Population screening at the FRAXA and FRAXE loci: molecular analyses of boys with learning difficulties and their mothers

Preliminary results on a large population-based molecular survey of FRAXA and FRAXE are reported. All boys with unexplained learning difficulties are eligible for inclusion in the study and data are presented on the first 1013 tested. Individuals were tested for the number of trinucleotide repeats at FRAXA and FRAXE and typed for four flanking microsatellite markers. Mothers of 760 boys were tested to determine the stability of the FRAXA and FRAXE repeats during transmission and to provide a population of control chromosomes. The frequency of FRAXA full mutations was 0.5%, which gives a population frequency of 1 in 4994, considerably less than previous reports suggest. No FRAXE full mutations were detected, confirming the rarity of this mutation. In the boys' X chromosomes, we detected one FRAXA premutation with 152 repeats and one putative FRAXE premutation of 87 repeats. No full or premutations were seen in the control chromosomes. A significant excess of intermediate alleles at both FRAXA and FRAXE was detected in the boys' X chromosomes by comparison with the maternal control chromosomes. This suggests that relatively large unmethylated repeats of sizes 41-60 for FRAXA and 31-60 for FRAXE may play some role in mental impairment. No instability was found in transmissions of minimal or common alleles in either FRAXA or FRAXE, but we saw two possible instabilities in transmission of FRAXA and two definite instabilities in transmission of FRAXE among 43 meioses involving intermediate or premutation sized alleles. We found no linkage disequilibrium between FRAXA and FRAXE but did find significant linkage disequilibrium between large alleles at FRAXE and allele 3 at the polymorphic locus DXS1691 situated 5 kb distal to FRAXE.      

FRAXA and FRAXE: the results of a five year survey

We report the results of a five year survey of FRAXA and FRAXE mutations among boys aged 5 to 18 with special educational needs (SEN) related to learning disability. We tested their mothers using the X chromosome not transmitted to the son as a control chromosome, and the X chromosome inherited by the son to provide information on stability of transmission. We tested 3738 boys and 2968 mothers and found 20 FRAXA and one FRAXE full mutations among the boys and none among the mothers. This gives an estimated prevalence of full mutations in males of 1 in 5530 for FRAXA and 1 in 23 423 for FRAXE.
We found an excess of intermediate and premutation alleles for both FRAXA and FRAXE. For FRAXA this was significant at the 0.001 level but the excess for FRAXE was significant only at the 0.03 level. We conclude that the excess of intermediate and premutation sized alleles for FRAXA may well be a contributing factor to the boys' mental impairment, while that for FRAXE may be a chance finding.
We studied approximately 3000 transmissions from mother to son and found five instabilities of FRAXA in the common or intermediate range and three instabilities of FRAXE in the intermediate range. Thus instabilities in trinucleotide repeat size for FRAXA and FRAXE are rare, especially among alleles in the common size range.


Keywords: FRAX syndromes; incidence; intermediate alleles; stability     

Molecular screening of fragile X (FRAXA) and FRAXE mental retardation syndromes in the Hellenic population of Greece and Cyprus: incidence, genetic variation, and stability.

This study presents the first large, population-based molecular investigation of the fragile X (FRAXA) and FRAXE mental retardation syndromes in the Hellenic populations of Greece and Cyprus. The aims of this population screening were to determine the prevalence of FRAXA and FRAXE syndromes among idiopathic mentally retarded (IMR) individuals, to estimate the incidence in the general population, and to investigate the molecular mechanism of instability and expansion of the FMR1-repeat. Ten FRAXA patients were identified to have either the full mutation (eight) or premutation (two) from a Hellenic population of 866 unrelated IMR individuals (611 males and 255 females, age range 3-25 years). No FRAXE patients were identified among the 611 IMR males. The incidence of FRAXA in the Hellenic population of Cyprus is estimated at 1 in 4,246 males. The repeat sites from the FMR1 and FMR2 alleles were accurately determined and showed similar distribution and frequencies with other population studies. The analysis of AGG interspersion within the FMR1-repeat in normal males revealed long, pure CGG repeats within the "gray zone" as well as variation within the 3' end showing polarity of instability. This finding supports the hypothesis that the AGG interspersion and the length of the pure repeat are major factors in determining allele stability. Analysis of FRAXAC1, DXS548, and FRAXAC2 identified particular alleles and haplotypes to have a significant association with either gray zone alleles or alleles >15 pure CGG repeats. We hypothesize that this subgroup of alleles and haplotypes are associated with long pure CGGs (>15 CGG) or 35 repeats and, having shared an evolutionary past, would have the tendency to expand.     

Studies of FRAXA and FRAXE in women with premature ovarian failure.

Recent reports suggest that women with FRAXA premutations have an increased likelihood of having premature ovarian failure (POF). We screened 147 women with idiopathic POF for the number of trinucleotide repeats at the FRAXA and FRAXE loci. We found six women with FRAXA premutations, including four familial and two sporadic cases, but no women with FRAXA full mutations. At the FRAXE locus there were no pre- or full mutations but there was an excess of small alleles with fewer than 11 repeats, including at least one small deletion at or near the triplet. The association of FRAXA premutations with POF confirms that premutation alleles can affect ovarian development or function or both.     

A new PCR assay useful for screening of FRAXE/FMR2 mental impairment among males

FRAXE (FMR2) is a fragile site associated with mental impairment located in Xq28, 600 kb distal to FRAXA (FMR1), the fragile X syndrome fragile site. The FRAXE mutation is an expansion of a CCG repeat that results in methylation of a nearby CpG island. FRAXE alleles could be divided into four categories: normal (6–30 CCG repeats), intermediate (31–60 CCG repeats), premutation (61–200 CCG repeats), and full mutation (over 200 repeats). We have developed a non‐isotopic polymerase chain reaction (PCR)‐based assay for the identification of FRAXE full mutation alleles among mentally impaired men. In this novel PCR test for the FRAXE locus, we used three primers to permit an amplification of a 223 bp monomorphic internal control fragment in addition to the amplification of a 419 bp (CCG)₁₆ FRAXE locus band. A linear series of 93 male patients referred for FRAXE testing but found to be negative for the (CCG)_n expansion in the FMR2 gene by Southern blotting analysis were retested by our PCR technique. In addition, we analyzed two positive controls consisting of a FRAXE fully mutated male and one male with a Xq terminal deletion. The developed PCR test showed accuracy of 100% in the normal individuals retested by PCR analysis, as well as in the two positive control samples utilized, in which the strategy of multiplex amplification worked as expected. Although not suitable for medical diagnosis of females and mosaics, it constitutes an important strategy for PCR typing and for FRAXE population screening. Hum Mutat 18:157–162, 2001. © 2001 Wiley‐Liss, Inc.     

Stability and haplotype analysis of the FRAXE region

FRAXE full mutations are rare and appear to be associated with mild mental retardation. As part of a screening survey of boys with learning difficulties to determine the frequency of full and premutations, we have collected data on the frequency of instability at FRAXE for about 4000 transmissions and the haplotype for over 7000 chromosomes. The distribution of FRAXE repeats was similar to other English populations but differed from two North American Caucasian series. Observed instability at FRAXE was rare but increased with increasing repeat number, and there were no expansions into the full mutation range, except in pedigrees ascertained through a full mutation. Haplotype analysis suggested division into five groups with each group having a characteristic distribution of FRAXE repeats. Fourteen of the 15 full mutations occurred on a single haplotype and this haplotype also had a significant excess of intermediate-sized alleles, suggesting that full mutations originate from large normal alleles. However, a related haplotype also had a significant excess of intermediates but we observed no full mutations on this haplotype, suggesting either loss or gain of stability determinants on it. We suggest that whilst triplet repeat size is a significant predisposing factor for expansion at FRAXE other genetic determinants are also likely to be important.     

Instability of the FMR2 trinucleotide repeat region associated with expanded FMR1 alleles

The fragile sites FRAXA and FRAXE, located ∼600 kb apart on Xq27.3 and Xq28, respectively, are due to a CGG trinucleotide repeat expansion. Although the expansion mechanism for these and other trinucleotide repeat disorders remains unknown, the similarities between the FRAXA and FRAXE regions suggest a possible association between the 2 sites. DNA from 953 individuals was analyzed to determine the distribution of FRAXE repeat sizes in this population and to ascertain potential association between FRAXA and FRAXE repeat sizes. Thirty-four FMR2 alleles ranging from 3–42 repeats were identified. No FRAXE expansions were found in this population, supporting previous findings that FRAXE expansions are rare. However, in the fragile X syndrome affected group, a FMR2 delection, 2 cases of FRAXE repeat instability and a FRAXE mosaic male were identified. Also, a previously identified, rare FMR2 polymorphism was observed. Statistical analyis showed no correlation between normal FRAXA and FRAXE repeat sizes studied, although there was a significant size difference in larger FMR2 alleles that segregated with expanded FMR1 alleles. These findings support the idea of an association between repeat expansion in the FMR1 gene and instability or deletions in the FMR2 gene. Am. J. Med. Genet. 73:447–455, 1997. © 1997 Wiley-Liss, Inc.     

Survey of the fragile X syndrome and the fragile X E syndrome in a special education needs population

To begin to understand the population dynamics of the fragile X (FRAXA) mutation and to learn more about the fragile X E (FRAXE) syndrome, we have initiated a survey of children in special needs education programs in the public school system. With respect to the FRAXA syndrome, we found approximately 1/1,000 full mutations among males. No large alleles at the FRAXE locus were observed among 462 individuals. The allele distributions at the two loci among Caucasians and among African Americans were examined as well as the level of heterozygosity. We found a significant difference in the FRAXA allele distribution among the two ethnic groups; the major difference was due to the lack of smaller alleles among the African Americans. No difference was found for the FRAXE allele distribution among the two groups. The level of heterozygosity was less than predicted by the allele distribution at both loci. This is probably due to unidentified large alleles among females with a test result of a single band. Alternatively, this excess may indicate that the population is not at equilibrium. © 1996 Wiley-Liss, Inc.     

Prevalence of the fragile X syndrome in Yugoslav patients with non-specific mental retardation

Mutations at two fragile sites, FRAXA and FRAXE, loci are caused by an expansion of a CGG/GCC trinucleotide repeat and are characterized by mental retardation. Here we report molecular screening survey of 97 unrelated individuals diagnosed with non-specific mental retardation (MR), which produced positive test for FRAXA in two boys and none positive for the FRAXE mutation. In addition, we studied allelic frequency distribution for the FRAXA locus in this group of mentally retarded patients, as well as in the 99 healthy subjects of Yugoslav population. The distribution of FMR1 CGG repeat size in both groups was similar: the most common allele contained 29 repeats (32.86% in the healthy population and 54.54% in MR population), followed by the allele with 28 CGG repeats (21.43% in the healthy and 12.2% in MR population). Premutation alleles with more than 45 repeats were not found in control nor in the MR group.     

FRAXE mutation analysis in three spanish families

Very little is known about the phenotype of FRAXE-positive individuals and the relation between the genotype/phenotype and genotype/cytogenetic expression. We describe three families with normal and mildly affected individuals and a severely retarded male expressing fragility at the FRAXE locus or presenting different expansions at the CGG FRAXE triplet. In addition, we analyze the FRAXE mutation in sperm DNA from a retarded male carrier with a handicapped daughter expressing fragility at the FRAXE locus. Mental status in FRAXE individuals is highly variable and, although mild mental retardation is observed in most cases, several carrier males are apparently normal. It seems that methylation is not as strictly associated with size of CGG triplets in the FRAXE locus as in FRAXA, and it is possible that normal carrier individuals with fully methylated increments in lymphocytes have a certain proportion of unmethylated alleles in the critical (i.e., neural) tissues. FRAXE mutation is apparently similar to FRAXA in that males with somatic large methylated increments are carriers of small unmethylated ones in germinal cells. © 1996 Wiley-Liss, Inc.     

Fragile X AGG analysis provides new risk predictions for 45–69 repeat alleles

We investigated the effect of AGG interruptions on fragile X repeat instability upon transmission of fragile X intermediate and small premutation alleles with 45–69 CGG repeats. The FMR1 repeat structure was determined for 375 mothers, 48 fathers, and 538 offspring (457 maternal and 81 paternal transmissions) using a novel PCR assay to determine repeat length and AGG interruptions. The number of AGG interruptions and the length of uninterrupted CGG repeats at the 3′ end were correlated with repeat instability on transmission. Maternal alleles with no AGGs conferred the greatest risk for unstable transmissions. All nine full mutation expansions were inherited from maternal alleles with no AGGs. Furthermore, the magnitude of repeat expansion was larger for alleles lacking AGG interruptions. Transmissions from paternal alleles with no AGGs also exhibited greater instability than those with one or more AGGs. Our results demonstrate that characterization of the AGG structure within the FMR1 repeat allows more accurate risk estimates of repeat instability and expansion to full mutations for intermediate and small premutation alleles. © 2013 Wiley Periodicals, Inc.     

FRAXE and mental retardation.

Mental impairment and instability of the CCG repeat at FRAXE is described in six kindreds. Cosegregation of FRAXA and FRAXE was found within one of these kindreds. Cytogenetic expression of FRAXE was shown to skip a generation when associated with a reduction in size of the CCG expansion when transmitted through a male; however, in general, transmission occurred through females and a copy number increased from one generation to the next. In these respects the behaviour of FRAXE paralleled that of FRAXA. A relationship between FRAXE and non-specific mental impairment is strongly suggested by the occurrence in these families of more mentally impaired male and female carriers, after removal of index cases, than could reasonably be expected by chance.     

A high-density SNP map for the FRAX region of the X chromosome. Single-nucleotide polymorphisms

Single-nucleotide polymorphisms (SNPs) are the most common type of genetic variation within the human genome, occurring approximately once every kilobase. However, for association studies, SNPs are not as informative as microsatellite markers and a large number of SNPs and substantial population sizes are required for linkage and mapping studies. A SNP map was generated for the FRAX region of the X chromosome, approximately 0.8 Mb proximal and 1.8 Mb distal to the FRAXA repeat, at a density of at least 1 SNP every 100 kb. SNPs were identified in a population of 28 women with a FRAXA expan-sion (including three women with a FRAXE expansion) on a background of different DXS548, CA1 and CA2 haplotypes, and a normal X chromosome with a different microsatellite haplotype. Fifty-four polymorphisms were identified in a total of 52 257 bp distributed over 2.6 Mb. This represented about 1 SNP every 1024 bp, which was consistent with a nondesert region (1 : 1000 bp). Because the SNPs identified in this study have haplotype and frequency data from an affected population, they should provide a useful resource for researchers to investigate the genetic mechanisms behind instability and expansion of both FRAXA and FRAXE triplet repeats.     

DNA diagnosis of FRAXA and FRAXE in Chinese children with neurodevelopmental disorders and fragile X syndrome

Fragile X (FraX) syndrome is the most common cause of inherited mental retardation. To see whether FRAXA or FRAXE can account for the etiology of some unexplained neurodevelopmental disorders in children, we screened for trinucleotide repeat expansion in a consecutive cohort of 73 Chinese children and their mothers seen in 1995 (group 1) referred for developmental assessment due to developmental delay, language delay, attention deficit hyperactivity disorder, autistic spectrum disorder, mental retardation and/or learning disability. We also screened DNA samples of all five previously diagnosed cytogenetically-positive FraX boys, their mothers and sisters (group 2). A control group of unrelated teenagers and adults were recruited from the community (group 3). In group 1, 3 families (2 mothers and a mother and her son) were found to carry a small premutation allele at FRAXA (premutation frequency = 2%, 3/153 independent X chromosomes), but none had any expansion at FRAXE. In group 2, all 5 FraX boys had full mutation at FRAXA and normal repeat length at FRAXE. In group 3, 1 male has a premutation allele out of 18 males and 59 females tested (premutation frequency of control = 0.7%, 1 out of 136 X chromosomes). For FRAXE screening in group 3, 2 females were carriers (1.5%, 2 out of 136 X chromosomes). Thus, FRAXA and FRAXE cannot account for the etiology of neurodevelopmental disorders in our cohort of Chinese children, and the prevalence of FRAXE mutation in normal Chinese population appears to be higher than reported in the Caucasians.     

FRAXA与FRAXE位点的快速基因筛查

Ｘ染色体长臂末端罕见脆性位点Ａ与Ｅ与分别由一个可遗传的三核苷酸重复序列的延长所致二种脆 性位占在细胞遗传学水平难以区别。为了建立快速筛查ＦＲＡＸＡ与ＦＲＡＸＥ位点的基因诊断法，本文采用毛细管ＰＣＲ－序列胶银染显示法对正常人，脆性Ｘ综合征家系及智力儿童三组样本进行了检测，结果显示正常人群的Ｐ（ＣＧＧ）ｎ与Ｐ（ＧＣＣ）ｎ均呈多态分面     

Possible founder effects for FRAXE alleles.

To determine if FRAXE alleles may have haplotype associations with nearby microsatellites, we analyzed 149 unrelated control Caucasian X chromosomes for FRAXE GCC alleles along with five nearby microsatellites. The microsatellites included three that are new; GT25, CA4, and CA5 located approximately 24, approximately 48, and approximately 50 kb proximal to the FRAXE GCC repeat, and two that were identified previously: DXS8091 and DXS1691, located approximately 90 and approximately 5 kb distal. No significant correlations between haplotypes for the proximal microsatellites were found. Significant correlations of FRAXE GCC repeats and distal microsatellite allele sizes, DXS8091 (r = 0.24) and DXS1691 (r = -0.40), were found. One haplotype, 18-19 of DXS8091-DXS1691, was present on 57% of chromosomes with > or =22 FRAXE repeats but present on only 10% with <22 repeats. We conclude that this distal haplotype association likely reflects a FRAXE allele founder effect. The lack of association or founder effects seen for the three newly identified proximal markers, located within 50 kb of FRAXE GCC, may reflect an unusually high rate of mutation for these microsatellites or a higher rate of recombination in the proximal region.     

The CAG repeat at the Huntington disease gene in the Portuguese population: insights into its dynamics and to the origin of the mutation

Huntington disease (HD) is caused by an expansion of a CAG repeat. This repeat is a dynamic mutation that tends to undergo intergenerational instability. We report the analysis of the CAG repeat in a large population sample (2,000 chromosomes) covering all regions of Portugal, and a haplotype study of (CAG)_n and (CCG)_n repeats in 140 HD Portuguese families. Intermediate class 2 alleles represented 3.0% of the population; and two expanded alleles (36 and 40 repeats, 0.11%) were found. There was no evidence for geographical clustering of the intermediate or expanded alleles. The Portuguese families showed three different HD founder haplotypes associated with 7-, 9- or 10-CCG repeats, suggesting the possibility of different origins for the HD mutation among this population. The haplotype carrying the 7-CCG repeat was the most frequent, both in normal and in expanded alleles. In general, we propose that three mechanisms, occurring at different times, may lead to the evolution from normal CAGs to full expansion: first, a mutation bias towards larger alleles; then, a stepwise process that could explain the CAG distributions observed in the more recent haplotypes; and, finally, a pool of intermediate (class 2) alleles more prone to give rise to expanded HD alleles.