Radiosensitivity of ataxia telangiectasia and Nijmegen breakage syndrome homozygotes and heterozygotes as determined by three-color FISH chromosome painting

A three-color chromosome painting technique was used to examine the spontaneous and radiation-induced chromosomal damage in peripheral lymphocytes and lymphoblastoid cells from 11 patients with ataxia telangiectasia (AT) and from 14 individuals heterozygous for an AT allele. In addition, cells from two homozygous and six obligate heterozygous carriers of mutations in the Nijmegen breakage syndrome gene (NBS) were investigated. The data were compared to those for chromosome damage in 10 unaffected control individuals and 48 cancer patients who had not yet received therapeutic treatment. Based on the well-documented radiation sensitivity of AT and NBS patients, it was of particular interest to determine whether the FISH painting technique used in these studies allowed the reliable detection of an increased sensitivity to in vitro irradiation of cells from heterozygous carriers. Peripheral blood lymphocytes and lymphoblastoid cells from both the homozygous AT and NBS patients showed the highest cytogenetic response, whereas the cells from control individuals had a low number of chromosomal aberrations. The response of cells from heterozygous carriers was intermediate and could be clearly differentiated from those of the other groups in double-coded studies. AT and NBS heterozygosity could be distinguished from other genotypes by the total number of breakpoints per cell and also by the number of the long-lived stable aberrations in both AT and NBS. Only AT heterozygosity could be distinguished by the fraction of unstable chromosome changes. The slightly but not significantly increased radiosensitivity that was found in cancer patients was apparently due to a higher trend toward rearrangements compared to the controls. Thus the three-color painting technique presented here proved to be well suited as a supplement to conventional cytogenetic techniques for the detection of heterozygous carriers of these diseases, and may be superior method.     

Tissue specificity of chromosomal rearrangements in ataxia-telangiectasia

Summary Cytogenetic studies of lymphocytes and fibroblasts from individuals with ataxia-telangiectasia (AT) demonstrate spontaneous chromosomal breakage. In the AT lymphocytes, this damage results in a high frequency of balanced rearrangements involving chromosome bands 7p14, 7q35, 14q12, and 14q32. The T-cell receptor , , and chain gene complexes and the immunoglobulin heavy chain gene complex, all of which may be functional in lymphocytes, have been localized to these bands. To assess the relationship between genes at these breakpoints and the entirety of the AT phenotype, we undertook a detailed cytogenetic analysis of fibroblasts and lymphocytes from seven AT homozygotes. Our findings indicate that the rearrangements present in the lymphocytes are not commonly observed in the fibroblasts, despite the increased instability of chromosomes from these cells relative to lymphocytes. Furthermore, the changes in the fibroblasts are neither consistent within nor between patients, suggesting that chromosome rearrangement occurs more randomly in this tissue. Therefore, differential site-specific damage in separate tissues may generate the distinct features of the disease in those tissues and may account for the pleiotrophic effects of the AT gene.     

Inversion (14)(q12qter) or (q11.2q32.3): The most frequently acquired rearrangement in lymphocytes

Summary In a large study of chromosome rearrangements occurring in human lymphocytes from normal subjects, inv (14)(q12qter) or (q11.2q32.3) is found to be the most frequent, affecting 0.15% of mitoses. The same inversion is observed in the lymphocytes of the chimpanzee, indicating the ancestry of this inversion. It is not induced by ionizing radiations, and its frequency may be increased in Fanconi anemia, but not in ataxia telangiectasia. It may represent one of the steps of the process of leukemogenesis.     

Gene conversion and transfer of genetic information within the inverted region of inversion heterozygotes

Prior studies of recombination which monitor exchange events in exceedingly short intervals (i.e., separable sites within a cistron) reveal that the basic event in recombination involves a non-reciprocal transfer of information, termed conversion. As a logical consequence of the model suggested by the work in Drosophila, the present investigation examined recombination between rosy mutant alleles (ry:3-52.0) in Drosophila melanogaster in a paracentric inversion (In(3R)P(18)) heterozygote, which placed the rosy region approximately at the center of the inverted region. Comparison of the results of this study with experiments carried out in standard chromosome homozygotes reveals a dramatic suppression of classical crossovers between the rosy mutant alleles in the inversion heterozygote. However, conversions continue to occur for all rosy mutant alleles in all heterozygous combinations in the inversion heterozygote. Moreover, the order of magnitude of conversion frequencies seen in the inversion heterozygote does not change from that seen in the standard chromosome homozygote study. The significance of these observations with reference to the role of rearrangements as barriers of information transfer is discussed. Particular attention is directed to the elaborate inversion polymorphisms seen in natural populations, and to notions concerning their role in the evolution of adaptive gene complexes.     

inv(9)(p24q13) in three sterile brothers

Only nine non-polymorphic constitutional pericentric inversions of chromosome 9 have been described. We report on a familial inv(9)(p24q13) associated with sterility in three brothers. The mother's chromosomes were normal in blood lymphocytes (n=130); the father was already deceased and his karyotype unknown. However, the presence of any of the maternal chromosomes 9 (as assessed by C-banding) in her carrier children is inconsistent with the assumption of maternal mosaicism. Two single sisters were also carriers. The same rearranged chromosome 9 in the three sterile brothers can hardly be regarded as a fortuitous association, especially when the breakpoints are almost identical to those of the sole inversion previously found in an azoospermic male. If their father was a carrier, the observed sterility may be the result of 'chromosome anticipation', a phenomenon already invoked for certain familial chromosomal rearrangements.     

Cytogenetic investigations in three cell types of a Saudi family with ataxia telangiectasia

Summary Chromosomal analyses were performed on lymphocytes, fibroblasts and lymphoblastoid cell lines derived from a Saudi family with ataxia telangiectasia (AT). The three siblings of a consanguineous marriage were all affected. The lymphocytes of the AT homozygotes (probands) showed an increase of 2- to 6-fold and 4- to 8-fold respectively, in the frequency of spontaneous and X-ray-induced chromosomal aberrations compared with controls, while the parents (obligate heterozygotes) of the patients showed no notable difference. The unirradiated lymphocytes from the oldest AT sibling, an 11-year-old boy (AT1), showed specific rearrangements involving chromosomes 7 and 14 [t(7;14)(q35;q12)] and 12 and 14 [t(12;14)(q23;q12)] in two different clones. The most severely affected sibling was a 9-year-old girl (AT2) who presented with a clone showing a novel rearrangement involving chromosomes 14 and 17, namely: del(14) (q31q32) and dup(17)(q21–q24). The lymphocytes from the third sibling, a 2-year-old boy (AT3), showed a t(2;14)(p24;q12). In addition, an inv(14)(q12q32) was observed in all three AT patients, while inv(7)(p14q35) was found only in patients 2 and 3. The lymphocytes from the AT parents and controls showed normal karyotypes. The breakpoints involving chromosomes 2,12 and 17, observed in our studies, have rarely been reported in other series of AT patients. No non-random chromosomal rearrangements were observed either in the skin fibroblasts or in the lymphoblastoid cell lines derived from the AT patients, although all cell lines showed an increase in both spontaneous and radiation-induced chromosomal breaks per cell. The present study constitutes the first report on a cytogenetic analysis of a Saudi family with three AT siblings.     

Paracentric inversion of chromosome 14 plus rare 9p variant in a couple with habitual spontaneous abortion.

A couple presenting with habitual spontaneous abortion both showed a chromosome rearrangement. The male had an apparently balanced paracentric inversion of chromosome 14 - 46,XY,inv(14) (q11q32). The female had a karyotype with a rare large short arm variant of chromosome 9 - 46,XX,var(9) (p11p21). Testing of a living normal child showed that he had inherited both rearrangements. Family testing showed the chromosome 9 variant in three generations, with all carriers being of normal phenotype and intelligence. This study confirms that the presence of more than one chromosomal rearrangement can be compatible with normal development. This is useful for genetic counselling. Nevertheless when such cases arise, each must be individually assessed.     

Karyotype instability with multiple 7/14 and 7/7 rearrangements

Summary Chromosomes were studied in a mentally retarded boy with microcephaly, growth retardation, facial erythema, café-au-lait spots, and IgA deficiency. In the lymphocytes there was a remarkable tendency to exchange parts of the chromosomes Nos. 7 and 14, the translocations almost exclusively taking place in bands 7p13, 7q32 and 14q11. Seven different types of rearrangements between Nos. 7 and 14, and some other chromosomal aberrations were found. No abnormalities could be detected in the bone marrow. The patient somewhat resembles those affected with ataxia-telangiectasia or with Bloom's syndrome, but on clinical and cytogenetic grounds these disorders could be excluded.7/14 Translocations similar to those found in our patient's lymphocytes have been reported to occur very rarely in the lymphocyte cultures of individuals with apparently normal chromosome constitution. A relationship between these phenomena may exist.     

Increased risk of trisomy 21 in offspring of carriers of balanced non-contributing autosomal rearrangements is not explained by interchromosomal effect

Only relatively recently the suggestion that interchromosomal effect (ICE) may be present in man had stopped to be argued. At once it became evident that this phenomenon is inherent to a proportion of balanced chromosome rearrangement carriers, predominantly to patients with fertility problems. It is important to establish whether ICE operates in genome of fertile rearrangement carriers and to determine what kind of rearrangement and how far increases a risk of aneuploidy offspring. Using own and literature data 1) we have assessed rates of inherited non-contributing balanced rearrangements in patients with trisomy 21 (T21) and rates of both mutant and inherited non-contributing balanced rearrangements in parents of offspring with T21 and 2) we have analyzed a parental origin of T21 in affected offspring of carriers of balanced rearrangement. We have found that carriers of balanced reciprocal translocation or inversion, but not robertsonian translocation, are at increased risk of T21 offspring. However these data do not support the existence of ICE in its common sense, i.e. as an effect of rearrangement on other chromosome’s segregation at the carrier’s meiosis. Probably the data obtained suggest an effect of paternal rearrangements on maternal chromosomes segregation after fertilization.     

Probable involvement of immunoglobulin superfamily genes in most recurrent chromosomal rearrangements from ataxia telangiectasia

Summary From the chromosomal analysis of 9461 lymphocytes from 57 patients affected by ataxia telangiectasia, it is concluded that bands 7p14, 7q35, 14q12, and 14qter, which are frequently recombined in rearrangements are also too frequently involved in rearrangements with a few other chromosome sites. Among these sites, the most frequently involved are bands 2p11, 2p12, 22q12, and 22q13.2, or the proximal parts of adjacent R-bands. The same rearrangements were observed in a large series of control lymphocytes but their frequencies were much lower than in ataxia telangiectasia. All these recurrent sites of rearrangements, except 22q13.2, are known to be near or at immunoglobulin genes or partially homologous genes like T-cell receptor genes and antigen Leu-2/T8. It is supposed that the rearrangements observed correspond to the visualization at the chromosomal level of illegitimate rearrangements between these genes, and by analogy, that another similar structure may exist on band 22q13.2.     

Evolution of chromosomal abnormalities in sequential cytogenetic studies of ataxia telangiectasia

Summary Sequential cytogenetic studies of four patients with ataxia telangiectasia showed the progressive development of lymphocyte clones, each marked with a rearranged chromosome 14. Initial studies had shown random chromosomal breaks and rearrangements. Later studies in all patients showed nonrandom rearrangement of chromosome 14 with a breakpoint at 14q12 and with the distal segment translocated to either chromosome 14 or 7. The proportion of circulating lymphocytes carrying the marker tended to increase with time, accounting for the majority of the lymphocytes eventually in one case. The marked lymphocyte clones evolved further, as a result of loss of the small centric portions of the rearranged chromosome 14 (14pter14q12).Perhaps the abnormal clones in ataxia telangiectasia escape immunologic surveillance and flourish in an immunologically impaired environment. Subsequent to the loss of the centric portion of the rearranged chromosome 14, the cells may acquire additional capabilities that enhance malignant transformation.     

Breakpoint mapping and array CGH in translocations: comparison of a phenotypically normal and an abnormal cohort

We report the analyses of breakpoints in 31 phenotypically normal and 14 abnormal carriers of balanced translocations. Our study assesses the differences between balanced translocations in normal carriers and those in abnormal carriers, focusing on the presence of genomic imbalances at the breakpoints or elsewhere in the genome, presence of cryptic chromosome rearrangements, and gene disruption. Our hypothesis is that all four features will be associated with phenotypic abnormalities and absent or much less frequent in a normal population. In the normal cohort, we identified neither genomic imbalances at the breakpoints or elsewhere in the genome nor cryptic chromosome rearrangements. In contrast, we identified candidate disease-causing imbalances in 4/14 abnormal patients. These were three breakpoint associated deletions and three deletions unrelated to the breakpoints. All six de novo deletions originated on the paternally inherited chromosome. Additional complexity was also present in one of these cases. Gene disruption by the breakpoints was present in 16/31 phenotypically normal individuals and in 5/14 phenotypically abnormal patients. Our results show that translocations in phenotypically abnormal patients are molecularly distinct from those in normal individuals: the former are more likely to be associated with genomic imbalances at the breakpoints or elsewhere and with chromosomal complexity, whereas the frequency of gene disruption is similar in both normal and abnormal translocation carriers.     

Ovarian dysgenesis due to an idic(X)(q2803)

A 17-year-old female patient with gonadal dysgenesis but no other turnerian features was found to have a 46,X,idic(X)(pter----q2803:q2803----pter) karyotype in her lymphocytes. Replication of the rearranged X was consistently late and symmetrical. It is postulated that the ovarian dysgenesis usually seen in nonmosaic carriers of Xq;Xq terminal rearrangements may be secondary to a nonreactivation of the abnormal chromosome before meiosis.     

A pachytene analysis of two male-fertile paracentric inversions in chromosome 1 of the mouse and in the male-sterile double heterozygote

Meiotic studies have been made at pachytene on two paracentric inversions in chromosome 1 of the mouse. Surface-spread preparations of primary spermatocytes have been analysed at the light microscope level in males heterozygous for the inversions In(1)1Rk and In(1)12Rk and in the double heterozygote In(1)1RK/In(1)12Rk. In singly heterozygous form, neither inversion produces any serious effect on male fertility. In the double heterozygote, spermatogenesis is arrested in the majority of cells at the spermatocyte stage and males are rendered totally sterile by azoospermia. In the double heterozygote, a complex loop, indicating the inversion bivalent, is found in 90% of pachytene cells analysed. In the In(1)1Rk/+ heterozygote, a looped bivalent was seen in 47 per cent of pachytene cells but in In(1)12Rk/+ no cells containing loops could be found. -80% of pachytene spermatocytes from the In(1)1Rk/In (1)12Rk double heterozygote showed apposition of the inversion bivalent to the sex bivalent. Such an association was rarely seen in pachytene cells of either of the fertile single heterozygotes. Spermatogenic failure in the double heterozygote may be related to interference, by the inversion bivalent, with X chromosome inactivation at meiotic prophase.     

Correlation of the clinical phenotype with a pericentric inversion of chromosome 9]

Pericentric inversion of chromosome 9 is one of the most common structural balanced chromosomal aberrations. It is considered as a paraphysiological variant of a normal karyotype and it is possible to find it as occasional report in healthy subjects. In the last ten years different signals have appeared in literature, concerning carriers of pericentric inversion of chromosome 9, who showed different anomalies of the clinical condition. Today it is difficult, because of the rarity of the data to establish if a true correlation exists between phenotypical anomalies in the subjects studied and the pericentric inversion, or if they are only casual associations. We are trying to find possible correlations between the chromosomal rearrangements and eventual congenital defects. We describe 11 subjects with pericentric inversion of chromosome 9 examined for the presence of dysmorphic signs, mental retardation and repeated miscarriage.     

Occurrence of <Emphasis Type="Italic">TRGV-BJ</Emphasis> hybrid gene in SV40-transformed fibroblast cell lines

Illegitimate V(D)J-recombination in lymphoid malignancies involves rearrangements in immunoglobulin or T-cell receptor genes, and these rearrangements may play a role in oncogenic events. High frequencies of TRGV-BJ hybrid gene (rearrangement between the TRB and TRG loci at 7q35 and 7p14-15, respectively) have been detected in lymphocytes from patients with ataxia telangiectasia (AT), and also in patients with lymphoid malignancies. Although the TRGV-BJ gene has been described only in T-lymphocytes, we previously detected the presence of TRGV-BJ hybrid gene in the genomic DNA extracted from SV40-transformed AT5BIVA fibroblasts from an AT patient. Aiming to determine whether the AT phenotype or the SV40 transformation could be responsible for the production of the hybrid gene by illegitimate V(D)J-recombination, DNA samples were extracted from primary and SV40-transformed (normal and AT) cell lines, following Nested-PCR with TRGV- and TRBJ-specific primers. The hybrid gene was only detected in SV40-transformed fibroblasts (AT-5BIVA and MRC-5). Sequence alignment of the cloned PCR products using the BLAST program confirmed that the fragments corresponded to the TRGV-BJ hybrid gene. The present results indicate that the rearrangement can be produced in nonlymphoid cells, probably as a consequence of the genomic instability caused by the SV40-transformation, and independently of ATM gene mutation.     

High recurrence of rearrangements involving chromosome 14 in an ataxia telangiectasia lymphoblastoid cell line and in its mutagen-treated derivatives

Summary The cytogenetic characterization of CH cell line obtained by Epstein-Barr-virus transformation of the lymphocytes of a patient affected by ataxia telangiectasia is reported. Control CH cells and 2 subcultures treated with the mutagens R7000 or NQO were developed in parallel and studied. A common chromosome anomaly, a der(14) t(11;14) (q13.2;q32), was found in all the studied karyotypes, indicating that it occurred either in vivo or early in vitro. In non-treated cultures, additional anomalies were present in 6 derived subclones. All R-7000 treated cells had the same karyotype corresponding to one of the subclones observed without prior treatment. All NQO-treated cells acquired 2 common anomalies, and could be differentiated into 2 subclones because of the addition of a t(7;14) or a t(11;14). Chromosome 14 was involved in various rearrangements after breakage in band q11.2 or q12 in 6/8 subclones. This was not correlated with tumorigenicity, which was clearly increased in mutagen-treated cells as tested by in vitro growth in semi-solid medium and in vivo by grafts into nude mice or growth on the chorio-allantoic membrane of chick embryos. The CH cell line and its derivatives appear to be a promising in vitro system, showing various stages progressing towards malignancy, and reproducing a number of chromosome anomalies spontaneously occurring in AT patients.     

Inv21p12q22del21q22 and intellectual disability

Chromosomal rearrangements are common in humans. Pericentric inversions are among the most frequent aberrations (1–2%). Most inversions are balanced and do not cause problems in carriers unless one of the breakpoints disrupts important functional genes, has near submicroscopic copy number variants or hosts “cryptic” complex chromosomal rearrangements. Pericentric inversions can lead to imbalance in offspring. Less than 3% of Down syndrome patients have duplication as a result of parental pericentric inversion of chromosome 21. We report a family with an apparently balanced pericentric inversion of chromosome 21. The proband, a 23-year-old female was referred for prenatal diagnosis at 16 weeks gestation because of increased nuchal translucency. She has a familial history of Down's syndrome and moderate intellectual disability, a personal history of four spontaneous abortions and learning difficulties. Peripheral blood and amniotic fluid samples were collected to perform proband's and fetus' cytogenetic analyses. Additionally, another six family members were evaluated and cytogenetic analysis was performed. Complementary FISH and MLPA studies were carried out. An apparent balanced chromosome 21 pericentric inversion was observed in four family members, two revealed a recombinant chromosome 21 with partial trisomy, and one a full trisomy 21 with an inverted chromosome 21. Array CGH analysis was performed in the mother and the brother's proband. MLPA and aCGH studies identified a deletion of about 1.7 Mb on the long arm of inverted chromosome 21q22.11. We believe the cause of the intellectual disability/learning difficulties observed in the members with the inversion is related to this deletion. The recombinant chromosome 21 has a partial trisomy including the DSCR with no deletion. The risk for carriers of having a child with multiple malformations/intellectual disability is about 30% depending on whether and how this rearrangement interferes with meiosis.     

A cytogenetic survey of men being investigated for subfertility.

A study of 336 men attending a subfertility clinic but otherwise unselected, and a further 12 men studied as a part of the investigation of the female partner, revealed 10 with major chromosome anomaly and 3 with unambiguous chromosomal variants. In addition to those with sex chromosome aneuploidy, an extra, small marker chromosome and D/D Robertsonian translocations, anomalies which have been reported in other studies, there were 7 men with rearrangements, including a paracentric inversion of chromosome 7 and an X/21 reciprocal translocation. These would have been difficult or impossible to identify without good banded preparations, suggesting that such rearrangement may be more frequent in association with subfertility than was appreciated.     

Cytogenetic investigations in a family with ataxia telangiectasia

Summary Cytogenetic findings on a family with ataxia telangiectasia (A-T) in which three of four sibs were affected are described. The affected individuals had approximately twice the level of spontaneous chromosome breakage of a normla control, while the parents and the normal sib had no significant increase. Lymphocytes from all three A-T homozygotes showed specific stable chromosomal rearrangements involving chromosomes 7 and 14. All of these abnormalities involved breakage at the usual four sites associated with A-T (7p14, 7q35, 14q12, and 14q32). Two rearrangements detected in the eldest and most severely affected patient were clones, one of which [t(14;14)(p11;q12)] is not commonly found in A-T cells. No chromosomal rearrangements were encountered in lymphocytes from the control, the parents, or the normal sib. Lymphocytes from the A-T patients also were found to be 7–11 times more sensitive to the induction of chromatid aberrations by X-irradiation than control cells. Lymphocytes from the parents and normal sib showed a moderately increased frequency of X-ray induced aberrations compared with that of the control.