these tissues was extracted. The CGG-repeat-length and methylation status in fetal tissues were determined by a combined strategy of Southern blotting and GC-Rich PCR. FMR1 expression was measured by real time PCR and Western blotting., Result: CGG-repeat-length in different tissues of each fetus was similar.A major methylated band in the full mutation range (540 CGG repeats) was detected in the brain, skin, testis and kidney tissues of Case 1. An unmethylated premutation band with 160 CGG repeats, and another two bands with 470 and 1 100 CGG repeats in the full mutation range were shown in the brain, skin, testis, lung, stomach, gut, liver, kidney, heart and blood of Case 2. However, the methylation status of CGG repeats in the mosaic fetus was heterogeneous among different tissues. The lowest premutation ratio was in the brain of the mosaic fetus compared with other tissues, and correspondingly FMR1 expression in its brain was minimum., Conclusion: This study clarify the tissue heterogeneity of CGG repeats and provides information for the genetic counseling and clinical diagnosis in fragile X syndrome. Based on the fact that the mosaic fetus' mother is a carrier of full mutation, it is speculated that the maternal CGG repeat has contracted before the differentiation of trilaminar germ disc.

Objective: To analyze (CGG)n repeats sequence and AGG interspersion correlated with unstable expansion of FMR1 gene in a general Chinese population., Methods: AmplideX FMR1 PCR Kit was used to amplify 380 X chromosomes from randomly selected 176 males and 102 females, 11 permutation carriers and 10 full mutation patients have served as controls. Results of capillary electrophoresis were analyzed with GeneMapper software Version 4.0. SPSS 11.0 software was used for statistical analysis., Results: The ratio of heterozygous females was 64.70%. The number of alleles in general males and females was 15 and 14, the classes of AGG pattern was 26 and 27, respectively. The range of alleles was between 17 to 45 CGG repeats in males and 21 to 44 CGG repeats in females, and 1 male was identified as gray zone carrier. The most frequent allele was 29 CGG repeats, which was followed by 30 and 36 repeats, while 28 CGG repeats were absent. The most common AGG pattern was 9A9A9, 99.21% of the population was detected with different forms and numbers of AGG interruption, and 6A interruption pattern was found in 10.02% samples especially in individuals with more CGG repeats. However, 57.58% of control samples had no AGG interruption, and none of the controls had 6A interruption pattern. No significant difference was observed in allele frequent distribution of (CGG)n repeats and AGG interspersion patterns between the males and females (P > 0.05), and AGGs was significantly different between general population and controls (P < 0.05)., Conclusion: AGGs and AGG pattern may have important roles in maintaining (CGG)n stability in general population of China, 9A9A6A9 may be a special pattern for preventing (CGG)n unstable expansion in Asian populations.

Objective: To investigate whether Chinese multiple system atrophy (MSA) patients have premutation of fragile X mental retardation 1 gene(FMR1)., Methods: FMR1 CGG repeats were analyzed in 157 MSA patients by polymerase chain reaction, agarose gel electrophoresis and capillary electrophoresis. The patients were collected from Movement Disorder & Neurogenetics Research Center of China-Japan friendship hospital. There were 83 male cases and 74 female cases, including 51 MSA-C patients, 12 MSA-P patients and 94 MSA-P+C patients., Results: No FMR1 CGG repeat premutation was detected in 157 MSA patients. The repeats ranged from 11-49, most common allele was 22. A MSA-C case carried 35/49 alleles did not have middle cerebellar peduncles(MCP) sign which was necessary for the diagnosis of fragile X associated tremor ataxia syndrome(FXTAS)., Conclusion: The FMR1 premutation in Chinese MSA patients might be very rare.

Objective: To establish a method of methylation-sensitive restriction enzymes based quantitative PCR (MSRE-qPCR) for analysis of CpG island DNA of FMR1 gene, and to assess its value for molecular diagnosis of fragile X syndrome., Methods: Thirty boys with mental retardation and abnormal repeats of 5'(CGG)n in the FMR1 gene and 20 mothers were analyzed by conventional PCR screening. Eag I was used to digest genomic DNA, and qPCR was performed to amplify CpG island in the FMR1 gene using both undigested and digested templates. Raw Ct values were obtained through quantitative PCR amplification. The degree of CpG island methylation was calculated by 2 - U+0394 U+0394 Ct. The result of MSRE-qPCR was verified by Southern blotting. 30 healthy females and 30 healthy males were used as controls to optimize the established MSRE-qPCR method., Results: The ranges of 2 - U+0394 U+0394 Ct value for normal methylation, partial methylation and full methylation were determined. Among the 30 patients, 3 were found to have partial methylation of CpG island of the FMR1 gene, and 27 were found to have full methylation (3/30 results were verified by Southern blotting). Only 7 mothers were found abnormal methylation of CpG island of FMR1 gene, whilst the remaining 13 mothers were normal., Conclusion: MSRE-qPCR is a quick and reliable method for quantitative analysis of CpG island methylation status in FMR1 gene, which may provide a new strategy for the diagnosis of fragile X syndrome.

Fragile X mental retardation 1 is the gene underlying fragile X syndrome (FXS). Its product, fragile X mental retardation protein, is closely involved with development of brain and neurons. PCR and Southern blotting have been the major methods for laboratory diagnosis of FXS. In this article, the progress in the molecular diagnosis of FXS is reviewed.

Objective: To analyze the expression of inducible nitric oxide synthase (iNOS) in the testis tissues of Fmr1 (fragile X mental retardation 1) knockout and wild-type male mice in different developmental stages, and provide background information for researches on fragile X syndrome., Methods: This study included 4, 6, 8 and 10 weeks old Fmr1 knockout and wild-type male mice, 6 in each age group. We identified the genotype of the mice by PCR, and detected and compared the expression of iNOS in the testis tissues of the Fmr1 knockout and wild-type mice by immunohistochemistry., Results: The iNOS expression was weakly positive in the Leydig cells of the 4-week-old mice, moderately positive in the 6-week-old ones, and strongly positive in 8- and 10-week-old ones, significantly weaker in the Fmr1 knockout than in the wild-type ones., Conclusion: The expression of iNOS significantly decreases in the testis of Fmr1 knockout mice, suggesting that iNOS may be involved in the pathogenesis of fragile X syndrome.

Fragile X-associated tremor/ataxia syndrome(FXTAS) is a neurodegenerative disease caused by FMR1 gene permutation(PM). The main clinical manifestations are intention tremor and/or ataxia, and the pathogenesis was related to RNA toxicity. In this paper, the research progress of clinical manifestatios, pathological characteristics, epidemiology and molecular mechanisms will be reviewed.

Epigenetics refers to changes in gene expression that do not entail a change in DNA sequence, namely phenotype changed, other than the genotype. These changes may remain through cell division for the remainder of the cell's life and may also last for multiple generations. It contains DNA methylation, histone modification, chromatin remodeling, gene imprinting and so on. Methylation is one of the chief epigenetic modify patterns in genomic DNA. DNA methylation via the interaction of methylation-CpG-binding proteins will affect the gene expression. Furthermore, the dysfuction of epigenetic mechanisms may lead to multiple genetic neurodevelopmental disorders and degenerative diseases. Here we mainly rewiewed the physiological and pathological significance of epigenetics regulations in neural development and neurodegeneration.

Objective: Fragile X syndrome (FXS) may be identified by many methods, such as PCR assay and Southern blot. However, each method has its limits or shortcomings. This study explored the reliability of the rapid, convenient and inexpensive hair root fragile X mental retardation protein (FMRP ) assay in the identification of FXS., Methods: FMRP in hair roots was determined by immunohistochemistry assay in 80 healthy children, in 40 children with mental retardation of unknown etiology and in 12 family members in one pedigree of FXS. FXS was confirmed by 7-deza-dGTP PCR., Results: There was a high expression of FMRP in hair roots (> or =80%) in healthy children. Two children were confirmed with FXS by 7-deza-dGTP PCR in 40 children with mental retardation of unknown etiology. FMRP expression was 10% and zero respectively in the two children. The other 38 children had FMRP expression of more than 80%. FMRP was not expressed in the two cases of FXS from the pedigree of FXS., Conclusions: Inexpensive, rapid and convenient hair root FMRP assay is reliable for the diagnosis of FXS and may be widely applied for screening and diagnosing FXS in children with mental retardation.

Variations of CGG repeats in the FRAXE fragile site were determined by PCR amplification followed by polyacrylamide gel electrophoresis and sequencing in randomly collected Qinba Mountain Area children whose intelligence level was determined by China-Wechsler Intelligence Scale for Children (C-WISC). Correlations between IQ score and the number of CGG repeats were analyzed. Results indicated a difference in the range of CGG repeats among populations from different regions, but no difference in the distribution of allele frequency in such a population. CGG repeats were not associated with IQ scores in randomly collected subjects (r=0.083, P>0.05), in males and females (r(m)=0.225, r(f)=-0.041, P>0.05), or in subjects with different intelligent levels (F=0.195, P>0.05). It can be inferred that variation in the size of CGG repeats was not associated with intellectual performance in the Qinba Mountain area children.

Objective: To search the relationship between fragile X male patients and expression of FMR-1 gene and to establish a rapid and simple method for screening male patients in general population., Methods: Two typical fragile X families demonstrated by cytogenetics method were analyzed by using RT-PCR., Results: No expression of FMR-1 gene in all male patients in the two families was noted, but all normal individuals showed expression of FMR-1 gene. There was an expression of HPRT gene as internal control in each of the individuals detected., Conclusion: There is a strong relationship between loss of expression of FMR-1 gene and clinical phenotype of the fragile X male patients. Further research of this method will introduce a more simple and rapid method for detecting male patients with fragile X syndrome. This method may be used for general population screening.

Objective: To gain more insight into biological role of the alternative splicing of the FMR1 gene, isoforms of the FMR1 mRNA from human fetal heart, spleen, liver and kidney were analyzed., Methods: RNAs were isolated from the tissues of an abortion fetus (approximately 6 months postconception). Alternative splicing patterns of the FMR1 mRNA were analyzed by RT-PCR and cloning strategy., Results: One isoform of the FMR1 mRNA was found to be dominant in all of the four tissues. The major isoform was as same as the dominant one in the fetal cortex but was different from the major isoform in adult brain. The difference between the major isoforms in the fetal tissues and adult brain was the splicing out or retaining the peptide encoded by exon 12 and exon 17., Conclusion: This result suggested a developmental switch of alternative splicing of the FMR1 gene. The difference between the major isoforms in the fetal tissues and adult brain suggested the two peptides may have special roles in related developmental stages.

Objective: To investigate the prevalence of fragile X syndrome in the Chinese., Methods: Fragile X syndrome was screened from 172,600 people living in Tongling city, Anhui province. Suspected patients were tested by PCR and Southern Blotting., Results: Seven of 88 mental handicap children were found with fra (X), including 3 female and 4 male patients. Six patients, 3 normal transmitting males and 5 carriers were found in detailed search in these 6 fra (X) families. Genetic linkage analyses of 2 of the families were reported here., Conclusion: The prevalence of fragile X syndrome in the Chinese is not apparently different from the previous reports on the Caucasian population.

A plasmid sublibrary of the 475 kb insert of YAC209G4 was constructed by using pBS II KS vector. The library of 3,500 clones having 100-600 bp inserts was screened with the probe of the 475 kb insert blocked with competitor DNA. Sixty unique single copy clones were found and sequenced. Checking with GenBank, 28 new STSs were obtained. Genbank accession numbers are U26560-26587. Three STSs were tested by PCR with appropriate primers using human genomic DNA as template and showed specific amplification bands as expected.

Fragile X syndrome [FRA(X)] as the most common form of inherited mental retardation in man has an incidence of one per 1250 and is associated with a fragile site at Xq27.3. A gene was identified at the fragile X locus and was designated Fragile X Mental Retardation-1 (FMR-1). FRA(X) resulted from expansion of (CGG)n trinucleotide repeat in 5' untranslated region of the human FMR-1 gene, and was associated with abnormal methylation of a CpG island 250 bp proximal to this (CGG)n repeat. Males with typical FRA(X) showed repression of FMR-1 transcription and absence of FMR-1 protein, which was believed to contribute to the fragile X phenotype. FMR-1 mRNA extracted from leukocytes in normal and clinically suspected males were detected by RT-PCR. The methylation status and CGG expansion were also studied by PCR and Southern blot. Two of 10 clinically suspected males were found devoid of FMR-1 expression and accompanied with hypermethylation of the CpG island and CGG trinucleotide repeat expansion.

PCR technique was applied to detect hypermethylation at BssH II recognition sites within CpG island of FMR-1 gene in normal, affected, and clinically suspected males with fragile X syndrome. Hypermethylation was found to occur in all 7 affected and 5 of 26 suspected, but none of 15 normal males. The results of PCR assay are in good coincidence with those of Southern blot and cytogenetic assays. This PCR assay will be a promising method in the diagnosis of fragile X syndrome in males with mental retardation.

Fragile X syndrome is characterized as an inherited unstable DNA sequence: the increasement of (CGG)n copy number. It suffered from the inactivation of FMR-1 (fragile X mental retardation 1), which is inhibited by amplification of (CGG)n and methylation of CpG island. The (CGG)n repeat variation in normal Chinese population was detected by PCR with sequencing gel analysis. We also analysed the amplification of (CGG)n and methylation of CpG island at Xq27.3 from 15 individuals in 6 families by Southern hybridization. These results indicate that abnormal methylation of CpG island always occurs in Fra (X) patients together with large amplification of (CGG)n. The (CGG)n could be either stable or amplified in springs of carrier females. These suggest that there would be a new genetic mechanism dynamic mutation.