Heritable fragility at 11q13 and 12q13

The chromosomes of two mentally retarded probands were investigated because they were suspected of having the fragile X syndrome. However two other fragilities were detected. In one patient a fra(11)(q13) was found and in the other a fra(12)(q13). Family studies revealed that both fragile sites were real heritable ones. Besides these two heritable fragile sites, the common fragile site at 3p14 was frequently observed. The effects of BUdR, FUdR and methotrexate on the frequency of the three fragilities were studied. The two heritable fragile sites differed from the common fragile site at 3p14 with respect to their inducibility by FUdR and methotrexate.     

Chromosome lesions which could be interpreted as “fragile sites” on the distal end of Xq

Chromosome lesions which could be interpreted as “fragile sites” on the distal end of the long arm of the X chromosome were identified during a cytogenetic study of 160 mentally retarded adult males with no apparent cause of their mental retardation and one normal adult female with a family history of fra (X) syndrome. Peripheral blood samples were cultured in either M199 or RPMI 1640 medium with FUdR or BrdU. Metaphases were examined for chromosome lesions or fragile sites on the distal end of Xq and 3 distinct sites were observed: Xq26, Xq27.2, and Xq27.3. Other chromosome lesions at Xq28 were observed and interpreted as nonspecific telomeric structural changes. Chromosome lesions were observed in cells from 14 of the 161 individuals. These included: 5 patients with an Xq26 site, 2 with the recently reported Xq27.2 site, 4 with the Xq27.3 site (characteristic of the fra (X) syndrome), 2 with nonspecific telomeric structural changes, and one individual with 2 lesions (a nonspecific telomeric structural change and an Xq26 site). Additional research is necessary to determine the frequency and clinical significance, if any, of lesions occurring in this region of the X chromosome and to distinguish among heritable fragile sites, constitutive fragile sites, and nonspecific telomeric structural changes.     

Constitutive fragile sites in fra(X) individuals

Recently, it was proposed that the constitutive fragile site at 3p14 be used as an "internal control" to indicate the effectiveness of the FUdR fragile site induction system. We have tested this hypothesis by determining the frequency of constitutive fragile sites at 1p31, 3p14, and 16q23 in cultures from 42 known fra(X) individuals. At least 50 cells were analyzed from each case. Seventy-four percent (31/42), 95% (40/42) and 90% (38/42) of the fra(X) individuals exhibited frequencies of less than 4% at constitutive fragile sites 3p14, 1p31 and 16q23, respectively. Of the 42 individuals tested, 12 or 28.6% showed no fragility at any of the 3 sites studied. On the other hand, at least one constitutive fragile site was observed in 50 cells studied from over 70% of the 42 people studied. It is suggested that "positive controls" continue to be used, while at the same time recording all fragile sites to identify a combination of constitutive fragile sites that may serve as an internal control indicator, and that DNA marker studies be used to complement cytogenetic testing.     

New heritable fragile site with spontaneous expression at 1q41

The report presents a family ascertained through recurrent spontaneous abortions in which a new heritable fragile site located at 1q41 is segregating. The fragile site is present in the mother and her son. It is expressed spontaneously in 100% of the metaphases from lymphocyte culture using standard conditions. The use of folate deficient medium and the addition of FUdR to the medium did not affect the appearance nor the level of expression of the fragile site. © 1995 Wiley-Liss, Inc.     

Chromosome abnormalities and rare fragile sites detected in azoospermia patients

Summary We have examined constitutional chromosome abnormalities and fragile sites in 40 patients with azoospermia. Chromosome abnormalities were found in four cases. Three cases showed a deletion of the long arm of the Y chromosome 46,X,del(Yq) and the other case had a ring of G group chromosome 46,XY,r(G). In a rare fragile sites test, four fragile site carriers were detected and three rare autosomal fragile sites were identified; fra(8)(q24.1), fra(11)(p15.1), and fra(17)(p12). The expression of these fragile sites were induced specifically by AT-specific DNA ligands, such as distamycin A and Hoechst 33258. In addition, one patient was found to be the case of double ascertainment of fragile sites, fra(8)(q24.1) and fra(17)(p12). The overall frequency of distamycin A-inducible fragile sites in azoospermia patients appeared to be higher than those reported for Japanese healthy subjects and cancer patients. However, no significant relation among fragile sites, clinical and histological findings has been detected so far.     

The fragile X(q27) form of X-linked mental retardation: FUdR as an inducing agent for fra(X)(q27) expression in lymphocytes,fibroblasts, and amniocytes

The effect of FUdR on the expression of fra(X)(q27) was examined in lymphocytes and/or fibroblasts from 16 affected males and 5 carriers from 10 families; six different culture media were used: F10, 5% serum, pH 7.3(37°C); medium 199, 5% serum, pH 7.6(37°C); folate-free 199, 5% serum, pH 7.6(37°C), and these three media with FUdR (0.05 μm). In lymphocytes there was no significant difference in the percentage of fra(X) expressing cells between any of the FUdR-containing media. The highest percentage of expressing cells seen in lymphocytes with FUdR was 56%. The average enhancement in males with FUdR in the 199 and folate-free 199 media was 30%. This relative enhancement with FUdR was very much higher in a few blood specimens delayed in transit and FUdR may prevent some of the false-negative results obtained from mailed specimens. FUdR did not induce the marker in four obligate carriers with previously negative results. The fibroblasts from affected males were grown in the six specific media for the last 48 hr. Two of the six media yielded reproducibly positive results. These were 199-FUdR and folate-free 199-FUdR with mean percentages of expressing cells of 12.8 ± 7.1% and 11.3 ± 6.1%, respectively. F10-FUdR, which contains thymidine, did not permit expression of the marker in fibroblasts and there was no difference in the percentage of fra(X) expression in 199-FUdR media with or without folate. It was concluded that FUdR shows promise as an agent to permit prenatal diagnosis of the condition and to enhance the detection of the marker in lymphocyte cultures.     

Simultaneous expression of the rare and common fragile sites on the X chromosome

The fragile X [fra(X)] syndrome is the most common inherited form of X-linked mental retardation and is associated with a rare folate sensitive fragile site on the X chromosome at band Xq27.3. Recently, a common fragile site located at chromosome band Xq27.2 was delineated (Sutherland & Baker 1990). In order to confirm the previous findings and to further investigate the conditions required for induction of both types of fragile sites, we studied the use of four experimental protocols. Samples from a control male, two fra(X) males and a fra(X) carrier female were studied. Both common and rare fragile sites were seen in the samples from the fra(X) subjects. Up to 4% of cells showed both common and rare fragile sites on the same X chromosome at the 500 band level. The rare and common fragile sites on the X chromosome could be clearly distinguished. From 1 to 3% of the control cells exhibited the common fragile site, while none exhibited the rare fragile site. These protocols should be useful in resolving questionable fra(X) syndrome diagnoses.     

The fragile X chromosome: Current methods

At the International Congress of Human Genetics (Jerusalem, September, 1981) a Workshop was held on the fragile X chromosome; it was entitled “The Fragile X Chromosome: Current Methods.” It was to focus on laboratory methods required to detect or to enhance the detection of the fragile X. The fragile site on the X chromosome is one of 13 proven fragile sites known in humans. (A diagram of these sites is provided.) The fragile site on the X is sensitive to the concentration of folic acid in the medium in which cells are cultured. Cytologically, gaps and breaks are increased at the X fragile site, which is located at band Xq27–28. To enhance expression in lymphocytes, prolongation of culture time to 96 hours, elevation of the pH, diminution of the colcemid effect, and air drying of slides are helpful. The need for methionine in low-folate media can be overridden by the addition of fluorodeoxyuridine (FUdR). Detection of the fragile X in males requires meticulous attention to methods of lymphocyte culture and metaphase preparation and then the examination of a sufficient number of mitoses, eg, 50–100 metaphases per individual. Detection of the fragile X in female carriers is often more difficult. Uniform detection of all obligate female carriers has not been achieved. Difficulty may correlate with increasing age or intelligence of females. Key methodologic advances with the fragile X include the addition of methotrexate, trifluorothymidine or, especially of FUdR to the culture medium. FUdR, for example, is helpful in demonstrating the fragile X in lymphoblastoid cell lines and fibroblasts. Both of these cell types now represent an opportunity to study the biochemistry of the fragile X. The success of the FUdR technique with skin fibroblasts heralds the feasibility of demonstrating the fragile X chromosome in cultured amniocytes. Since the Workshop, it has been reported that with FUdR the fragile X could in fact be detected in 46,XY amniotic fluid cells.     

Chromosome lesions which could be interpreted as "fragile sites" on the distal end of Xq

Chromosome lesions which could be interpreted as "fragile sites" on the distal end of the long arm of the X chromosome were identified during a cytogenetic study of 160 mentally retarded adult males with no apparent cause of their mental retardation and one normal adult female with a family history of fra (X) syndrome. Peripheral blood samples were cultured in either M199 or RPMI 1640 medium with FUdR or BrdU. Metaphases were examined for chromosome lesions or fragile sites on the distal end of Xq and 3 distinct sites were observed: Xq26, Xq27.2, and Xq27.3. Other chromosome lesions at Xq28 were observed and interpreted as nonspecific telomeric structural changes. Chromosome lesions were observed in cells from 14 of the 161 individuals. These included: 5 patients with an Xq26 site, 2 with the recently reported Xq27.2 site, 4 with the Xq27.3 site (characteristic of the fra (X) syndrome), 2 with nonspecific telomeric structural changes, and one individual with 2 lesions (a nonspecific telomeric structural change and an Xq26 site). Additional research is necessary to determine the frequency and clinical significance, if any, of lesions occurring in this region of the X chromosome and to distinguish among heritable fragile sites, constitutive fragile sites, and nonspecific telomeric structural changes.     

Distribution of autosomal fragile sites in specimens cultured for prenatal fragile X diagnosis

We reviewed the distribution of autosomal fragile sites (FS) and spontaneous chromosome breaks or gaps (CB) at chromosome locations other than those recognized as FS from 100 amniotic fluid samples (AF), 19 chorionic villus samples (CVS), and 5 percutaneous umbilical blood samples (PUBS) referred for fragile X [fra(X)] analysis. We present data on the degree of expression of autosomal fragility in AF, CVS, and PUBS samples, and the relationship between degree of expression and induction system. The most common observed FS were: 3p14, 9p32, and 6q26 in AF; 9q32, 3q27, and 8q22 in CVS; and 3p14, Xq22, and 16q23 in PUBS cases. Distribution of FS and CB, when compared by induction system, was not found to be identical. Our data also indicate that the presence of any particular FS cannot be used as an indicator for the effectiveness of the fra(X) induction system in prenatal samples.     

Fragile (X) X-linked mental retardation I: Relationship between age and intelligence and the frequency of expression of fragil (X)(q28)

Members of eight Saskatchewan families with fragile (X) X-linked mental retardation were studied in an attempt to relate frequency to age and intelligence. The mean IQ of 37 affected men was 35 (range 10–66). The mean IQ of 32 carriers was 88 (range 57–119), and the mean IQ of 13 females who remain at risk for being carriers, have no affected sons, and who failed to demonstrate the fra(X) was 100 (range 78–126). We demonstrated a significant inverse relationship between age and frequency of the fra(X) in carriers and in affected males. However, we demonstrated a more highly significant inverse relationship between frequency of the fra(X) and IQ in carriers but to a lesser extent in affected males. Of 32 carriers, only 3 (9.4%) did not demonstrate the fra(X) after addition of 5-fluoro-2′-deoxyuridine (FUdR) to the folic acid and thymidine-reduced culture medium. From these data we would recommend that chromosome studies in individuals at risk for fra(X) X-linked mental retardation be carried out at the youngest age and that the addition of FUdR to culture medium is useful in carrier identification. It is clear that, in at least the carriers, a lower expression of the fra(X) is highly significantly correlated to higher intelligence.     

Infantile autism, fragile (X) (q27.3) and RFLP analysis in an extended Swedish family

In an extended family with eight individuals with infantile autism, in association with other developmental disorders and fragile (X) (q27.3), DNA techniques were used to investigate linkage between X chromosomal probes and the disorder. F9 was not informative and recombination was found between fragile X and DXS15, DXS51 and DXS52.     

Autosomal folate sensitive fragile sites in normal and mentally retarded individuals in Greece

The frequencies of autosomal folate sensitive fragile sites were compared in populations of mentally retarded fra(X) negative (N = 220) and normal children (N = 76) in Greece. In addition, the frequency of autosomal fragile sites was studied in 20 known fra(X) children in order to test if the fra(X) syndrome is associated with general chromosome instability. The frequencies of both common and rare autosomal fragile sites did not differ significantly between the mentally retarded and the normal children, although the rate of expression was considerably higher in the retarded group. Autosomal fragile sites were not increased in the fra(X) patients. Fra(3)(p14) was by far the most frequent one in all groups. The frequency of fra(6)(q26) was found to be considerably higher among the mentally retarded children, this difference being almost statistically significant. Further cytogenetic studies of normal and retarded individuals are required in order to elucidate this point further.     

Bromodeoxyuridine induces chromosomal fragile sited in the canine genome

Peripheral blood lymphocytes from 3 clinically normal domestic dogs were cultured for bromodeoxyuridine (BrdU) induction of fragile site expression. BrdU induced fragile site expression in cells from all 3 dogs. The mean percent of cells with fragile sites and the mean number of fragile sites per cel were significantly increaed in all BrdU incubated cultures compared to control cultures. The frequency of BrdU fragile site expression did not vary significantly among the dogs. Lymphocytes from all 3 dogs expressed BrdU induced autosomal fragile sites. Two BrdU induced fragile sites were identified on the long arm of chromosome 1, one of which was close to or coincident with a previously identified folate sensitive fragile site on this canine chromosome. Lymphocytes from the 2 female dogs also expressed BrdU induced fragile sites on the X chromosome, but BrdU failed to induce fragile sites on the X chromosome from the one male dog in the study. The 2 BrdU-induced fragile sites identified on the long arm of the X chromosome were close to, or coincident with 2 previously described folate-sensitive common fragile sited on the canine X chromosome. This is the fist report of induction of BrdU-inducible fragile sites in the genome of the domestic dog. © 1993 Wiley-Liss, Inc.     

Frequency of tri- or multiradial configurations in fragile X identification

Using the FUdR system for fragile X induction, we have observed no triradial or bisatellited configurations at fra (X) (q27.3) in over 5,000 fra(X) chromosomes examined from over 150 fra(X) individuals. Based on our observations, and those of Turner and Jacobs (1983) and Daniel et al (1984), we hypothesize that triradial configurations may not occur at Xq27 with FUdR induction. To test this hypothesis we cultured whole blood simultaneously in parallel folate-deficient and FUdR fra(X) induction systems, and systematically examined fra(X) chromosomes for triradials. Neither autosomes nor X chromosomes exhibited any apparent triradial figures in the FUdR system, while 1.4% of the fra(X) chromosomes in TC 199 exhibited a triradial. Also we observed one autosomal triradial at 4q35. We conclude that triradial configurations occur in low frequencies in the folate deficient system and seldom if ever in the FUdR system.     

Co-cultivation studies in the expression of fragile(X) (q27) in lymphocytes

Co-cultivation studies in different combinations have been carried out between cells which have been found to be positive for the fragile (X) (q27) and control cells. As no increase or decrease was discernible in the level of Fra(X) detected in 'positive' cells, and no fragility was introduced into the 'negative' cells, it was concluded that there had been no intercellular co-operation, and a diffusible expression or suppression factor was not involved.     

Fra(X)(q27.2), the common fragile site, observed in only one of 760 cases studied for the fragile X syndrome.

Cell cultures from 760 whole blood, amniotic fluid, chorionic villus sample, and peripheral umbilical blood sample specimens were exposed to multiple fra(X)(q27.3) induction systems (none had aphidicolin). Fifty-three exhibited the rare fragile site, fra(X)(q27.3) or FRAXA, none of which demonstrated the common fragile site or FRAXD at band Xq27.2. Only one cell in one of the negative whole blood FUdR-treated cultures from a mentally retarded male showed FRAXD. Therefore, it appears that FRAXD occurs very rarely in cultures treated to induce FRAXA since only one positive cell was observed in over 88,000 analyzed. It appears that very low frequencies of fra(X)(q27) can be accounted for only in part by the presence of the common fragile site since only one of 9 cases, each with one fra(X)(q27) positive cell, exhibited FRAXD and the others were FRAXA. After confirmation of FRAXA with direct DNA testing in a large number of low frequency cases, it should be possible to rely on the detection of very low frequencies of fra(X)(q27.3), e.g., 1% with at least 2 positive cells.     

Fragile site expression in families with von Hippel-Lindau disease

Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder that predisposes to diverse tumors including renal cell carcinoma. Six affected and four unaffected subjects from five families were studied to determine the frequency of fragile site expression. Peripheral lymphocyte cultures from each subject were treated with low folate, 5-fluorodeoxyuridine (FUdR), and FUdR plus caffeine for fragile site induction. A site was considered to be fragile if it was expressed at least two times in half of the affected or unaffected subjects. Of the established sites, four were expressed in the unaffected group (3p14, 6p22, 8q22, and Xp22) and six were expressed in the affected group (3p14, 4q31, 5q31, 7q32, Xp22, and Xq22). Only 3p14 and Xp22 were expressed in both groups. There were four new sites: three (3q26, 6p21, 7p15) in the unaffected group and one (16q24) in the affected group. The 3p14 site was expressed twice as frequently in affected versus unaffected subjects. This finding is of interest because of reports of the involvement of 3p14 in hereditary renal cell carcinoma and in VHL.     

The common fragile site in band q27 of the human X chromosome is not coincident with the fragile X

The common fragile site on the end of the long arm of the human X chromosome has been shown to be at a different location from the rare fragile site which produces the fragile X syndrome of intellectual handicap. The different locations can be clearly seen in chromosomes at about the 550 band level of resolution. This finding should help resolve difficulties in fragile X cytogenetics where expression of the common fragile site can lead to false positive diagnoses.     

Aphidicolin-sensitive specific common fragile sites: A biomarker of exposure to pesticides

In this work, we analyzed the aphidicolin-sensitive common fragile sites in seven females and four males occupationally exposed to pesticides and in ten controls. The same males had been monitored one year earlier in a previous study by the same authors. Results showed enhanced expression in exposed subjects at eight bands, namely, 6q25, 7p22, 7q22, 7q32, 13q14, 14q24, 16q22, and 16q23. Most of these bands were fragile sites andbreakpoints involved in chromosome rearrangements found in hematopoietic tumors. Moreover, six of these bands were already detected, with enhanced expression, in the first monitoring carried out on male subjects. These results indicated that fragile sites analysis is a reproducible cell response to human exposure to pesticides. Environ. Mol. Mutagen. 29:250-255, 1997 © 1997 Wiley-Liss, Inc.