A cytogenetic study in 120 Turkish children with intellectual disability and characteristics of fragile X syndrome

We review the evidence for the frequency of the fragile X syndrome (FXS), other X-linked abnormalities, and other chromosomal disabilities of Turkish pediatric psychiatry outpatients with intellectual disability. Reported clinical features and genetic findings were used in cytogenetic screenings to estimate the prevalence of the fragile X (fra X) and other chromosomal aberrations in 120 patients with mental retardation, language disorders, attention deficit hyperactivity, or developmental delay, in comparison with 30 healthy children. Data on the clinical, intellectual and behavioral findings in 14 fra X positive children (11.7%) is presented. Ten of the 120 patients (8.3%) had enlargement of the heterochromatin region of chromosome 9. Other chromosomal aberrations and autosomal fragile sites (FS) were also observed. There was a statistically significant difference in the autosomal and X-linked FS between the study and control groups (p < 0.05). The tests for the fra X chromosome are likely to be of diagnostic benefit in young children with autism or developmental delay, particularly in speech, and who have large and prominent ears.     

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.     

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.     

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.     

Recent experience in prenatal fra(X) detection

At least 35 cases of prenatal fra(X) diagnosis have been confirmed and reported. Amniotic fluid, fetal blood and chorion -ic villus samples have exhibited fra(X) (q27.3) in cultures from 26 males and 9 females. Here we have detected fra(X) in female and male amniotic fluid specimens, AF1/fra(X),X and AF2/fra(X),Y, respectively, and a male CVS/fra(X),Y using both FUdR and excess thymidine (THY) to demonstrate the marker chromosome. Both FUdR and THY detected fra(X) and usually FUdR was superior to THY with the exception of placental cultures. It was important to examine more than one culture per protocol since no fra(X) was observed in one AF2 FUdR culture while another exhibited 19.2% expression. Similarly, confirmation studies in lung fibroblast cultures for AF2 exhibited 4.3% fra(X) in one lab while another found negative results. A similar observation in whole blood cultures was also made recently by us. In addition, we have recently experienced our first false negative fra(X),X prenatal diagnosis. We have observed another case where only one cell in 300 exhibited fra(X) where the male fetus was 50% at-risk and was referred to us after the 20th week of gestation by sonography. On the basis of our experience we recommend the following: 1) the excess THY fra(X) induction system is effective but not superior to FUdR; 2) at least two duplicate cultures per induction system should be analyzed for the marker chromosome to avoid the possibility of false-negative diagnosis; 3) where fra(X) is not demonstrated or is present in very low frequencies in CVS and/or amniotic fluid cultures, complementary DNA marker studies and/or fetal blood cultures must be made available; 4) gestational age dating by ultrasonography is recommended as early as possible.     

Improved prenatal detection of fra(X)(q27.3): methods for prevention of false negatives in chorionic villus and amniotic fluid cell cultures

The reliable detection of fra(X)(q27.3) in prenatal samples is important for providing genetic counseling. We have identified 5 new cases of prenatal fragile X [fra(X)] detection in 3 chorionic villus sample (CVS) and 2 amniotic fluid (AF) cell cultures. In 4 of the 5 cases, either excess thymidine (THY) or a combination of THY and 5-fluorodeoxyuridine (FUdR) was clearly superior to FUdR alone as fra(X) inducers. Amniocytes from one case were cultured only in RPMI-1640 and later exposed to FUdR or THY separately. They showed only 2% fra(X) while parallel cultures initiated in Chang medium and incubated in RPMI for at least 7 days (recovery) before fra(X) induction exhibited strikingly increased fra(X) frequencies. Chang medium alone will not allow fra(X) induction in AF (Jenkins EC, Brown WT [1986]: "Genetic Disorders and the Fetus: Diagnosis, Prevention and Treatment." New York: Plenum Press, pp 185-204). Now, using CVS cells, we report that only 1% and 0% fra(X) were detected using FUdR or THY in cells cultured in RPMI for 4 days after removal from Chang medium. Cells with 7 days "recovery" in RPMI exhibited increases from 2 to 6%. Therefore, we have found that Chang medium is very helpful when the appropriate recovery time in another medium is allowed before fra(X) induction. Some false negative reports can be attributed to: induction in Chang medium alone; lack of sufficient recovery time after initiating cells in Chang before induction; and unavailability of the excess THY fra(X) induction system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distamycin A-inducible fragile sites and cancer proneness

To determine the baseline frequency of autosomal rare fragile sites in cancer patients, we conducted a population cytogenetic study of 370 patients with leukemias, solid tumors, and other neoplastic disorders. Twenty carriers of rare fragile sites were detected in this patient group. The rare autosomal fragile sites were at fra(8)(q24), fra(11)(p15), fra(16)(p12.1), fra(16)(q22), and fra(17)(p12). All of these fragile sites were found to be distamycin A inducible. Compared with a population incidence in healthy subjects (44 of 845, 5.21%), the overall incidence of distamycin A-inducible fragile sites was not higher in the patient group (20 of 370, 5.41%). Analysis of these individual fragile sites and particular diseases, however, suggests that the distamycin A-inducible fragile sites may play a role in the etiology of leukemia, myeloproliferative disorders, and benign tumors.     

Nature of distamycin A-inducible fragile sites

Five rare distamycin A-inducible fragile sites have been identified on human chromosomes: fra(8)(q24.1), fra(11)(p15.1), fra(16)(p12.1), fra(16)(q22), and fra(17)(p12). All of these fragile sites are located at the junction of Giemsa-positive (G) and negative (R) bands and their expression can be induced by a variety of AT specific DNA ligands. Analysis of family data indicate that the distamycin A-inducible fragile sites segregate as a simple codominant trait with complete penetrance, and probands receive these fragile site genes equally from mothers and fathers. Based on current knowledge of chromosome instability, the nature of distamycin A-inducible fragile sites is discussed. Distamycin A-inducible fragile sites appear to be unique chromosomal regions particularly susceptible to fragility under certain stress conditions. They may also be hot spots for recombination, gene amplification, and integration of foreign genomes.     

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.     

In situ nick translation of the fragile X region

At the present time, the molecular nature of the fragile site at Xq27.3 is not well understood. To examine the sensitivity of this region to DNAase I, in situ nick translation was performed on metaphase chromosomes from a fragile X (fra(X] positive individual. In this technique DNAase I is used to nick regions of chromosomal DNA that are in "open" conformation. Biotinylated dUTP was incorporated by nick translation at these sites. The incorporation was identified by double antibody labeling and avidin-horseradish peroxidase staining. Spreads, which had been stained with this technique, were photographed and subsequently trypsin-Giemsa G banded (post-GTG banded) for chromosome identification. In 36 of 44 (82%) fra(X) positive male cells, the region distal to fra(X) (q27.3) was prominently stained in contrast to its light staining appearance in GTG preparations. The fragile site itself was outlined more clearly than can be achieved by GTG or homogeneous staining. When autosomal fragile sites were induced by the addition of 1.5 microM aphidicolin 17 hours prior to harvest, 24 of 27 (89%) fragile sites on the ends of autosomes were prominently stained in regions distal to the break. Because the fra(X) and autosomal fragile regions behaved similarly, this suggests that they have a similar conformation. Thus, while autosomal and Xq27.3 fragile sites are strongly induced by different means, the organization of these sites and the regions distal to them appear to be similar.     

Fragile X mental retardation: prevalence in a group of institutionalized patients in Italy and description of a novel EEG pattern

We have studied the prevalence of the fra (X) and of the autosomal fragile sites fra (10) (q25) and fra (16) (q22) in patients from an institute for the mentally retarded in Italy. We found six cases (1.9%) of fra (10) (q25) and 9 (2.9%) of fra (16) (q22). The study of the fra (X) was restricted to a subgroup of 91 males who did not have other chromosome anomalies or variants, and led to the discovery of 4 fra (X) cases. These 4 had the Martin-Bell syndrome; 3 of them were epileptic and had a characteristic EEG pattern originating during sleep from the temporal lobe not previously described in fra (X) mental retardation.     

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.     

Experience with multiple approaches to the prenatal diagnosis of the fragile X syndrome: amniotic fluid, chorionic villi, fetal blood and molecular methods

We have had experience with 160 prenatal diagnosis cases for the fragile X syndrome [fra(X)] or Martin-Bell Syndrome. In 140, amniotic fluid was utilized; 98 had a documented family history of fra(X). The 94 completed cases included 4 no growth; 56 males of which 7 were fra(X)-positive and 2 false-negative; 38 females of which 5 were fra(X) positive. There was no fra(X) positive result when a family history of mental retardation was not documented as fra(X). Molecular methods (RFLPs) were utilized in 10 amniotic fluid and 5 chorionic villus specimens (CVS). Percutaneous umbilical blood sampling was used in 2 negative cases and 1 fra(X) positive case because of timing, tissue culture failure or confirmation of another method. CVS were received in 13 cases, and RFLPs were utilized in 5 of the CVS cases. There was no positive fra(X) CVS chromosome result in males, 1 positive result in a female, but 2 false negatives were detected by RFLPs. On the basis of the results, it can be concluded that cytogenetic and molecular methods are complementary and best used together and that multiple approaches can enhance the efficiency and reliability of fra(X) prenatal diagnosis.     

The use of early simultaneous percutaneous umbilical blood sampling (PUBS) and amniocentesis for prenatal fragile X chromosome diagnosis

Early simultaneous percutaneous umbilical blood sampling (PUBS) and amniocentesis for prenatal diagnosis were undertaken for the first time in a 17-week gestation fetus at risk for the fragile X [fra (X)] syndrome. Metaphase spreads from 300 fetal lymphocytes were examined within 5 days following PUBS, while approximately 5 weeks were required for the analysis of 148 amniocytes. The chromosomes were interpreted as normal (46,XX) and the fetus as fragile X-negative at the time of prenatal diagnosis. This was cytogenetically confirmed after delivery of a healthy term female infant. Our results suggest that early PUBS may become a useful adjunct to amniocentesis because of shorter culture time and earlier diagnosis.     

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.     

Noninvolvement of chromosome 16 in karyotype evolution of acute myeloid leukemia in a patient with a heritable fragile site on 16q22

A fragile site on the long arm of chromosome #16 (q22) was detected in a 24-year-old man with pancytopenia. During the course of the disease he developed an inverted duplication of region q11-12 of chromosome #1 and a translocation between chromosomes #9 and #13: t(9;13)(p22;q32). These abnormalities, as well as an additional iso-like marker chromosome that consisted of one normal 9p and the abnormal 9p arm, were detected in Epstein-Barr nuclear antigen-positive B-cell cultures. Two years later, evolution of the abnormal clone with loss of chromosome #7 and, subsequently, chromosome #22 occurred in connection with development of acute myeloid leukemia. Although the heritable fragile site on chromosome #16 was present in all cell populations investigated, it was not involved in the evolution of the abnormal karyotype. This fragile chromosome #16 also was found in 4 of 11 family members in whom chromosome analysis was performed, thus suggesting this aberration was inherited in a dominant autosomal pattern. The incidence of the heritable fragile site in normal and leukemic cells of the patient, as well as stimulated blood cultures of his relatives, are reported. In addition, the possible relationship between this constitutional chromosome breakage syndrome and the occurrence of leukemia is analyzed.     

Cytogenetic guidelines for fragile X studies tested in routine practice.

Several organizations have proposed guidelines for fra(X) studies on peripheral blood lymphocytes. To evaluate these guidelines, we reviewed 1,033 consecutive specimens referred for fra(X) analysis. Each specimen was cultured with medium 199 and RPMI 1640 with 5-fluorodeoxyuridine or excess thymidine. The karyotype and expression of fra(X) were established from 20 GTL- or QFQ-banded cells and by screening of up to 130 more banded cells. We found anomalies other than fra(X) in 37 (3.6%) of the patients. We found 4% or more fra(X) cells in 38 (3.7%) cases from 36 unrelated families, including 33 (3.9%) of 850 males and 5 (2.7%) of 183 females. Another 4 females had 1 to 3% fra(X) cells. Six specimens were fra(X)-positive in only one stress system, and 32 were positive in 2 systems. To find the first 2 fra(X) cells in males, we needed to study up to 36 cells in 31 cases, 50 in one case, and 57 in another. To find the first 2 fra(X) cells in females, we needed to study up to 17 cells in 4 cases and 57 in another. A strong family history of fra(X) occurred in 5 patients, and each one was fra(X)-positive. Some manifestations of the fragile X syndrome occurred in 507 cases, 17 (3%) of which were fra(X)-positive. Abnormalities considered unlikely to be the fragile X syndrome occurred in 103 cases, 3 (3%) of which were fra(X)-positive. Use of chromosome breakage and fra(3)(p14) as quality control indicators of the fra(X) stress systems was found to be unreliable.(ABSTRACT TRUNCATED AT 250 WORDS)     

SV40-transformed fragile (X) amniocytes.

We report SV-40 transformation of female and male fragile X [fra(X)] amniocytes. In the transformants, fra(X) (q27.3) was detected in the 7th passage (9 cell generations) in fra(X) female amniocytes. It was conserved until at least the 20th passage (42 cell generations) although the frequency was reduced or became difficult to detect due to karyotypic evolution in the later generations. Similarly, for the male fra(X) amniocyte line, fra(X) was demonstrated at the 2nd passage (3 cell generations) and persisted until at least the 13th passage (29 cell generations). The prolonged period of reproductive potential of these transformed lines ranging from at least 29-42 generations suggests that the cryopreservation of significant quantities of early passage fra(X) transformed amniocytes will assure a reliable and continuous supply of positive control cells. These lines may be used for fra(X) prenatal diagnostic studies thereby improving the ability to quality control the particular fra(X) induction system being used.     

Cytogenetic guidelines for fragile X studies tested in routine practice

Several organizations have proposed guidelines for fra(X) studies on peripheral blood lymphocytes. To evaluate these guidelines, we reviewed 1,033 consecutive specimens referred for fra(X) analysis. Each specimen was cultured with medium 199 and RPMI 1640 with 5-fluorodeoxyuridine or excess thymidine. The karyotype and expression of fra(X) were established from 20 GTL- or QFQ-banded cells and by screening of up to 130 more banded cells. We found anomalies other than fra(X) in 37 (3.6%) of the patients. We found 4% or more fra(X) cells in 38 (3.7%) cases from 36 unrelated families, including 33 (3.9%) of 850 males and 5 (2.7%) of 183 females. Another 4 females had 1 to 3% fra(X) cells. Six specimens were fra(X)-positive in only one stress system, and 32 were positive in 2 systems. To find the first 2 fra(X) cells in males, we needed to study up to 36 cells in 31 cases, 50 in one case, and 57 in another. To find the first 2 fra(X) cells in females, we needed to study up to 17 cells in 4 cases and 57 in another. A strong family history of fra(X) occurred in 5 patients, and each one was fra(X)-positive. Some manifestations of the fragile X syndrome occurred in 507 cases, 17 (3%) of which were fra(X)-positive. Abnormalities considered unlikely to be the fragile X syndrome occurred in 103 cases, 3 (3%) of which were fra(X)-positive. Use of chromosome breakage and fra(3)(p14) as quality control indicators of the fra(X) stress systems was found to be unreliable. Our findings support most of the proposed guidelines for fra(X) studies but indicate a need for modifications of others. © 1992 Wiley-Liss, Inc.     

Prenatal detection of fra(X)(q27.3) in female identical twins: reliability of low level cytogenetic prenatal expression in females.

Recently, we detected fra(X)(q27.3) in amniocyte cultures from female identical twins. The pregnant woman did not exhibit fra(X)(q27.3) in whole blood cultures but was the sister of 2 affected brothers. DNA marker analyses showed that she was a carrier of FRAXA. Amniotic fluid cultures (AFCs) from twins A and B exhibited the fragile X [fra(X)] chromosome, but the level of cytogenetic expression was very low in twin A's AFCs. DNA marker studies indicated both twins were carriers of FRAXA. Peripheral umbilical blood sample (PUBS) cultures exhibited fra(X)(q27.3) at a frequency of about 10% for both twins. DNA fingerprinting indicated that the twins were identical, confirming the clinical impression, with a very thin separating amniotic membrane. To our knowledge, this is the only report of prenatal fra(X)(q27.3) detection in female identical twins, and the second report of identical twin detection [Rocchi et al., 1985]. We have diagnosed prenatally fra(X)(q27.3) in 5 female fetuses using AFCs. The average fra(X) frequency was 4% for these positive female fetuses with a range of 0.5% to 8.5%. Follow-up whole blood studies confirmed our original results at an average fra(X) frequency of 25%. In conclusion: 1. Low frequencies, perhaps 1 or 2%, or a few positive cells in AFCs, are likely to increase in magnitude when confirmed in whole blood cultures either pre- or postnatally. 2. It appears likely that the risk is low for false positive results in AFCs when low frequencies of fra(X)(q27.3) are encountered.