Localisation of the endpoints of deletions in the 5'' region of the Duchenne gene using a sequence isolated by chromosome jumping.

We have used chromosome jumping technology to move from within a large intron sequence in the Duchenne muscular dystrophy (DMD) gene to a region adjacent to exons of the gene. The single copy jump clone, HH1, was used to characterise deletions in patients previously shown to be deleted for DNA markers in the 5' end of the gene. 12 out of 15 such patients have breakpoints which lie between HH1 and the genomic locus J-47. Thus the vast majority of the deletions in these patients have proximal breakpoints in a similar region distal to the 5' end of the gene. HH1 was mapped with respect to the X;1 translocation in a DMD female and was shown to lie at least 80 kb from the starting point of the chromosome jump, HIP25.     

Molecular analysis of X-autosome translocations in females with Duchenne muscular dystrophy.

To further an understanding of the mechanism of constitutional chromosomal rearrangement, the translocation breakpoints of two X-autosome translocations carried by females with Duchenne or Becker muscular dystrophy have been mapped, cloned and sequenced. Breakpoints were mapped to specific introns within the dystrophin gene and intron sequences spanning the two breakpoints were cloned and used as probes to identify DNA fragments containing the translocation junctions. The junction-containing fragments were cloned after amplification by inverse PCR or single-specific-primer PCR. Sequence through the junctions and the autosomal regions spanning the breakpoints identified the mechanism of rearrangement as non-homologous exchange with minor additions or deletions (0-8 nucleotides) at the breakpoints. Paternal origin of these X-autosome translocations, coupled with evidence for non-transmission of X-autosome translocations through male meiosis suggested that the translocations were the result of a post-meiotic rearrangement in spermiogenesis.     

Common sequence motifs at the rearrangement sites of a constitutional X/autosome translocation and associated deletion.

Reciprocal chromosome translocations are common de novo rearrangements that occur randomly throughout the human genome. To learn about causative mechanisms, we have cloned and sequenced the breakpoints of a cytologically balanced constitutional reciprocal translocation, t(X;4)(p21.2;q31.22), present in a girl with Duchenne muscular dystrophy (DMD). Physical mapping of the derivative chromosomes, after their separation in somatic cell hybrids, reveals that the translocation disrupts the DMD gene in Xp21 within the 18-kb intron 16. Restriction mapping and sequencing of clones that span both translocation breakpoints as well as the corresponding normal regions indicate the loss of approximately 5 kb in the formation of the derivative X chromosome, with 4-6 bp deleted from chromosome 4. RFLP and Southern analyses indicate that the de novo translocation is a paternal origin and that the father's X chromosome contains the DNA that is deleted in the derivative X. Most likely, deletion and translation arose simultaneously from a complex rearrangement event that involves three chromosomal breakpoints. Short regions of sequence homology were present at the three sites. A 5-bp sequence, GGAAT, found exactly at the translocation breakpoints on both normal chromosomes X and 4, has been preserved only on the der(4) chromosome. It is likely that the X-derived sequence GGAATCA has been lost in the formation of the der(X) chromosome, as it matches an inverted GAATCA sequence present on the opposite strand exactly at the other end of the deleted 5-kb fragment. These findings suggest a possible mechanism which may have juxtaposed the three sites and mediated sequence-specific breakage and recombination between nonhomologous chromosomes in male meiosis.     

Mapping of 12 translocation breakpoints in the Xp21 region with respect to the locus for Duchenne muscular dystrophy

Over 20 females have been reported to carry reciprocal X; autosome translocations with breakpoints in Xp21 and to suffer from Duchenne muscular dystrophy (DMD). We have positioned nine of these breakpoints with respect to the Duchenne gene by mapping probes from the DMD region against a panel of somatic cell hybrids, each containing one of the translocation chromosomes from a different female patient; further information has also been obtained by in situ hybridization, including the breakpoint location in a tenth DMD patient. We have also characterized two translocation breakpoints that lie in the same chromosomal region but which are not associated with the expression of DMD. All the DMD-associated translocation breakpoints examined lie at several sites within the DMD locus and between the two non-DMD breakpoints.     

Genetic counselling in carriers of reciprocal chromosomal translocations involving short arm of chromosome X

A central concept in genetic counselling is the estimation of the probability of occurrence of unbalanced progeny at birth and other unfavourable outcomes of pregnancy (miscarriages, stillbirths and early death). The estimation of the occurrence probability for individual carriers of four different X-autosome translocations with breakpoints at Xp, namely t(X;5)(p22.2;q32), t(X;6)(p11.2;q21), t(X;7)(p22.2;p11.1), and t(X;22)(p22.1;p11.1), is presented. The breakpoint positions of chromosomal translocations were interpreted using GTG, RBG and FISH-wcp. Most of these translocations were detected in women with normal phenotype, karyotyped because of repeated miscarriages and/or malformed progeny. A girl with very rare pure trisomy Xp22.1-->pter and a functional Xp disomy was ascertained in one family and her clinical picture has been described in details. It has been suggested that not fully skewed X chromosome inactivation of X-autosome translocation with breakpoint positions at Xp22 (critical segment) could influence the phenotype and risk value. Therefore, the X inactivation status was additionally evaluated by analysis of replication banding patterns using RBG technique after incorporation of BrdU. In two carriers of translocations: t(X;5)(p22.2;q32) and t(X;7)(p22.2;p11.1), late replication state of der(X) was observed in 5/100 and 10/180 analysed cells, respectively. In these both cases the breakpoint positions were clustered at the critical segment Xp22.2. In two other cases, one with the breakpoint position within [t(X;22)(p22.1;p11.1)] and one outside the critical region [t(X;6)(p11.2;q21)], fully skewed inactivation was seen. Therefore, we suggest that neither the distribution of the breakpoint positions nor fully skewed inactivation influenced the phenotype of observed t(X;A) carriers. The occurrence probabilities of the unbalanced progeny were calculated according to Stene and Stengel-Rutkowski along with application of updated available empirical data. In the studied group the values of occurrence probability for unbalanced offspring at birth ranged from 2.1% to 17%. Information on the magnitude of the individual figures may be important for women carrying a reciprocal X;A translocation when deciding upon further family planning.     

Mapping of Xp21 translocation breakpoints in and around the DMD gene by pulsed field gel electrophoresis

Balanced translocations with a breakpoint in the Xp21 region are likely to disrupt the giant Duchenne muscular dystrophy (DMD) locus and can be demonstrated in females suffering from the disease. Pulsed field gel electrophoresis allows the positioning of these breakpoints by detecting junction fragments on the derived chromosomes; DNA probes hybridizing to these fragments may be located as many as several hundred kilobases away from the breakpoints. By using this approach, 11 translocation breakpoints from the Xp21 region have been analyzed. The localization of three previously examined breakpoints was confirmed. Six other breakpoints, including a breakpoint flanking the DMD gene and not associated with the DMD phenotype, could be positioned relative to SfiI sites on a 3.5-Mb restriction map of the region.     

The problem of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal X-linked muscular disorder. The biochemical defect remains unknown, but the gene responsible has been mapped to band Xp21. The gene has now been cloned in two laboratories solely from knowledge of its map location. L. M. Kunkel and his colleagues isolated genomic sequences (PERT 87) from within a large deletion causing DMD, whereas our group isolated genomic sequences (XJ) spanning the junction of an X-autosome translocation causing the disease. Chromosome walking by both groups has led to the isolation of over 400 kilobases of the PERT 87 and XJ region. Subclones of PERT 87 and XJ reveal restriction fragment length polymorphisms that segregate with the DMD gene in 95% of meioses, and fail to hybridize with DNA from about 8% of male patients. Selected subclones of PERT 87 and XJ contain exons that hybridize to muscle-derived complementary DNA (cDNA) clones. The cDNA clones detect a large (16 kilobase) message. Analysis of deletions, mutations and translocations suggests a DMD gene of between two million and three million base pairs. The clones obtained so far are useful for attempts to generate antibody against the gene product and for carrier identification and prenatal diagnosis.     

Molecular analysis of the Duchenne muscular dystrophy region using pulsed field gel electrophoresis

Genetic and molecular studies show that the Duchenne muscular dystrophy (DMD) locus at Xp21 is large and complex. We have analyzed this region using pulsed field gel electrophoresis (PFGE) and have determined physical distances between Xp21 probes. The sum of the sizes of the Sfil restriction fragments detected by these probes is greater than 4000 kb. The deletion endpoints in two DMD patients were detected by observing changes in these restriction fragments. In addition, the Xp21 breakpoint for the X;1 translocation in an affected female was mapped. These results demonstrate the applicability of PFGE for analysis of Xp21, and should facilitate the mapping of other translocations and deletions in this region, some of which lead to glycerol kinase deficiency and adrenal hypoplasia as well as DMD.     

Further studies of gene deletions that cause Duchenne and Becker muscular dystrophies

Fetal muscle cDNA clones covering at least 11.4 kb of the Duchenne muscular dystrophy (DMD) gene sequence were used to identify a deletion-prone region in DNA from DMD and Becker muscular dystrophy (BMD) patients. Of 36 BMD cases, 17 (47%) had deletions and all of the deletions began in the same intron of the gene. Of 107 DMD patients, 27 (25%) were deleted for this region, and 19 deletions originate in the same intron. Using a cDNA probe for an adjacent region of the gene, 32 new deletions were detected in DMD patients (total 44%). No new BMD deletions were detected. The DMD deletions were very heterogeneous. Thus two cDNA probes covering 2.4 kb could detect 53% of these deletions. Considering the whole locus, DMD and BMD are caused by a deletion of the gene sequence in at least 67% of cases.     

X-inactivation patterns in lymphocytes and skin fibroblasts of three cases of X-autosome translocations with abnormal phenotypes

Summary X-inactivation patterns were studied by replication analyses both in lymphocytes and skin fibroblasts of two patients carrying balanced X-autosome translocations, t(X;10)-(pter;q11) and t(X;17)(q11;q11), and one patient with an unbalanced translocation t(X;22)(p21;q11). Preferential late replication of the normal X chromosome was found in lymphocytes of both patients carrying balanced translocations and in skin fibroblasts of the patient carrying the translocation t(X;17). However, skin fibroblasts of the patient with a translocation t(X;10) showed preferential late replication of the abnormal der(X) chromosome with no spreading of late replication to the autosomal segment. In the case of unbalanced translocation t(X;22) there was preferential late replication of the der(X) chromosome both in lymphocytes and skin fibroblasts. The abnormal phenotype of the patients is discussed in relation to the observed X-inactivation patterns and the variability of the patterns in different tissues.     

The first BCR gene intron contains breakpoints in Philadelphia chromosome positive leukemia.

The hallmark of chronic myelogenous leukemia (CML) is a translocation between chromosomes 9 and 22 - the Philadelphia (Ph') translocation. The translocation is also found in acute lymphocytic leukemia (ALL) albeit in a lower percentage of patients. The breakpoint on chromosome 22 is located within the BCR gene: in CML, breakpoints are clustered within 5.8 kb of DNA, the major breakpoint cluster region (Mbcr). In ALL, breakpoints have been reported within the Mbcr but also in more 5' regions encompassing the BCR gene. To characterize the latter breakpoints, we have molecularly cloned and mapped the entire gene, which encompasses approximately 130 kb of DNA. Mbcr negative, Ph'-positive ALL breakpoints were not distributed at random within the gene but rather were found exclusively within the 3' half of the first BCR gene intron. In contrast to the Mbcr, which is limited to a region of 5.8 kb, this part of the intron has a size of 35 kb. Translocation breakpoints in this region appear to be specific for ALL, since it was not rearranged in clinically well-defined CML specimens nor in any other tumor DNA samples examined.     

Characterization of a B-lymphocyte t(2;14) (p11;q32) translocation from an ataxia telangiectasia patient conferring a proliferative advantage on cells in vitro

Patients with the recessively inherited disorder ataxia telangiectasia (AT) are particularly prone to the development of both B-cell and T-cell tumours. Specific translocations involving T-cell gene rearrangements and an unknown locus 3' of IGH have been described in AT T-cell clone and tumour cells. We describe here a t(2;14)(p11;q32) translocation which was observed in nonmalignant short-term-cultured B lymphocytes from an AT patient. In vivo, the clone of cells grew from 1% to 6% of the total cell population over a period of 2 yr. Clonal translocations may therefore be associated with AT B cells, as well as AT T cells. B lymphocytes were transformed with Epstein-Barr virus, and the t(2;14) translocation cell was cellularly cloned. Using Southern filter analysis and in situ hybridisation to define more clearly the positions of the breakpoints, we show that the translocation at 14q32 involves a deletion within the IGH chain gene of at least J1, J2, DQ52, and sequences 1.5 kb 5' of DQ52 and that the breakpoint is either adjacent to the non-deleted JH sequences or upstream of these sequences, within the D or V regions, but proximal to all members of the VHII family of genes. The breakpoint at 2p11 is outside and proximal to IGK with respect to the centromere in an unknown gene. Sub-lines with an initially low proportion of translocation cells eventually became monoclonal in vitro for these cells. This suggests they have a growth advantage in vitro.     

Localization of DNA sequences to a region within Xp11.21 between incontinentia pigmenti (IP1) X-chromosomal translocation breakpoints.

Incontinentia pigmenti (IP) is an X-linked dominant disorder characterized by developmental anomalies of the tissues and organs derived from embryonic ectoderm and neuroectoderm. An IP locus, designated IP1, probably resides in Xp11.21, since five unrelated patients with nonfamilial IP have been identified who possess constitutional de novo reciprocal X;autosome translocations involving Xp11.21. We have used a series of somatic cell hybrids containing the rearranged chromosomes derived from three of the five IP1 patients, along with other hybrid cell lines, to map probes in the vicinity of the IP1 locus. Five anonymous DNA loci--DXS422, DXS14, DXS343, DXS429, and DXS370--have been mapped to a region within Xp11.21, between two IP1 X-chromosomal translocation breakpoints; the IP1 t(X;17) breakpoint is proximal (centromeric) to this region, and the IP1 t(X;13) and t(X;9) X-chromosomal breakpoints lie distal to it. While no IP1 translocation breakpoint has yet been identified by pulsed-field gel electrophoretic (PFGE) analysis, an overlap between three probes--p58-1, 7PSH3.5, and cpX210--has been detected, placing these probes within 125 kb. Four probes--p58-1, 7PSH3.5, cpX210, and 30CE2.8--have been helpful in constructing a 1,250-kb PFGE map of the region between the breakpoints; these results suggest that the IP1 X-chromosomal translocation breakpoints are separated by at least this distance. The combined somatic cell hybrid and PFGE analyses we report here favor the probe order DXS323-(IP1 t(X;13), IP1, t(X;9]-(DXS422, DXS14, DXS343, DXS429, DXS370)-(IP1 t(X;17), DXZ1). These sequences provide a starting point for identifying overlapping genomic sequences that span the IP1 translocation breakpoints; the availability of IP1 translocation breakpoints should now assist the cloning of this locus.     

Sequence analysis of the breakpoint regions of an X;5 translocation in a female with Duchenne muscular dystrophy.

X;autosome translocations in females with Duchenne muscular dystrophy (DMD) provide an opportunity to study the mechanisms responsible for chromosomal rearrangements that occur in the germ line. We describe here a detailed molecular analysis of the translocation breakpoints of an X;autosome reciprocal translocation, t(X;5)(p21;q31.1), in a female with DMD. Cosmid clones that contained the X-chromosome breakpoint region were identified, and subclones that hybridized to the translocation junction fragment in restriction digests of the patient's DNA were isolated and sequenced. Primers designed from the X-chromosomal sequence were used to obtain the junction fragments on the der(X) and the der(5) by inverse PCR. The resultant clones were also cloned and sequenced, and this information used to isolate the chromosome 5 breakpoint region. Comparison of the DNA sequences of the junction fragments with those of the breakpoint regions on chromosomes X and 5 revealed that the translocation arose by nonhomologous recombination with an imprecise reciprocal exchange. Four and six base pairs of unknown origin are inserted at the exchange points of the der(X) and der(5), respectively, and three nucleotides are deleted from the X-chromosome sequence. Two features were found that may have played a role in the generation of the translocation. These were (1) a repeat motif with an internal homopyrimidine stretch 10 bp upstream from the X-chromosome breakpoint and (2) a 9-bp sequence of 78% homology located near the breakpoints on chromosomes 5 and X.     

Altered chromatin modifications in AML1/RUNX1 breakpoint regions involved in (8;21) translocation

The RUNX1/AML1 gene is the most frequent target for chromosomal translocation, and often identified as a site for reciprocal rearrangement of chromosomes 8 and 21 in patients with acute myelogenous leukemia. Virtually all chromosome translocations in leukemia show no consistent homologous sequences at the breakpoint regions. However, specific chromatin elements (DNase I and topoisomerase II cleavage) have been found at the breakpoints of some genes suggesting that structural motifs are determinant for the double strand DNA-breaks. We analyzed the chromatin organization at intron 5 of the RUNX1 gene where all the sequenced breakpoints involved in t(8;21) have been mapped. Using chromatin immunoprecipitation assays we show that chromatin organization at intron 5 of the RUNX1 gene is different in HL-60 and HeLa cells. Two distinct features mark the intron 5 in cells expressing RUNX1: a complete lack or significantly reduced levels of Histone H1 and enrichment of hyperacetylated histone H3. Strikingly, induction of DNA damage resulted in formation of t(8;21) in HL-60 but not in HeLa cells. Taken together, our results suggest that H1 depletion and/or histone H3 hyperacetylation may have a linkage with an increase susceptibility of specific chromosomal regions to undergo translocations.     

Physical mapping of nine Xq translocation breakpoints and identification of XPNPEP2 as a premature ovarian failure candidate gene

Women with balanced translocations between the long arm of the X chromosome (Xq) and an autosome frequently suffer premature ovarian failure (POF). Two "critical regions" for POF which extend from Xq13-->q22 and from Xq22-->q26 have been identified using cytogenetics. To gain insight into the mechanism(s) responsible for ovarian failure in women with X;autosome translocations, we have molecularly characterized the translocation breakpoints of nine X chromosomes. We mapped the breakpoints using somatic cell hybrids retaining the derivative autosome and densely spaced markers from the X-chromosome physical map. One of the POF-associated breakpoints in a critical region (Xq25) mapped to a sequenced PAC clone. The translocation disrupts XPNPEP2, which encodes an Xaa-Pro aminopeptidase that hydrolyzes N-terminal Xaa-Pro bonds. XPNPEP2 mRNA was detected in fibroblasts that carry the translocation, suggesting that this gene at least partially escapes X inactivation. Although the physiologic substrates for the enzyme are not known, XPNPEP2 is a candidate gene for POF. Our breakpoint mapping data will help to identify additional candidate POF genes and to delineate the Xq POF critical region(s).     

X-Autosome translocations: Cytogenetic characteristics and their consequences

Summary To define the principal characteristics of X-autosome translocations, the authors present a study of 105 cases, five of which are personal observations. The autosomal pairs 15, 21, and 22 are affected by t(X-Aut) more often than would be expected. The distribution of breakpoints on the X chromosome does not differ significantly from the expected distribution. The analysis of different patterns of inactivation seems to confirm that the inactivation could occur at random, but would be followed by a cellular selection favoring the better genetic balance. An estimate of the incidence of t(X-Aut) is proposed, based upon the conclusions that only one chromosome is susceptible to translocation in meiosis in both males and females and that all affected men will be sterile, as will be 50% of women.