Data on the CGG repeat at the fragile X site in the non-retarded Japanese population and family suggest the presence of a subgroup of normal alleles predisposing to mutate

The fragile X mutation is the result of amplification in the repeat number of p(CGG) _n in FMR-1; alleles with more than 52 repeats have been shown to be so unstable as to mutate in the repeat number in almost every transmission. To improve our understanding of mutations in normal alleles of FMR-1, the following studies were carried out in the Japanese population: a study on length variation in the repeat to determine the allele distribution of the repeat length in a non-retarded population, family studies to observe new mutations in normal allele, and haplotype analyses with microsatellite markers flanking the repeat to confirm estimated mutation rates and founder chromosomes in the fragile X syndrome. Analysis of the p(CGG) _n in 370 unrelated males detected 24 distinct alleles with repeats of 18–44. A comparison with previously reported data suggests the presence of racial/ethnic differences in the allele distribution. No premutation allele was found in 824 unrelated X chromosomes examined by the polymerase chain reaction and Southern blot analysis. Family studies detected one new mutation in a total of 303 meioses. However, the mutation rate was not in accordance with the expected or observed heterozygosities in the population or with linkage disequilibrium observed between the repeat numbers and the haplotypes of the markers flanking the CGG. The haplotype in the chromosome in which the new mutation was found was the same as that frequently found in the Japanese fragile X chromosomes, and the variance in the CGG repeat number was wider in chromosomes with the haplotypes frequently found in the fragile X chromosome than in those with the other haplotypes. These observations suggest that a subgroup is present in normal alleles and that this subgroup is more liable to mutate than others.     

Linkage disequilibrium between the fragile X mutation and two closely linked CA repeats suggests that fragile X chromosomes are derived from a small number of founder chromosomes

In order to investigate the origin of mutations responsible for the fragile X syndrome, two polymorphic CA repeats, one at 10 kb (FRAXAC2) and the other at 150 kb (DXS548) from the mutation target, were analyzed in normal and fragile X chromosomes. Contrary to observations made in myotonic dystrophy, fragile X mutations were not strongly associated with a single allele at the marker loci. However, significant differences in allelic and haplotypic distributions were observed between normal and fragile X chromosomes, indicating that a limited number of primary events may have been at the origin of most present-day fragile X chromosomes in Caucasian populations. We propose a putative scheme with six founder chromosomes from which most of the observed fragile X–linked haplotypes can be derived directly or by a single event at one of the marker loci, either a change of one repeat unit or a recombination between DXS548 and the mutation target. Such founder chromosomes may have carried a number of CGG repeats in an upper-normal range, from which recurrent multistep expansion mutations have arisen.     

Linkage disequilibrium and linkage analysis of the glucose-6-phosphatase gene

Recent studies have indicated that the four most common mutations account for 78% of mutant alleles in the glucose-6-phosphatase (G6Pase) gene. A significant fraction of mutant alleles remain unidentified. Thus, informative polymorphic markers are necessary for linkage analysis in carrier testing and prenatal diagnosis in families where mutations can not be identified. The common mutations appear to be ethnic-specific, suggesting that the individual mutations may have a common founder. With the recent discovery of the nucleotide 1176 polymorphism, we have studied whether these mutations are in linkage disequilibrium with the polymorphism. The results of polymerase chain reaction/allele-specific oligonucleotide analysis show that nucleotide 1176 C is in linkage disequilibrium with mutations R83 C and R83H, and with the splicing mutation 727G→T. The 1176 T polymorphism is in linkage disequilibrium with 459insTA. A GT repeat polymorphism has also been found. However, its heterozygosity is low. The 1176 nucleotide polymorphic marker can be used in carrier and prenatal diagnosis of GSD1a families that have unidentified mutations and are informative for this marker. Received: 27 January 1998 / Accepted: 17 April 1998     

Linkage homogeneity near the fragile X locus in normal and fragile X families

The fragile X syndrome locus, FRAXA, is located at Xq27. Until recently, few polymorphic loci had been genetically mapped close to FRAXA. This has been attributed to an increased frequency of recombination at Xq27, possibly associated with the fragile X mutation. In addition, the frequency of recombination around FRAXA has been reported to vary among fragile X families. These observations suggested that the genetic map at Xq27 in normal populations was different from that in fragile X populations and that the genetic map also varied within the fragile X population. Such variability would reduce the reliability of carrier risk estimates based on DNA studies in fragile X families. Five polymorphic loci have now been mapped to within 4 cM of FRAXA--DXS369, DXS297, DXS296, IDS, and DXS304. The frequency of recombination at Xq26-q28 was evaluated using data at these loci and at more distant loci from 112 families with the fragile X syndrome. Two-point and multipoint linkage analyses failed to detect any difference in the recombination fractions in fragile X versus normal families. Two-point and multipoint tests of linkage homogeneity failed to detect any evidence of linkage heterogeneity in the fragile X families. On the basis of this analysis, genetic maps derived from large samples of normal families and those derived from fragile X families are equally valid as the basis for calculating carrier risk estimates in a particular family.     

Different haplotypes for cystic fibrosis-linked DNA polymorphisms in Polish and Dutch populations

Summary We analyzed DNA from 34 Polish and 63 Dutch cystic fibrosis (CF) patients and their families using the polymorphic markers XV2c and KM19, which are in linkage disequilibrium with the CF mutation. Strong linkage disequilibrium was found in the Dutch population sample, but the haplotypes of the Polish chromosomes showed a significantly less extreme disequilibrium. Our data and previous studies indicate that the highest degree of homogeneity of the CF defect and hence the best possible use of the XV2c/KM19/CF linkage disequilibrium for CF carrier detection/exclusion is in populations of northern European origin.     

Molecular heterogeneity of the fragile X syndrome.

The fragile X syndrome is an X-linked disorder which has been shown to be associated with the length variation of a DNA fragment containing a CGG trinucleotide repeat element at or close to the fragile site. Phenotypically normal carriers of the disorder generally have a smaller length variation than affected individuals. We have cloned the region in cosmids and defined the area containing the amplified sequence. We have used probes from the region to analyse the mutation in families. We show that the mutation evolves in different ways in different individuals of the same family. In addition we show that not all fragile X positive individuals show this amplification of DNA sequence even though they show expression of the fragile site at levels greater than 25%. One patient has alterations in the region adjacent to the CGG repeat elements. Three patients in fragile X families have the normal fragment with amplification in a small population of their cells. These observations indicate that there is molecular heterogeneity in the fragile X syndrome and that the DNA fragment length variation is not the only sequence responsible for the expression of the fragile site or the disease phenotype.     

The fragile X syndrome in Finland: demonstration of a founder effect by analysis of microsatellite haplotypes

Microsatellite markers RS46 (DXS548) and FRAXAC2 flanking the fragile X mutation, an expansion of a (CGG)_n repeat within the FMR-1 gene, were typed in 60 unrelated northern and eastern Finnish fragile X families and in a control population from the same geographical region. A significant difference was found in allelic and haplotypic distributions between the normal X and fragile X chromosomes. Evidence for a strong founder effect was detected, with the haplotype 196-153 being present on 80% of the fragile X chromosomes, but on only 8% of the normal X chromosomes. In addition to this major haplotype, four minor haplotypes were found on the fragile X chromosomes. These results suggest that the majority of present-day fragile X mutations in Finland may have a common initial ancestor, probably from the 16th century.     

A high proportion of novel mutations in BRCA1 with strong founder effects among Dutch and Belgian hereditary breast and ovarian cancer families

We have identified 79 mutations in BRCA1 in a set of 643 Dutch and 23 Belgian hereditary breast and ovarian cancer families collected either for research or for clinical diagnostic purposes. Twenty-eight distinct mutations have been observed, 18 of them not previously reported and 12 of them occurring more than once. Most conspicuously, a 2804delAA mutation has been found 19 times and has never been reported outside the Netherlands. A common haplotype spanning ≥375 kb could be identified for each of the nine examined recurrent mutations, indicating the presence of multiple BRCA1 founder mutations in the Dutch population. The 2804delAA mutation has been estimated to have originated ~32 generations ago. No specific breast or ovarian cancer phenotype could be assigned to any of the common mutations, and the ovarian cancer incidence among 18 families with the 2804delAA mutation was heterogeneous.     

Mutation and polymorphism analysis of the TRKA (NTRK1) gene encoding a high-affinity receptor for nerve growth factor in congenital insensitivity to pain with anhidrosis (CIPA) families

The human TRKA gene encodes a high-affinity tyrosine kinase receptor for nerve growth factor. Congenital insensitivity to pain with anhidrosis (CIPA) is an autosomal recessive genetic disorder reported from various countries and characterized by anhidrosis (inability to sweat), the absence of reaction to noxious stimuli, and mental retardation. We have found that TRKA is the gene responsible for CIPA. We have studied TRKA in 46 CIPA chromosomes derived from 23 unrelated Japanese CIPA families. including three that have been previously reported, and identified 11 novel mutations. Four (L93P, G516R, R648 C, and D668Y) are missense mutations that result in amino acid substitutions at positions conserved in the TRK family, including TRKA, TRKB, and TRKC. Three (S131 fs, L579 fs, and D770 fs) are frameshift mutations. Three (E164X, Y359X, and R596X) are nonsense mutations. The other is an intronic branch-site (IVS7-33T-->A) mutation, causing aberrant splicing in vitro. We also report the characterization of eight intragenic polymorphic sites, including a variable dinucleotide repeat and seven single nucleotide polymorphisms, and describe the haplotypic associations of alleles at these sites in 106 normal chromosomes and 46 CIPA chromosomes. More than 50% of CIPA chromosomes share the frameshift mutation (R548 fs) that we described earlier. This mutation apparently shows linkage disequilibrium with a rare haplotype in normal chromosomes, strongly suggesting that it is a common founder mutation. These findings represent the first extensive analysis of CIPA mutations and associated intragenic polymorphisms; they should facilitate the detection of CIPA mutations and aid in the diagnosis and genetic counseling of this painless but severe genetic disorder with devastating complications.     

Fragile X syndrome: diagnosis using highly polymorphic microsatellite markers.

We describe two highly polymorphic microsatellite AC repeat sequences, VK23AC and VK14AC, which are closely linked to the fragile X at Xq27.3. Both VK23AC (DXS297) and VK14AC (DXS292) are proximal to the fragile site. Two-point linkage analysis in 31 fragile X families gave (a) a recombination frequency of 1% (range 0.00%-4%) with a maximum lod score of 32.04 for DXS297 and (b) a recombination frequency of 7% (range of 3%-15%) with a maximum lod score of 12.87 for DXS292. Both of these polymorphisms are applicable to diagnosis by linkage in families with fragile X syndrome. A multipoint linkage map of genetic markers at Xq27.3 was constructed from genotyping these polymorphisms in the CEPH pedigrees. The DXS292 marker is in the DXS98-DXS297 interval and in 3 cM proximal to DXS297.     

Molecular analysis of the (CGG)n expansion in the FMR-1 gene in 59 Spanish fragile X syndrome families

The fragile X mental retardation syndrome is caused by an expansion of a trinucleotide repeat (CGG)_n in the FMR-1 gene. Molecular genetic study of fragile X provides accurate diagnosis and facilitates genetic counseling in families with affected members. We present here the molecular study of 59 Spanish fragile X syndrome families using probe StB 12.3 and the polymerase chain reaction (PCR) of the (CGG)_n repeat sequence of the FMR-1 gene. The results obtained have allowed us to characterize 455 individuals, including eight prenatal diagnoses. The clinical diagnosis of fragile X in 89 affected males was confirmed, 137 female carriers were identified (48 of whom were mentally retarded), 176 individuals at risk were found not to have the expansion, and 12 cases of normal transmitting males (NTM) were detected. In the sample studied, no de novo mutations were detected, nor any mutation different from that described for the (CGG)_n expansion. One nonmentally retarded male was detected as having an unmethylated CpG island for the FMR-1 gene, but with more than 200 CGG repeats (high functioning male). The analysis of the (CGG)_n repeat in 208 normal chromosomes gave an allele distribution similar to that in other Caucasoid population groups, with alleles of 29 and 30 CGG repeats accounting for 46% of the chromosomes. The combination of Southern analysis and PCR of the (CGG)_n repeat is highly efficient for diagnosis, compared with cytogenetic techniques, especially in the detection of female carriers, NTMs, and prenatal diagnosis, enabling accurate genetic counseling to be provided in all cases.     

Two novel polymorphic sequences in the glucocerebrosidase gene region enhance mutational screening and founder effect studies of patients with Gaucher disease

Gaucher disease, an inherited glycolipid storage disorder, is caused by a deficiency of the catabolic enzyme glucocerebrosidase (EC 3.2.1.45). The gene for human glucocerebrosidase is located on chromosome 1q21 and has a highly homologous pseudogene situated 16 kb downstream. We report two novel polymorphic sequences in the glucocerebrosidase gene region: the first consists of a variable number of dinucleotide (CT) repeats located 3.2 kb upstream from the glucocerebrosidase gene, and the second is a tetranucleotide (AAAT) repeat found between the glucocerebrosidase gene and its pseudogene, 9.8 kb downstream from the functional gene. These polymorphic sequences, along with a previously reported PvuII polymorphism in intron 6 of the glucocerebrosidase gene, were analyzed in patients with Gaucher disease (n=106) and in two normal control populations, one of Ashkenazi Jewish ancestry (n=72) and the second comprising non-Jewish individuals (n=46). In these samples, strong linkage disequilibrium was found between mutations N370S, c.84–85insG, and R463C and specific haplotypes; no significant linkage disequilibrium was found when examining haplotypes of patients with the L444P mutation. Studies of these polymorphic sites in several instances also led to the recognition of genotyping errors and the identification of unusual recombinant alleles. These new polymorphic sites provide additional tools for mutational screening and founder effect studies of Gaucher disease. Received: 5 December 1998 / Accepted: 14 January 1999     

Mutation of the start codon in the FRDA1 gene: linkage analysis of three pedigrees with the ATG to ATT transversion points to a unique common ancestor

Friedreich ataxia (FRDA) is a progressive neurodegenerative disorder caused by loss-of-function mutations in the gene encoding frataxin. Most patients with FRDA have trinucleotide repeat expansions in both alleles of the FRDA1 gene. In patients heterozygous for the expansion the second allele may be inactivated by a point mutation. We identified the ATG→ATT (M1I) mutation of the start codon in three independent families. Individuals with symptoms of FRDA in these families are compound heterozygous for the repeat expansion and the ATG mutation. To look for a common founder of the M1I mutation, a detailed linkage analysis employing six polymorphic chromosome 9 markers was performed. We found complete haplotype identity for two of the three chromosomes with the point mutation. The third case shows partial conformity and may be the result of a single recombination event. Received: 13 February 1998 / Accepted: 18 March 1998     

Connexin 26 (GJB2) mutations in the Turkish population: implications for the origin and high frequency of the 35delG mutation in Caucasians

Mutations in the Connexin 26 (GJB2/Cx26) gene are responsible for more than half of all cases of prelingual non-syndromic recessive deafness in many Caucasian populations. To determine the importance of Cx26 mutations as a cause of deafness in Turks we screened 11 families with prelingual non-syndromic deafness, seven (64%) of which were found to carry the 35delG mutation. We subsequently screened 674 Turkish subjects with no known hearing loss and found twelve 35delG heterozygotes (1.78%; 95% confidence interval: 0.9%-3%) but no examples of the 167delT mutation. To search for possible founder effects, we typed chromosomes carrying the 35delG mutation for closely linked polymorphic markers in samples from Turkey and United States and compared the allele frequencies with those of hearing subjects. The data showed a modest degree of disequilibrium in both populations. Analyses of two pedigrees from Turkey demonstrated both conserved and different haplotypes, suggesting possible founder effects and multiple origins of the 35delG mutation.     

Testing models of selection and demography in Drosophila simulans

We analyze patterns of nucleotide variability at 15 X-linked loci and 14 autosomal loci from a North American population of Drosophila simulans. We show that there is significantly more linkage disequilibrium on the X chromosome than on chromosome arm 3R and much more linkage disequilibrium on both chromosomes than expected from estimates of recombination rates, mutation rates, and levels of diversity. To explore what types of evolutionary models might explain this observation, we examine a model of recurrent, nonoverlapping selective sweeps and a model of a recent drastic bottleneck (e.g., founder event) in the demographic history of North American populations of D. simulans. The simple sweep model is not consistent with the observed patterns of linkage disequilibrium nor with the observed frequencies of segregating mutations. Under a restricted range of parameter values, a simple bottleneck model is consistent with multiple facets of the data. While our results do not exclude some influence of selection on X vs. autosome variability levels, they suggest that demography alone may account for patterns of linkage disequilibrium and the frequency spectrum of segregating mutations in this population of D. simulans.     

COL4A3 founder mutations in Greek-Cypriot families with thin basement membrane nephropathy and focal segmental glomerulosclerosis dating from around 18th century

Mutations in the COL4A3/COL4A4 genes of type IV collagen account for about 40% of cases of thin basement membrane nephropathy, a condition that is estimated to affect 1% or more of the general population. We recently described 10 Cypriot families with familial hematuria and thin basement membrane nephropathy in the presence of focal segmental glomerulosclerosis, with founder mutations on COL4A3 gene. Seven of the families carried mutation G1334E on haplotype K, and another three carried mutation G871C on haplotype Ky. In this report we performed extension of the haplotypes with additional polymorphic markers, 12 for haplotype K and 22 for haplotype Ky, to estimate the linkage disequilibrium value between the mutation and flanking noncommon markers. Haplotype Ky extended to 13.71 Mb, but we did not attempt further analysis owing to the small number of chromosomes. Haplotype K extended to 3.83 Mb, thereby suggesting that it was a much older event compared to mutation G871C. Mutation G1334E was calculated to be about 5-10 generations old with a possible origin between 1693 and 1818 AD, during the Ottoman ruling of the island. Both mutations are clustered in specific geographic regions with apparently formerly isolated populations, although mutation G1334E has been detected elsewhere on the island. The identification of founder mutations in large families with microscopic hematuria greatly facilitates presymptomatic diagnosis and provides useful information on the history of the population, while it may also assist in association studies in search for disease modifier genes.     

Pathology of unstable sequence of genome: fragile-X-syndrome]

Fragile X syndrome is the most frequent form of inherited mental retardation and is associated with a fragile site at Xq27-3. This fragile site is an unstable microsatellite repeat, p(CCG). In fragile X syndrome families, this sequence exhibits variable amplification, the length of which correlates with phenotype. Affected persons have both a "full mutation" and abnormal DNA methylation. Subjects with smaller increase of this sequence, called "premutation", have little or no risk retardation, but are at high risk of having affected children or grandchildren. The passage from "premutation" to "full mutation" status occurs only with transmission from the mother. The unusual segregation patterns in fragile X pedigrees is referred to as the Sherman paradox, now elucidated by genotypic analysis. We present here a brief review of this pathology and illustrate the use of this new diagnostic test in our laboratory.     

Linkage disequilibrium and haplotype analysis in German Friedreich ataxia families

The main mutation causing Friedreich ataxia (FRDA) is the expansion of a GAA repeat localized within the intron between exon 1 and exon 2 of the gene X25. This expansion has been observed in 98% of FRDA chromosomes. To analyze frequencies of markers tightly linked to the Friedreich ataxia gene and to investigate wheter a limited number of ancestral chromosomes are shared by German FRDA families, a detailed analysis employing nine polymorphic markers was performed. We found strong linkage disequilibria and association of FRDA expansions with a few haplotypes. FRDA haplotypes differ significantly from control haplotypes. Our results confirm that GAA repeat expansions in intron 1 of the frataxin gene are limited to a few chromosomes and indicate an obvious founder effect in German patients. Based on these analyses, we estimate a minimum age of the mutation of 107 generations.     

Recurrent and Founder Mutations in the Netherlands: the Long-QT Syndrome

Background and objective

The long-QT syndrome (LQTS) is associated with premature sudden cardiac deaths affecting whole families and is caused by mutations in genes encoding for cardiac proteins. When the same mutation is found in different families (recurrent mutations), this may imply either a common ancestor (founder) or multiple de novo mutations. We aimed to review recurrent mutations in patients with LQTS.

Methods

By use of our databases, we investigated the number of mutations that were found recurrently (at least three times) in LQT type 1–3 patients in the Netherlands. We studied familial links in the apparently unrelated probands, and we visualised the geographical distribution of these probands. Our results were compared with published literature of founder effects in LQTS outside the Netherlands.

Results

We counted 14 recurrent LQT mutations in the Netherlands. There are 326 identified carriers of one of these mutations. For three of these mutations, familial links were found between apparently unrelated probands.

Conclusion

Whereas true LQT founder mutations are described elsewhere in the world, we cannot yet demonstrate a real founder effect of these recurrent mutations in the Netherlands. Further studies on the prevalence of these mutations are indicated, and haplotype-sharing of the mutation carriers is pertinent to provide more evidence for founder mutation-based LQTS pathology in our country.     

A fragile X case with an amplification/deletion mosaic pattern

Fragile X syndrome is the most common cause of hereditary mental retardation. The FMR1 gene, which is involved in fragile X syndrome, contains a polymorphic CGG repeat, which expands in affected patients. Expanding triplet repeats have been shown to be a new type of mutation, termed "dynamic mutation", responsible for more than 12 genetic diseases. These mutations occur as multiple steps rather than as a single event. The first step leads to an unstable allele that then becomes increasingly unstable generally achieving further increases in copy or occasionally contraction. In this report, we describe a fragile X boy with both a hypermethylated full mutation and a deletion of 905 bp encompassing the CGG repeat. The upstream breakpoint is 438 bp 5' to the CGG repeat and the downstream breakpoint is 420 bp 3' of the triplet repeats. The deletion includes the ATG starting codon for translation of the FMR1 gene. This was confirmed by using FMRP immunocytochemistry both on blood smears and hair roots. The deleted region is flanked by a ccgg direct repeat next to the breakpoints; this may have had a critical role in the formation of a secondary DNA structure leading to the deletion.