Characterization of familial partial 10p trisomy by chromosomal microdissection,FISH, and microsatellite dosage analysis

Unbalanced translocations are a frequent cause of multiple congenital anomalies in children. Translocations as small as 2–5 Mb of DNA are detectable by G-banding under optimal conditions. Some of these small translocations are visible but cannot be characterized cytogenetically due to the lack of characteristic banding on Giemsa preparations. We have combined chromosomal microdissection and fluorescence in situ hybridization (FISH) to identify the origin of a small translocated segment in three members of a family with a derivative chromosome 9 and multiple anomalies, including several ophthalmologic anomalies. We microdissected the abnormal region of the derivative 9 chromosome and used this DNA to generate a FISH probe. This probe hybridized to distal 10p on the metaphase spread of the proband, indicating the origin of the translocated segment. A whole 10p FISH probe confirmed the origin by hybridizing to the translocated segment of the derivative chromosome. FISH was then performed with a whole chromosome 9 painting probe and excluded the presence of a reciprocal, balancing translocation. We then studied the chromosome 10 partial duplication with microsatellite markers to better characterize the chromosomal segment that caused these phenotypic features. By examining the involved areas with distal 10p and 9p microsatellite markers, we were able to demonstrate a minimum of 9 Mb of trisomic 10p DNA with a chromosomal breakpoint between 10p14– 10p15. We then compared this family’s clinical findings to those of individuals with partial 10p trisomy who had been reported in the literature. The clinical phenotypes seen in this family are similar to, but milder than, the phenotypes of persons with the larger partial trisomies of 10p that were diagnosable by cytogenetic analysis alone. This study shows that microdissection and DNA markers can be used to precisely define small translocations that are difficult to identify by conventional G-banded chromosome analysis. Received: 29 February 1996 / Revised: 19 April 1996     

Mapping by chromosome sorting of several gene probes, including c-myc, to the derivative chromosomes of a 3;8 translocation associated with familial renal cancer

In eight members of a single family a constitutional translocation t(3;8) (p14.2;q24.1) is associated with the development of renal cancer. Chromosomes isolated from a cell line established from a subject with this translocation were analysed in flow with a fluorescence-activated cell sorter (FACS II). Nearly six million chromosomes from the flow karyotype region containing the der(8) and 5.5 million from the region containing the der(3) were sorted, the DNA extracted, digested with EcoRI, size fractionated by electrophoresis, and transferred to nitrocellulose. Hybridization with gene probes for c-mos, which has been localized to 8q11-q22 and somatostatin, which has been mapped to 3q28, confirmed that the sorted fractions contained, respectively, the der(8) and der(3) chromosomes. The cellular oncogenes c-raf-1 (3p25) and c-myc (8q24) were found to be translocated to the der(8) and der(3) chromosomes, respectively. The possible role that the relocation of c-myc might have on the development of renal carcinoma in carriers of this 3;8 translocation was further studied by analysis of the region surrounding the c-myc gene. By the use of cosmid cloning, no rearrangement 31 Kb 5'(or 19 Kb 3') of the translocated gene was found, indicating that the break-point is not immediately adjacent to c-myc. In an associated study, the DNA fragment D3S2 from chromosome 3 was found to map to 3p14.2-pter. This assignment in conjunction with published somatic cell hybrid data enabled D3S2 to be mapped more precisely to the interval 3p14.2-3p21.     

Molecular characterization of a Y;15 translocation segregating in a family

Summary We have used Y-specific and Y-derived DNA probes for in situ hybridization and Southern blotting analysis to characterize a Y;15 translocation showing normal Mendelian inheritance in a family. Cytogenetically there appeared to be an unbalanced translocation of Yqh to 15p; this translocation may be considered as a prototype of those translocations between Yq and the short arm of an acrocentric chromosome which have a population incidence of approximately 1 in 2,000. Our molecular studies showed that, in all probability, the breakpoints were near the border between Yq11.23 and Yq12, and in 15p11, respectively; the translocation is abbreviated t(Y;15)(q12;p11). Using the Y-specific probe pY431 in a quantitative Southern hybridization assay, normal females had no hybridization, female carriers and normal men had the same amount, and male carriers had twice that amount. Cytogenetic analysis and quantitative in situ hybridization using probes pY431 and pY3.4 were consistent with the hypothesis that the portion of Yq translocated to 15p comprised all of Yq12 and none of Yq11. The absence of Southern hybridization with probes specific for Yp and Yq11 confirmed this observation. Even though the family was ascertained through two brothers who both had schizophrenia and were carriers of the translocation, the clinical evaluation of a total of nine individuals with the translocation and five without it did not suggest its association with an abnormal phenotype.     

Molecular and cytogenetic characterization of a de novo t(5p;21q) in a patient previously diagnosed as monosomy 21.

Genomic single-copy DNA fragments were used to characterize an undetected chromosome translocation in an individual whose metaphase chromosome analysis revealed apparent monosomy 21. Eight RFLPs detected by six probes were used to identify homologous sequences from chromosome 21 in DNA digests from the proband and her parents. These family studies showed that the proband was disomic for the distal region of 21q. Reverse banding and in situ hybridization of chromosome 21-specific probes to metaphase chromosomes from the proband revealed a de novo translocation with breakpoints at 5p13 or 14 and 21q11 or 21. In situ hybridization permitted orientation of the translocated portion of chromosome 21 on the derivative chromosome 5 and, in conjunction with molecular analysis and previous mapping studies, refined the physical map for the long arm of chromosome 21.     

A family with Huntington disease and reciprocal translocation 4;5.

We report the clinical and cytogenetic findings in a family in which a balanced reciprocal translocation between the long arm of chromosome 4 and the short arm of chromosome 5 is segregating together with Huntington disease in 2 generations. In situ hybridization studies revealed that the linked human DNA marker is located on the short arm of the normal and translocated chromosome 4 in the region 4p16. The association between Huntington disease and the translocation in this family may represent a chance occurrence. However, it is also possible that there is an undetected rearrangement of DNA on chromosome 4 involving the gene for Huntington disease but not affecting the site of the linked marker. Finally, the likelihood that this represents heterogeneity cannot be excluded.     

携带抗黄矮病基因的中间偃麦草染色体2Ai_2特异的分子标记

The wheat-Thinopyrum intermedium addition lines Z1,Z2 contain a pair of Th. intermedium chromosomes 2Ai-2 carrying the gene with resistance to barley yellow dwarf virus (BYDV). Genomic in situ hybridization (GISH) was used to analyze the chromosome constitution of Z1,Z2 by using genomic DNA probes from Th. intermedium and Pseudoroegneria strigosa. The results showed that the chromosome constitution of either Z1 or Z2 composes of 42 wheat chromosomes and two Th. intermedium chromosomes (2Ai-2). The 2Ai-2 chromosome is St-E intercalary translocation, in which the E genomic chromosome segment translocated into the middle region of the long arm of chromosome belonging to St genome. With the genomic DNA probe of Ps. strigosa, the GISH pattern specific to the 2Ai-2 chromosome may be used as a molecular cytogenetic marker. A detailed RFLP analysis on Z1, Z2 and their parents was carried out by using 12 probes on the wheat group 2 chromosomes. Twenty RFLP markers specific to the 2Ai-2 chromosome were identified. Two RAPD markers of OPR16 –350 and OPH09 -1580, specific to the 2Ai-2 chromosome, were identified from 280 RAPD primers. These molecular markers could be used to assisted-select translocation lines with small segment of the 2Ai-2 chromosome and provide tools to localize the BYDV resistance.     

携带抗黄矮病基因的中间偃麦草染色体2Ai—2特异的分子标记

小麦－中间偃麦草二体异附加系Ｚ１、Ｚ２具有一对携带抗黄矮病基因的中间偃麦草染色体２Ａｉ－２。利用中间偃麦草（Ｔｈｉｎｏｐｙｒｕｍ ｉｎｔｅｒｍｅｄｉｕｍ （Ｈｏｓｔ） Ｂａｋｗｏｔｈ ａｎｄ Ｄｅｗｅｙ）和拟鹅冠草（Ｐｓｅｕｄｏｒｏｅｇｎｅｉａ ｓｔｒｉｇｏｓａ）基因组ＤＮＡ作探针,对Ｚ１、Ｚ２进行基因组原位杂交分析。结果表明,Ｚ１、Ｚ２附加的一对中间偃麦草染色体２Ａｉ－２为Ｓｔ－Ｅ染色体,Ｅ组染     

Different risks in two familial translocations t(9;12) with similar breakpoints.

The risk of offspring with unbalanced karyotypes born to carriers of reciprocal chromosomal translocation (RCT) is important to evaluate for further family planning and prenatal diagnosis. The authors describe two families with carriers of similar RCT concerning breakpoint positions and discuss the different individual risks for abnormal progeny. These translocations were studied by GTG, RBG and CBG banding. They have the same breakpoint on 9p, i.e. 9p22, and a different one on 12p, i.e. terminal (pter----p13) and intermediate (p11.2), respectively. The risk value of 27% for family 1 was obtained directly from the large enough pedigree (high risk) a risk value of about 5% was estimated for family 2, according to the guidelines of Stene and Stengel-Rutkowski (1988). The data show that similar translocations with only slight differences in the breakpoints position have different risks for unbalanced progeny. Results of these empiric findings may be used directly in genetic counselling of a family with RCT leading to a single imbalance of the same segment.     

Characterization of a new 4BS.7HL wheat-barley translocation line using GISH, FISH, and SSR markers and its effect on the β-glucan content of wheat

A spontaneous interspecific Robertsonian translocation was revealed by genomic in situ hybridization (GISH) in the progenies of a monosomic 7H addition line originating from a new wheat 'Asakaze komugi' × barley 'Manas' hybrid. Fluorescence in situ hybridization (FISH) with repetitive DNA sequences (Afa family, pSc119.2, and pTa71) allowed identification of all wheat chromosomes, including wheat chromosome arm 4BS involved in the translocation. FISH using barley telomere- and centromere-specific repetitive DNA probes (HvT01 and (AGGGAG)(n)) confirmed that one of the arms of barley chromosome 7H was involved in the translocation. Simple sequence repeat (SSR) markers specific to the long (L) and short (S) arms of barley chromosome 7H identified the translocated chromosome segment as 7HL. Further analysis of the translocation chromosome clarified the physical position of genetically mapped SSRs within 7H, with a special focus on its centromeric region. The presence of the HvCslF6 gene, responsible for (1,3;1,4)-β-D-glucan production, was revealed in the centromeric region of 7HL. An increased (1,3;1,4)-β-D-glucan level was also detected in the translocation line, demonstrating that the HvCslF6 gene is of potential relevance for the manipulation of wheat (1,3;1,4)-β-D-glucan levels.     

Genetic recombination in a chromosomal translocation t(2;8)(p11;q24) of a Burkitt's lymphoma cell line, KOBK101

We analyzed a chromosomal translocation, t(2;8)(p11;q24), in a Burkitt's lymphoma cell line, KOBK101. The translocation reciprocally occurred between a site about 150 bp upstream from the J5 segment in the Ig kappa-encoding gene on chromosome 2 and the A-rich end of an Alu repetitive element located far downstream from the c-myc gene on chromosome 8. Short segments of both parental chromosomes were deleted at the rearrangement site. A sequence related to the heptamer recognition signal for the V-J recombination of Ig genes and a topoisomerase I-recognition sequence were detected at the breakpoints. The V-J recombination occurred on both chromosome 2 and the translocated chromosome 2p- at the J3 and J4 segments, respectively. The J region on the translocated chromosomes was mutated, as compared with that on the untranslocated chromosome, while the Alu element and its upstream sequence were conserved. These results suggest the following aspects to the chromosomal translocation of this cell line. A V-J recombination seems to have occurred at the proximal end of the J4 segment first, and then the translocation took place in the region between the J4 and J5 segments. The translocation may have been mediated by the functions of topoisomerase I and the Alu repetitive sequence located at the breakpoint, although the possibility cannot be ruled out that the recombination machinery for Ig gene rearrangements functioned irregularly.     

Deletions in patients with classical choroideremia vary in size from 45 kb to several megabases

Making use of the p1bD5 probe (DXS165), we have isolated several markers from the choroideremia locus by chromosomal jumping, preparative field-inversion gel electrophoresis, and cloning of a deletion junction fragment. With these clones we were able to identify and characterize eight deletions in 69 choroideremia patients investigated. The deletions are heterogeneous, in both size and location. The smallest deletion (patient LGL1134) comprises approximately 45 kb of DNA, whereas the largest ones (patients 25.6 and LGL2905) span a DNA segment of at least 5 megabases, which is comparable in size to the smallest deletion detected in a TCD patient (patient XL45) showing a complex phenotype. The TCD deletions encompass variable parts of 150-200-kb DNA segment that is flanked by p1bD5 (DXS165) at the centromeric side and by pZ 11 at the telomeric side. The deletions in patients 33.1, LGL1101, and LGl1134 do not span a translocation breakpoint which was previously mapped on the X chromosome of a female with TCD. The clones isolated from the TCD locus are valuable diagnostic markers for deletion analysis of patients or carrier females. In addition, they should be useful for the isolation of expressed sequences that are part of the TCD gene.     

Differences in the genetic structure of Bacillus subitilis strains carrying the trpE26 mutation and strain 168.

It was previously shown that in strains of Bacillus subtilis bearing the trpE26 mutation a chromosome segment (from trpD to ilvA) is translocated to a position near the thr region. Further PBS1-mediated transduction data have now revealed that these strains also possess an inversion of part of the chromosome from the origin of replication, down to the tre locus on one side and the cysB locus on the other. These data concern evidence of linkage of tre-12- to markers in the translocation (hisH2, tyrA1, and metB3) as well as linkage of the cysB3 marker to thi-86, gly-133, and catA. They explain the previously observed absence of linkage of markers in the translocated segment to cysB3. The model proposed for the formation of merodiploids in trpE26 strains, which calls for the fusion of two genetic elements, is not incompatible with this new finding.     

Characterization of a wheat–Thinopyrum bessarabicum (T2JS-2BS·2BL) translocation line

Thinopyrum bessarabicum (2n = 2x = 14, JJ or E^bE^b) is an important genetic resource for wheat improvement due to its salinity tolerance and disease resistance. Development of wheat–Th. bessarabicum translocation lines will facilitate its practical utilization in wheat improvement. In this study, a novel wheat–Th. bessarabicum translocation line T2JS-2BS·2BL, which carries a segment of Th. bessarabicum chromosome arm 2JS was identified and further characterized using sequential chromosome C-banding, genomic in situ hybridization (GISH), dual-color fluorescent in situ hybridization (FISH) and DNA markers. The translocation breakpoint was mapped within bin C-2BS1-0.53 of chromosome 2B through marker analysis. Compared to the Chinese Spring (CS) parent and to CS-type lines, the translocation line has more fertile spikes per plant, longer spikes, more grains per spike and higher yield per plant, which suggests that the alien segment carries yield-related genes. However, plants with the translocation are also taller, head later and have lower 1,000-kernel weight than CS or CS-type lines. By using markers specific to the barley photoperiod response gene Ppd-H1, it was determined that the late heading date was conferred by a recessive allele located on the 2JS segment. In addition, four markers specific for the translocated segment were identified, which can be used for marker-aided screening.     

蓝粒小麦易位系的荧光原位杂交鉴定

普通小麦（Triticum aestivum L.)和长穗偃麦草（Agropyron elongatum (Host)Beauv=Elytriga elongatum(Host)Nevski=Thinopyrum ponticum (Host)Barkworth and Dewey,2n=10x=70)杂交后选育出的蓝粒小麦异代换系（蓝５８）,２n=42其中９９０６中被易位蓝粒片段的相对长度约占易位小麦染色体短臂的１／３,而９９０２中被易位蓝粒片段的相对长度约占易位小麦染色体长臂的１／２,并将９９０２的蓝粒易位片段定位在小麦Ｄ组染色体上;（２）９９１５易位附加和９９０４易位－易位附加,其体细胞染色体数均为４４,其中９９１５的体细胞染色体只有一对发生了易位,另外队了两条长穗偃麦草染色体;而９９０４有两对染色体发生了易位,并易位系中控制蓝粒性状的长穗偃麦草染色体片段的定位和蓝粒小麦易位系的应用进行了讨论。     

Identification of Blue-grained Wheat Translocation Lines Using Fluorescence in Situ Hybridization

The blue-grained wheat substitution line (blue 58) originated from wild hybridization between Triticum aestivum L. and Agropyron elongatum (Host) Beauv= Elytrigia elongatum (Host) Nevski= Thinopyrum ponticum (Host) Barkworth and Dewey (2n=10x=70) was irradiated and four translocation lines were screened by fluorescence in situ hybridization from the offsprings. The results obtained include the following: (1) both the two translocation lines, 9906 and 9902, have 42 chromosomes. The length of the translocated blue-grained segment was approximately one-third of the short-arm and one-half of the long-arm of the translocated wheat chromosome in 9906 and 9902, respectively, and the blue-grained translocated segment in 9902 was located on D genome; (2) both 9915 and 9904 have 44 chromosomes. One pair of chromosomes was translocated and two chromosomes from Th. ponticum were added in 9903, while two pairs of chromosomes were translocated in 9904 by blue-grained wheat segment. The location and application of blue-grained wheat translocation lines were discussed.     

Activation-Induced Cytidine Deaminase Alters the Subcellular Localization of Tet Family Proteins

Activation-induced cytidine deminase (Aid), a unique enzyme that deaminates cytosine in DNA, shuttles between the nucleus and the cytoplasm. A recent study proposed a novel function of Aid in active DNA demethylation via deamination of 5-hydroxymethylcytosine, which is converted from 5-methylcytosine by the Ten-eleven translocation (Tet) family of enzymes. In this study, we examined the effect of simultaneous expression of Aid and Tet family proteins on the subcellular localization of each protein. We found that overexpressed Aid is mainly localized in the cytoplasm, whereas Tet1 and Tet2 are localized in the nucleus, and Tet3 is localized in both the cytoplasm and the nucleus. However, nuclear Tet proteins were gradually translocated to the cytoplasm when co-expressed with Aid. We also show that Aid-mediated translocation of Tet proteins is associated with Aid shuttling. Here we propose a possible role for Aid as a regulator of the subcellular localization of Tet family proteins.     

A cell hybrid and recombinant DNA library that facilitate identification of polymorphic loci in the vicinity of the Huntington disease gene.

Somatic cell hybrids were selected that retain a derivative chromosome 5 from an individual in which the p15.1-pter segment of chromosome 5 is replaced with the p15.1-pter segment of chromosome 4. Hybrids that retain this derivative chromosome exclusively were found to be positive for G8, a DNA marker closely linked to the Huntington disease gene on chromosome 4p. From one such hybrid, a segregant was isolated that had deleted the entire q arm of the derivative chromosome but retained the p arm intact as its only detectable human DNA. A complete recombinant DNA library was prepared from this cell line, and the inserts in approximately 1/3 of the recombinant phage with human DNA were shown to be derived from 4pter-4p15.1, which represents only approximately 1% of the total human genome. The cell hybrid and DNA library represent a rapid and efficient means to identify and isolate many polymorphic DNA markers close to and flanking the Huntington disease gene.     

Identification of DNA sequences flanking the breakpoint of human t(14q21q) Robertsonian translocations.

We have employed molecular probes and in situ hybridization to investigate the DNA sequences flanking the breakpoint of a group of t(14q21q) Robertsonian translocations. In all the families studied, the probands were patients with Down syndrome who carried a de novo t(14q21q) translocation. The DNA probes used were two alphoid sequences, alphaRI and alphaXT, which are specific for the centromeres of chromosomes 13 and 21 and of chromosomes 14 and 22, respectively; a satellite III sequence, pTRS-47, which is specific for the proximal p11 region of chromosomes 14 and 22; and a newly defined satellite III DNA, pTRS-63, which is specific for the distal p11 region of chromosome 14. The two alphoid probes detected approximately the same amount of autoradiographic signal on the translocated chromosomes as was expected for chromosomes 14 and 21 of the originating parent, suggesting that there has been no loss of these centromeric sequences during the translocation events. Results with the two satellite III probes indicated that the domain corresponding to pTRS-47 was retained in the translocated chromosomes, whereas the domain for pTRS-63 was lost. These results have allowed us to place the translocation breakpoint between the pTRS-47 and pTRS-63 domains within the p11 region of chromosome 14.     

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.     

Breakpoints in Robertsonian translocations are localized to satellite III DNA by fluorescence in situ hybridization.

We characterized 21 t(13;14) and 3 t(14;21) Robertsonian translocations for the presence of DNA derived from the short arms of the translocated acrocentric chromosomes and identified their centromeres. Nineteen of these 24 translocation carriers were unrelated. Using centromeric alpha-repeat DNA as chromosome-specific probe, we found by in situ hybridization that all 24 translocation chromosomes were dicentric. The chromatin between the two centomeres did not stain with silver, and no hybridization signal was detected with probes for rDNA or beta-satellite DNA that flank the distal and proximal ends of the rDNA region on the short arm of the acrocentrics. By contrast, all 24 translocation chromosomes gave a distinct hybridization signal when satellite III DNA was used as probe. This result strongly suggests that the chromosomal rearrangements leading to Robertsonian translocations occur preferentially in satellite III DNA. We hypothesize that guanine-rich satellite III repeats may promote chromosomal recombination by formation of tetraplex structures. The findings localize satellite III DNA to the short arm of the acrocentric chromosomes distal to centromeric alpha-repeat DNA and proximal to beta-satellite DNA.