Distal Breakpoint Research Articles

BAC clones and STS markers near the distal breakpoint of the fourth t-inversion, In(17)4d, in the H2-M region on mouse chromosome 17.

The H2-M region is the most distal part of the mouse major histocompatibility complex (Mhc) and is likely to include the distal breakpoint of the fourth t-inversion, In(17)4d. The conserved synteny breakpoint between mouse and human is located in the H2-M region between D17Leh89, a putative olfactory receptor gene, and Pgk2 (phosphoglycerate kinase 2). To analyze the H2-M region, we screened a mouse bacterial artificial chromosome (BAC) library, using the D17Mit64, D17Tu49, D17Leh89, D17Leh467, and Pgk2 markers. Thirty-eight BAC clones were obtained and mapped in five clusters, and 25 sequence-tagged site (STS) markers were newly developed. The regions surrounding D17Tu49 and D17Leh467 are abundant in L1 repeat sequences and may, therefore, be candidates for the breakpoints of conserved synteny and t-inversion. D17Leh89 was linked to D17Mit64 by two contiguous BAC clones. The Aeg1 (acidic epididymal glycoprotein 1) and Aeg2 genes were mapped close to Pgk2, on the same BAC clones. The genetic length between D17Leh89-D17Mit64 and Pgk2-Aeg can be estimated as 0.5-0.7 centiMorgan (cM), and the most distal class I gene, H2-M2, can be placed 0.3-1.0 cM proximal to the t-inversion breakpoint. A recombinational hotspot is suggested to be located between Aeg and Tpxl in an interspecific cross of (C57BL/6J x Mus spretus).

The mechanisms involved in formation of deletions and duplications of 15q11-q13.

Haplotype analysis was undertaken in 20 cases of 15q11-q13 deletion associated with Prader-Willi syndrome (PWS) or Angelman syndrome (AS) to determine if these deletions arose through unequal meiotic crossing over...

Expressed copies of the MN7 (D15F37) gene family map close to the common deletion breakpoints in the Prader-Willi/Angelman syndromes

Approximately 70% of patients with Prader-Willi syndrome or Angelman syndrome have a similar sized de novo deletion of 3–4 Mb in the proximal region of 15q. The distal breakpoints appear to cluster between the P gene (OCA2) and D15S24, whereas two deletion breakpoint clusters have been identified on the proximal side (one centromeric to D15S541 and one between D15S541 and D15S9). Based on the identification of a gene family in 15q11→q13 (MN7, D15F37), we have previously proposed that the presence of multiple copies of this sequence may be related to the instability of this region. Using fluorescence in situ hybridization and YAC mapping, we have found that at least one D15F37 locus is centromeric to D15S9 and at least two are between OCA2 and D15S24. As determined by cDNA cloning and sequence analysis, each of the individual loci is expressed. The close proximity of the D15F37 loci and the deletion breakpoints suggests that the common deletions arise by unequal crossover events at or near these loci.

Cat eye syndrome chromosome breakpoint clustering: identification of two intervals also associated with 22q11 deletion syndrome breakpoints

The supernumerary cat eye syndrome (CES) chromosome is dicentric, containing two copies of 22pter→q11.2. We have found that the duplication breakpoints are clustered in two intervals. The more proximal, most common interval is the 450–650 kb region between D22S427 and D22S36, which corresponds to the proximal deletion breakpoint interval found in the 22q11 deletion syndrome (DiGeorge/velocardiofacial syndrome). The more distal duplication breakpoint interval falls between CRKL and D22S112, which overlaps with the common distal deletion interval of the 22q11 deletion syndrome. We have therefore classified CES chromosomes into two types based on the location of the two breakpoints required to generate them. The smaller type I CES chromosomes are symmetrical, with both breakpoints located within the proximal interval. The larger type II CES chromosomes are either asymmetrical, with one breakpoint located in each of the two intervals, or symmetrical, with both breakpoints located in the distal interval. The co-localization of the breakpoints of these different syndromes, plus the presence of low-copy repeats adjacent to each interval, suggests the existence of several specific regions of chromosomal instability in 22q11.2 which are involved in the production of both deletions and duplications. Since the phenotype associated with the larger duplication does not appear to be more severe than that of the smaller duplication, determination of the type of CES chromosome does not currently have prognostic value.

Detailed microsatellite analysis of chromosome 3p region in non‐papillary renal cell carcinomas

Multiple hemi- and homozygous interstitial deletions at chromosome 3p have been found in different types of cancer, including non-papillary renal cell carcinoma (RCC). To determine the frequency and size of deletions, we have analyzed paired normal and tumor DNA obtained from short-term cultures of 104 non-papillary RCCs with 29 microsatellite markers covering the entire chromosome 3p region. Deletion mapping provided evidence for terminal deletion, with the most distal breakpoint between loci D3S1606 and D3S3666 in 94 cases, whereas constitutional heterozygosity was retained at all loci in 4 RCCs. In 6 cases, interstitial deletions were detected. Deletion mapping detected the smallest overlapping region between loci D3S3666 and D3S1560, which corresponds to an approx. 55 cM genetic distance. Int. J. Cancer 73:225–229, 1997. © 1997 Wiley-Liss, Inc.

Detailed microsatellite analysis of chromosome 3p region in non-papillary renal cell carcinomas.

Multiple hemi- and homozygous interstitial deletions at chromosome 3p have been found in different types of cancer, including non-papillary renal cell carcinoma (RCC). To determine the frequency and size of deletions, we have analyzed paired normal and tumor DNA obtained from short-term cultures of 104 non-papillary RCCs with 29 microsatellite markers covering the entire chromosome 3p region. Deletion mapping provided evidence for terminal deletion, with the most distal breakpoint between loci D3S1606 and D3S3666 in 94 cases, whereas constitutional heterozygosity was retained at all loci in 4 RCCs. In 6 cases, interstitial deletions were detected. Deletion mapping detected the smallest overlapping region between loci D3S3666 and D3S1560, which corresponds to an approx. 55 cM genetic distance.

A novel gene encoding an SH3 domain protein is mutated in nephronophthisis type 1.

Juvenile nephronophthisis (NPH), an autosomal recessive cystic kidney disease, is the primary genetic cause of chronic renal failure in children. About two thirds of patients with NPH carry a large homozygous deletion at the gene locus NPH1 on 2q13. We here identify a novel gene. NPHP1, which extends over most of this common deletion. The 4.5-kb transcript encodes a protein with an SH3 domain, which is highly conserved throughout evolution. The 11-kb interval between the 3' end of NPHP1 and an inverted repeat containing the distal deletion breakpoint was found to contain the first exon of a second gene, MALL. In patients with a hemizygous deletion of the NPH1 region, additional point mutations were found in NPHP1 but not in MALL.

Analysis of 1;17 translocation breakpoints in neuroblastoma: implications for mapping of neuroblastoma genes.

Deletions and translocations resulting in loss of distal 1p-material are known to occur frequently in advanced neuroblastomas. Fluorescence in situ hybridisation (FISH) showed that 17q was most frequently involved in chromosome 1p translocations. A review of the literature shows that 10 of 27 cell lines carry 1;17 translocations. Similar translocations were also observed in primary tumours. Together with the occurrence of a constitutional 1;17 translocation in a neuroblastoma patient, these observations suggest a particular role for these chromosome re-arrangements in the development of neuroblastoma. Apart from the loss of distal 1p-material, these translocations invariably lead to extra copies of 17q. This also suggested a possible role for genes on 17q in neuroblastoma tumorigenesis. Further support for this hypothesis comes from the observation that in those cell lines without 1;17 translocations, other chromosome 17q translocations were present. These too lead to extra chromosome 17q material. Molecular analysis of 1;17 translocation breakpoints revealed breakpoint heterogeneity both on 1p and 17q, which suggests the involvement of more than 2 single genes on 1p and 17q. The localisation of the different 1p-breakpoints occurring in 1;17 translocations in neuroblastoma are discussed with respect to the recently identified candidate tumor suppressor regions and genes on 1p. In this study, we focused on the molecular analysis of the 17q breakpoints in 1;17 translocations. Detailed physical mapping of the constitutional 17q breakpoint allowed for the construction of a YAC contig covering the breakpoint. Furthermore, a refined position was determined for a number of 17q breakpoints of 1;17 translocations found in neuroblastoma cell lines. The most distal 17q breakpoint was identified in cell line UHG-NP and mapped telomeric to cosmid cCI17-1049 (17q21). This suggests that genes involved in a dosage-dependent manner in the development of neuroblastoma map in the distal segment 17q22-qter. Future studies aim at the molecular cloning of 1;17 translocation breakpoints and at deciphering the mechanisms leading to 1;17 translocations and possibly to the identification of neuroblastoma genes at or in the vicinity of these breakpoints.

Cytogenetic, fluorescence in situ hybridisation, and clinical evaluation of translocations with concomitant deletion at 13q14 in chronic lymphocytic leukaemia.

Deletions and translocations of 13q14 are the most frequent structural chromosome abnormalities found in chronic lymphocytic leukaemia (CLL). We have identified 13q14 translocations in the blood of 30 of 450 (6.6%) CLL patients by conventional cytogenetics, using tetradecanoyl phorbol 12-myristate 13-acetate (TPA) as a mitogen. The translocations are characterised by multiple partner chromosomes and a high incidence, 6 of 30 cases, of complex rearrangements. Seven cases were also studied by fluorescence in situ hybridisation (FISH) using four previously ordered YACs, to define the breakpoints further. Deletions with varying proximal and distal breakpoints were found in six cases. Two of the cases had deletions of the cytogenetically normal chromosome 13 at q14, and in one case the 13q14 translocation was a secondary genetic event. No difference in the clinical features between the patients with 13q14 translocation and 54 patients with 13q14 deletions or four patients with both a translocation and a deletion was observed. These data suggest that the genetic consequence of 13q14 translocations in CLL is the loss of a tumour suppressor gene.

FHIT gene and the FRA3B region are not involved in the genetics of renal cell carcinomas

The FHIT gene locus at 3p14.2 covers about 500 kb, including the fragile site FRA3B and the constitutional t(3;8) breakpoint associated with the development of multiple renal cell carcinomas (RCC). A terminal deletion of the short arm of chromosome 3 with the most distal breakpoint in the FRA3B region is the characteristic genetic event in nonpapillary RCC. Since aberrant FHIT transcripts have been observed in gastrointestinal and other tumors, this gene has been suggested to function as a tumor suppressor. To evaluate the role of FHIT and the FRA3B region in the genetics of RCC, we analyzed FHIT expression by RT-PCR and performed microsatellite deletion mapping in the FHIT region. In addition to two cases from a t(3;8) family, only three out of 100 sporadic nonpapillary RCC showed a breakpoint within the FHIT region, whereas 94 tumors showed a deletion breakpoint proximal to the FHIT gene. FHIT transcripts of normal size were observed in 33 out of 34 tumors. Direct sequencing of eight PCR products revealed a normal FHIT sequence without mutations in the coding region. An established cell line from a renal cancer xenograft showed a smaller FHIT transcript. Sequence analysis revealed a mixture of several splicing variants of the FHIT gene. Since only three out of 100 sporadic nonpapillary RCC had a deletion breakpoint within the FRA3B/FHIT region, and since all but one renal cell tumor showed a normal FHIT transcript, we can exclude the involvement of the FHIT gene and the FRA3B region in the genetics of renal cell cancer. Genes Chromosom. Cancer 20:9–15, 1997. © 1997 Wiley-Liss, Inc.

FHIT gene and the FRA3B region are not involved in the genetics of renal cell carcinomas

The FHIT gene locus at 3p14.2 covers about 500 kb, including the fragile site FRA3B and the constitutional t(3;8) breakpoint associated with the development of multiple renal cell carcinomas (RCC). A terminal deletion of the short arm of chromosome 3 with the most distal breakpoint in the FRA3B region is the characteristic genetic event in nonpapillary RCC. Since aberrant FHIT transcripts have been observed in gastrointestinal and other tumors, this gene has been suggested to function as a tumor suppressor. To evaluate the role of FHIT and the FRA3B region in the genetics of RCC, we analyzed FHIT expression by RT-PCR and performed microsatellite deletion mapping in the FHIT region. In addition to two cases from a t(3;8) family, only three out of 100 sporadic nonpapillary RCC showed a breakpoint within the FHIT region, whereas 94 tumors showed a deletion breakpoint proximal to the FHIT gene. FHIT transcripts of normal size were observed in 33 out of 34 tumors. Direct sequencing of eight PCR products revealed a normal FHIT sequence without mutations in the coding region. An established cell line from a renal cancer xenograft showed a smaller FHIT transcript. Sequence analysis revealed a mixture of several splicing variants of the FHIT gene. Since only three out of 100 sporadic nonpapillary RCC had a deletion breakpoint within the FRA3B/FHIT region, and since all but one renal cell tumor showed a normal FHIT transcript, we can exclude the involvement of the FHIT gene and the FRA3B region in the genetics of renal cell cancer.

Counselling dilemmas associated with the molecular characterisation of two Angelman syndrome families.

We report the molecular characterisation of two families with Angelman syndrome referred for prenatal diagnosis, in which atypical molecular findings resulted in counselling dilemmas. The first is a familial case...

Delimiting the Wolf-Hirschhorn syndrome critical region to 750 kilobase pairs.

Wolf-Hirschhorn syndrome (WHS) is a multiple anomaly condition characterized by mental and developmental defects, resulting from the absence of the distal segment of one chromosome 4 short arm (4p16.3). Owing to the complex and variable expression of this disorder, it is thought that the WHS is a contiguous gene syndrome with an undefined number of genes contributing to the phenotype. The 2.2 Mbp genomic segment previously defined as the critical region by the analyses of patients with terminal or interstitial deletions is extremely gene dense and an intensive investigation of the developmental role of all the genes contained within it would be daunting and expensive. Further refinement in the definition of the critical region would be valuable but depends on available patient material and accurate clinical evaluation. In this study, we have utilized fluorescence in situ hybridization to further characterize a WHS patient previously demonstrated to have an interstitial deletion and demonstrate that the distal breakpoint occurs between the loci FGFR3 and D4S168. This reduces the critical region for this syndrome to less than 750 kbp. This has the effect of eliminating several genes previously proposed as contributing to this syndrome and allows further research to focus on a more restricted region of the genome and a limited set of genes for their role in the WHS syndrome.

Constitutional inversion of chromosome 7 and hematologic cancers

Nonrandom aberrations of chromosome 7 have been described in various hematopoietic disorders. We describe here two unrelated families with the same constitutional inversion of chromosome 7 [inv(7)(q11.2q22)]. The probands in both families had acute leukemia and cytogenetic analysis revealed that the inversion was the sole cytogenetic abnormality in the bone marrow at diagnosis. There is a history of hematologic diseases in one of these families that included a son who is a carrier of this constitutional inversion. The distal inversion breakpoint lies within the common region of chromosome loss identified in some myeloid diseases. These observations raise the possibility that this inherited chromosome rearrangement could result in a mutation of a tumor suppressor gene and possibly represent a predisposing event for the development of leukemia in these individuals.

Aphidicolin-Induced FRA3B Breakpoints Cluster in Two Distinct Regions

The common fragile site at chromosomal band 3p14.2 (FRA3B) is the most sensitive single site in the human genome to induced chromosomal lesions. This fragile site may predispose chromosome 3p to breakage that is commonly observed in lung, renal, and many other cancers. We previously used aphidicolin induction of FRA3B expression in a chromosome 3-only somatic cell hybrid to generate a series of hybrids with breakpoints in the 3p14.2 region. These breakpoints were localized to two distinct clusters, separated by 200 kb, that lie on either side of a region of frequent breakage within FRA3B as observed by FISH analysis. Seven proximal aphidicolin-induced breakpoints were localized at or near the end of a THE element. The THE-1 element is flanked by LINE andAlurepetitive elements. The eight distal aphidicolin-induced breakpoints clustered in a region capable of forming multiple hairpin-like structures. Thus repetitive elements and hairpin-like structures may be responsible for chromosome fragility in this region.

51 Analysis of prognostic factors for low risk myelodysplastic syndrome; a proposal of new scoring system

Chromosomal deletions occurring in myeloid malignancies have sometimes been reported either with no breakpoints or as terminal deletions. It is of importance to deduce whether these deletions are actually terminal or interstitial because this has implications for their biologic consequences and the mechanism of their development. Chromosomal deletions have been observed in 38 patients with myeloid malignancies. Two or more deletions occurred in six cases, and in seven cases this was part of a complex abnormality. In all, 45 deletions were observed. In all cases analyzed, the deletions consistently were interstitial. Of the 38 cases, 16 were myelodysplastic syndromes (MDS) [refractory anemia (RA), three; RA with ringed sideroblasts (RARS) three; RA with excess of blasts (RAEB) eight; RAEB in transformation (RAEB-t) one; and unclassified, one], 11 cases were acute nonlymphocytic leukemia (ANLL), and 11 were other myeloproliferative disorders [polycythemia rubra vera (PRV) seven; essential thrombocytopenia (ET), three; unclassified, one]. In general, no uniformity of breakpoints could be identified other than del(9)(q13q22.2) most of which occurred with t(8;21) and del(20)(q11.2q13.3 or 13.1). The breakpoints corresponded to or were adjacent to fragile sites in 49% (proximal 64%, distal 33%). These data emphasize that chromosomal deletions in myeloid malignancies are interstitial. The uniformity of breakpoints in del 9q and del 20q supports the concept that in some instances the exact breakpoints may be important through juxtaposition of genes rather than loss of critical regions. The data also suggest that there may be different mechanisms for the development of proximal and distal breakpoints.

A 4 Mb cryptic deletion associated with inv(8)(q13.1q24.11) in a patient with trichorhinophalangeal syndrome type I.

We report on an 11 year old girl with trichorhinophalangeal syndrome type I (TRPS1), postaxial polydactyly of the fingers, and a de novo paracentric inversion on the long arm of...

Eleven X Chromosome Breakpoints Associated with Premature Ovarian Failure (POF) Map to a 15-Mb YAC Contig Spanning Xq21

Eleven balanced X-autosome translocations associated with premature ovarian failure (POF) were mapped to a YAC contig spanning most of Xq21 and constructed between the DXS223 and DXS1171 loci. The contig corresponds to a genomic region of about 15 Mb and contains the whole X-Y homologous region. The most proximal and most distal breakpoints associated with POF were mapped 15 Mb apart. The remaining breakpoints were localized along this large region, in the X-specific and in the X-Y homologous region. Four of the YACs contained two breakpoints in the same or in flanking STS intervals. Our results confirm the cytological findings and suggest that a minimum number of eight different genes in Xq21 may be involved with ovary development. Interruption of such loci could be the cause of POF.

Long-range genomic rearrangements upstream of Kit dysregulate the developmental pattern of Kit expression in W57 and Wbanded mice and interfere with distinct steps in melanocyte development.

Mutations in the murine dominant white spotting (W) locus cause pleiotropic developmental defects that affect hematopoietic cells, melanocytes, germ cells and the interstitial cells of Cajal in the gut. W mutations either alter the coding sequence of the Kit receptor tyrosine kinase, resulting in a receptor with impaired kinase activity, or affect Kit expression. Here we describe the molecular and cell-type-specific developmental defects of two of the latter class of regulatory W alleles, W57 and Wbanded(bd). In both mutants, the temporal and spatial patterns of Kit expression are dysregulated during embryogenesis and in adult animals. In Wbd mice, ectopic expression of Kit in the dermatome of the somites at days 10.8 and 11.8 of development seemed to interfere with melanoblast development. In contrast, the W57 allele leads to an intrinsic pigmentation defect by downregulating developmental Kit expression in trunk melanoblasts, but not melanoblasts around the otic vesicle. Both mutations affect transcriptional initiation of the Kit gene. The W57 allele is associated with a 80 kb deletion 5' of the Kit-coding region while Wbd is associated with a 2.8 Mb genomic inversion of chromosome 5 with the distal breakpoint between Kit and the platelet-derived growth factor receptor alpha (Pdgfra) gene, and the proximal breakpoint between the genes for the GABA receptor beta 1 (Gabrb1) and the Tec tyrosine kinase, juxtaposing the Kit and Tec tyrosine kinase genes. Neither W57 nor Wbd affect genomic sequences previously suggested in in vitro experiments to control cell-type-specific expression of Kit. These results link specific mechanisms of cellular and developmental defects to long-range genomic rearrangements that positively and negatively affect Kit transcription in different cell lineages as well as in different subpopulations of the same lineage.

Refined molecular characterization of the breakpoints in small inv dup(15) chromosomes.

Inv dup(15) is the most common supernumerary marker chromosome in humans. To investigate the mechanism responsible for this frequent chromosome rearrangement, we characterized the breakpoints in 18 individuals with small inv dup(15) chromosomes [i.e., negative for the Prader-Willi (PWS)/Angelman syndrome (AS) critical region]. Since two proximal breakpoint regions ("hotspots") for PWS/AS deletions have been previously identified with the most proximal 15q markers D15S541/S542 and S543, we hypothesized that formation of the small inv dup(15) chromosomes may involve one or both of these breakpoint hotspots. By analysis with S542, both breakpoint regions were found to be involved in approximately equal frequencies. In ten cases, the inv dup(15) was negative for S542 (Class I), indicating the breakpoint is between the centromere and the most proximal marker on chromosome 15. For the other eight cases, S542 was positive by fluorescence in situ hybridization (5/5) and/or microsatellite analysis (7/7), but S543 was negative (Class II). These two breakpoint regions appear to be the same as the two proximal breakpoints reported in the common PWS/AS deletions. To initiate cloning and sequencing of the Class II breakpoint, the gap in the yeast artificial chromosome (YAC) contig between S541/S542 and S543 was filled by screening the CEPH YAC and mega-YAC libraries. YACs 705C2 and 368H3 were found to bridge this gap, and therefore contain the more distal breakpoint region. The finding of consistent breakpoints in small inv dup(15), like that found in PWS/AS deletions, provides strong evidence for hotspots for chromosome breakage in this region. In addition, our results show that two extra copies (tetrasomy) of the region from 15cen to the euchromatic region containing S542 are present in individuals with Class II breakpoints. Since most individuals carrying a small inv dup(15) are phenotypically normal, the euchromatin region included in the small inv dup(15) chromosomes does not appear to contain genes with clinically significant dosage effects.