A new type of nonhomologous synapsis in T(X;4)1R1 translocation male mice

The synaptonemal complexes of T(X;4)1R1 (abbreviated R1) translocation heterozygotes have been examined by electron microscopy and compared with those of two X-7 translocations: R5 and R6. The X chromosome breakpoint of R1 is estimated to lie between 78 and 82% from the proximal end of the X, in the same general region as the R5 and R6 breakpoints. The position of the autosomal breakpoint of R1, like that of R6, is about 30% from the proximal end of the respective autosome. R1 is also similar to R6 in that there is extensive nonhomologous synapsis both in quadrivalents and heteromorphic bivalents. We have recently found that the location of breakpoints with respect to the position of the G-bands appears to be related to the synaptic behavior seen in translocation heterozygotes. If both breaks of a reciprocal translocation lie in G-light bands, as was the case with R5, synapsis is confined to homology. However, if one break lies in or immediately adjacent to a G-dark band, there is nonhomologous synapsis, as occurs with R1 and R6. Comparison of the synaptic behavior of R1 with R5 and R6 leads to the conclusion that this G-band-related nonhomologous synapsis is of a different type than the "synaptic adjustment" phenomenon that has been described by Moses (1977a). This G-band-related nonhomologous synapsis is not substage-specific, but competes with homologous synapsis during zygotene-early pachytene.     

Axial shortening during pachynema unrelated to nonhomologous synapsis

The pachytene behavior of chromosomes participating in quadrivalent formation in male mice heterozygous for T(X;4)7Rl or T(X;4)8Rl was analyzed in electron micrographs of microspread spermatocytes. In each population of nuclei from the translocation heterozygotes, the longest 4X axes were approximately the proportional length expected from the respective contributions of the 4 and the X estimated from breakpoint positions in mitotic chromosomes. However, the 4X axis of these translocation quadrivalents undergoes extensive shortening. In both R7 and R8 the shortest 4X axis observed in the population of nuclei was approximately the length of the normal 4 axis. This equalization of axial lengths suggests that there may be an interchromosomal interaction between synapsed chromosomes. In R8, axial shortening of the 4X occurs as pachynema progresses. In both translocations, shortening is accompanied by twisting of the 4X around the 4. Both axial shortening and twists are characteristics exhibited by chromosomal axes of unequal length as part of the meiotic phenomenon described as "synaptic adjustment" (Moses, 1977). Synaptic adjustment involves, in addition, nonhomologous synapsis, which is delayed until the latter part of pachynema. However, axial shortening in R7 and R8 is not accompanied by nonhomologous synapsis. In R7, nonhomologous synapsis does not occur; in R8, it is confined to quadrivalents in which the 4X axis is near its maximum length (i.e., early). This behavior suggests that axial shortening and nonhomologous synapsis during the progression of pachynema (previously considered collectively under the term "synaptic adjustment") are not necessarily coupled events.     

X-linked anhidrotic ectodermal dysplasia and de novo t(X;1) in a female

Summary A de novo translocation (X;1)(q13.1;p36.33) was found in a 2-year-old girl with typical clinical features of X-linked anhidrotic ectodermal dysplasia (EDA). The breakpoint at Xq13.1 is approximately the same as has been described in 2 other EDA females with X;autosome translocations.     

Differential Role of Autophagy in CD4 T Cells and Macrophages during X4 and R5 HIV-1 Infection

Background

HIV-1 can infect and replicate in both CD4 T cells and macrophages. In these cell types, HIV-1 entry is mediated by the binding of envelope glycoproteins (gp120 and gp41, Env) to the receptor CD4 and a coreceptor, principally CCR5 or CXCR4, depending on the viral strain (R5 or X4, respectively). Uninfected CD4 T cells undergo X4 Env-mediated autophagy, leading to their apoptosis, a mechanism now recognized as central to immunodeficiency.

Methodology/Principal Findings

We demonstrate here that autophagy and cell death are also induced in the uninfected CD4 T cells by HIV-1 R5 Env, while autophagy is inhibited in productively X4 or R5-infected CD4 T cells. In contrast, uninfected macrophages, a preserved cell population during HIV-1 infection, do not undergo X4 or R5 Env-mediated autophagy. Autophagosomes, however, are present in macrophages exposed to infectious HIV-1 particles, independently of coreceptor use. Interestingly, we observed two populations of autophagic cells: one highly autophagic and the other weakly autophagic. Surprisingly, viruses could be detected in the weakly autophagic cells but not in the highly autophagic cells. In addition, we show that the triggering of autophagy in macrophages is necessary for viral replication but addition of Bafilomycin A1, which blocks the final stages of autophagy, strongly increases productive infection.

Conclusions/Significance

Taken together, our data suggest that autophagy plays a complex, but essential, role in HIV pathology by regulating both viral replication and the fate of the target cells.     

Genetic mapping of cytological and isozyme markers on chromosomes 1R, 3R, 4R and 6R of rye

Cytogenetic maps involving chromosomes 1R, 3R, 4R and 6R have been developed from the analysis of offspring of crosses between multiple heterozygous rye plants. The maps include isozyme loci GpiR1, Mdh-R1 and Pgd2 (located in chromosome 1R), Mdh-R2 (located in chromosome 3R), Pgm-R1 (located in chromosome 4R) and Aco-R1 (located in chromosome 6R). Various telomeric and interstitial C-bands of these four chromosomes, the centromere split of chromosome 3R, and translocation TR01 were used as cytological markers. By means of electron microscope analysis of spread pachytene synaptonemal complexes, the breakpoint of TR01 was physically mapped in chromosome arms 4RS and 6RL. From the linkage data, conclusions were derived concerning the cytological locations of the isozyme loci and the physical extent of the evolutive translocations involving chromosome arm 6RL.     

Incontinentia pigmenti (type 1) and X;5 translocation.

The authors present a 5-year-old girl with total absence of speech, dysmorphic features, pigmented lesions on the legs, an abnormal EEG and otherwise normal intelligence representing a mild form of type 1 Incontinentia pigmenti associated with an (X;5) (p11.2;q35.2) apparently balanced translocation prenatally diagnosed. The seven previous translocation type 1 IP patients are reviewed and all have the same Xp11 breakpoint. Somatic cell hybrids have been made to further study this breakpoint and further define the putative type 1 IP gene.     

R5 human immunodeficiency virus type 1 (HIV-1) replicates more efficiently in primary CD4+ T-cell cultures than X4 HIV-1

In this report, we present evidence that R5 human immunodeficiency virus type 1 (HIV-1) replicates more efficiently in primary CD4+ T cells than X4 HIV-1. By comparing CD3/CD28-costimulated CD4+ T-cell cultures infected by several X4 and R5 HIV-1 strains, we determined that R5-infected CD4+ T cells produce more virus over time than X4-infected CD4+ T cells. In the first comparison, we found that more cells were infected by the X4-tropic strain LAI than by the R5-tropic strain JR-CSF and yet that higher levels of viral production were detected in the R5-infected cultures. The differential viral production was partially due to the severe cytopathic effects of the X4 virus. We also compared cultures infected with the isogenic HIV-1 strains NL4-3 (X4) and 49.5 (R5). We found that fewer cells were infected by the R5 strain, and yet similar levels of viral production were detected in both infected cultures. Cell death played less of a role in the differential viral production of these strains, as the cell viability remained comparable in both X4- and R5-infected cultures over time. The final comparison involved the primary R5-tropic isolate KP1 and the primary dual-tropic isolate KP2. Although both strains infected similar numbers of cells and induced comparable levels of cytopathicity, viral production was considerably higher in the R5-infected culture. In summary, these data demonstrate that R5 HIV-1 has an increased capacity to replicate in costimulated CD4+ T cells compared to X4 HIV-1.     

Thyroid hormone promotes insulin-induced glucose uptake by enhancing Akt phosphorylation and VAMP2 translocation in 3T3-L1 adipocytes

The purpose of this study was to test a hypothesis that T3 promotes glucose uptake via enhancing insulin-induced Akt phosphorylation and VAMP2 translocation in 3T3-L1 adipocytes. T3 significantly enhanced insulin-induced phosphorylation of Akt, cytoplasma to cell membrane translocations of vesicle-associated membrane protein 2 (VAMP2) and glucose transporter 4 (GLUT4), and glucose uptake in adipocytes. Akt inhibitor X abolished the promoting effects of T3, suggesting that Akt activation is essential for T3 to enhance these insulin-induced events in adipocytes. Knockdown of VAMP2 using siRNA abrogated the effects of T3 on insulin-induced GLUT4 translocation and glucose uptake, suggesting that VAMP2 is an important mediator of these processes. These data suggest that T3 may promote glucose uptake via enhancing insulin-induced phosphorylation of Akt and subsequent translocations of VAMP2 and GLUT4 in 3T3-L1 adipocytes. Akt phosphorylation is necessary for the promoting effects of T3 on insulin-stimulated VAMP2 translocation. Further, VAMP2 is essential for T3 to increase insulin-stimulated translocation of GLUT4 and subsequent uptake of glucose in adipocytes.     

Humanized mice dually challenged with R5 and X4 HIV-1 show preferential R5 viremia and restricted X4 infection of CCR5+CD4+ T cells

CCR5-tropic (R5) immunodeficiency virus type 1 (HIV-1) strains are highly transmissible during the early stage of infection in humans, whereas CXCR4-tropic (X4) strains are less transmissible. This study aimed to explore the basis for early phase R5 and X4 HIV-1 infection in vivo by using humanized mice dually challenged with R5 HIV-1_NLAD8-D harboring DsRed and X4 HIV-1_NL-E harboring EGFP. Whereas R5 HIV-1 replicated well, X4 HIV-1 caused only transient viremia with variable kinetics; however, this was distinct from the low level but persistent viremia observed in mice challenged with X4 HIV-1 alone. Flow cytometric analysis of HIV-1-infected cells revealed that X4 HIV-1 infection of CCR5⁺CD4⁺ T cells was significantly suppressed in the presence of R5 HIV-1. X4 HIV-1 was more cytopathic than R5 HIV-1; however, this was not the cause of restricted X4 HIV-1 infection because there were no significant differences in the mortality rates of CCR5⁺ and CCR5⁻ cells within the X4 HIV-1-infected cell populations. Taken together, these results suggest that restricted infection of CCR5⁺CD4⁺ T cells by X4 HIV-1 (occurring via a still-to-be-identified mechanism) might contribute to the preferential transmission of R5 HIV-1 during the early phase of infection.     

Karyotypes and X chromosome inactivation in segregants of a murine X-autosome translocation, T(X;4)37H

Karyotypes and X chromosome inactivation were studied in embryos obtained from female mice carrying T(X;4)37H translocation on day 6 to 8 of gestation by a BrdU-acridine orange method. A total of 18 different karyotypes were found in 477 embryos examined: 90.0% embryos were products expected from 2:2 alternate or adjacent 1 disjunction. 3:1 and adjacent 2 disjunctions accounted for approximately 8.0% and 0.7% conceptuses, respectively. In the embryo proper of balanced T37H/ + conceptuses, inactivation was random with respect to the normal X and the larger translocation X (4x) chromosome. In all the cells with the 4x inactive, the late replication apparently did not spread to the attached autosomal portion, although black/brown coat variegation implies spreading of inactivation into the autosomal region. The X chromosome segment deprived of the inactivation center remained active in all the cells examined and it exerted deleterious effects on embryonic or fetal development. Observation in embryos having two maternally derived X chromosomes showed that they were indeed resistant to inactivation in early extraembryonic cell lineages, and two copies of active X chromosomes in the trophectoderm fatally affected embryonic development due to inability to form the extraembryonic ectoderm and ectoplacental cone from the polar trophectoderm. In unbalanced X aneuploids the X chromosomes with the deletion were preferentially inactivated due to strong selection against nullisomy X.     

Array-CGH characterization of a de novo t(X;Y)(p22;q11) in a female with short stature and mental retardation

We report the clinical and molecular investigations in a girl with 46,X,-X,+der(X)t(X;Y)(p22;q11) de novo karyotype who presented an intricate phenotype characterized by mental retardation and facial dysmorphisms in combination with short stature. The structure of the derivative X chromosome was studied using BAC array-CGH which disclosed the Xp22 breakpoint between the STS and the VCX3A gene and the presence of the Yq11.1qter chromosome. It is common that females with Xp;Yq translocations present only short stature and are normal in every other aspect. Thus, this would be the first case in which a girl with Xp;Yq translocation presents an unusual phenotype with intermediate male clinical features with Xp;Yq translocations. The risk of developing gonadoblastoma in females with Y chromosome material is also discussed and, to this effect, different explanations related to this apparent variation are also presented.     

Breakpoint mapping and haplotype analysis of three reciprocal translocations identify a novel recurrent translocation in two unrelated families: t(4;11)(p16.2;p15.4)

The majority of constitutional reciprocal translocations appear to be unique rearrangements arising from independent events. However, a small number of translocations are recurrent, most significantly the t(11;22)(q23;q11). Among large series of translocations there may be multiple independently ascertained cases with the same cytogenetic breakpoints. Some of these could represent additional recurrent rearrangements, alternatively they could be identical by descent (IBD) or have subtly different breakpoints when examined under higher resolution. We have used molecular breakpoint mapping and haplotyping to determine the origin of three pairs of reciprocal constitutional translocations, each with the same cytogenetic breakpoints. FISH mapping showed one pair to have different breakpoints and thus to be distinct rearrangements. Another pair of translocations were IBD with identical breakpoint intervals and highly conserved haplotypes on the derived chromosomes. The third pair, t(4;11)(p16.2;p15.4), had the same breakpoint intervals by aCGH and fosmid mapping but had very different haplotypes, therefore they represent a novel recurrent translocation. Unlike the t(11;22)(q23;q11), the formation of the t(4;11)(p16.2;p15.4) may have involved segmental duplications and sequence homology at the breakpoints. Additional examples of recurrent translocations could be identified if the resources were available to study more translocations using the approaches described here. However, like the t(4;11)(p16.2;p15.4), such translocations are likely to be rare with the t(11;22) remaining the only common recurrent constitutional reciprocal translocation.     

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.     

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.     

Molecular cytogenetic analysis of radiation-induced wheat-rye terminal and intercalary chromosomal translocations and the detection of rye chromatin specifying resistance to Hessian fly

Radiation-induced wheat-rye chromosome translocation lines resistant to Hessian fly, Mayetiola destructor (say), were analyzed by in situ hybridization using total genomic and highly repetitive rye DNA probes pSc119 and pSc74. In situ hybridization analysis revealed the exact locations of the translocation breakpoints and allowed the estimation of the sizes of the transferred rye segments. T6BS·6BL-6RL and T4BS· 4BL-6RL are terminal translocations with either most of the complete long arm of rye chromosome 6R or only the distal 57% of the 6RL arm attached to the long arms of wheat chromosomes 6B and 4B, respectively. The breakpoint in T6BS·6BL-6RL is located at a fraction length (FL) of 0.11 in the long arm of T6BS 6BL-6RL and at FL 0.46 in the long arm of T4BS·4BL-6RL. Ti4AS·4AL-6RL-4AL is an intercalary translocation with the breakpoint located at FL 0.06 in the long arm of wheat chromosome 4A. The inserted 6RL segment, with the Hessian fly resistance gene, has a size of 0.7 m, and is the smallest and, so far, the first radiation-induced intercalary translocation identified in wheat.by R. Apples     

Breakpoints of t(4;11) translocations in the human MLL and AF4 genes in ALL patients are preferentially clustered outside of high-affinity matrix attachment regions.

Chromosomal translocations t(4;11) are based on illegitimate recombinations between the human MLL and AF4 genes, and are associated with high-risk acute leukemias of infants and young children. Here, the question was asked, whether a correlation exists between the location of translocation breakpoints within both genes and the location of S/MARs. In "halo mapping experiments" (to define SARs), about 20 kb of MLL DNA was found to be attached to the nuclear matrix. Similar experiments performed for the translocation partner gene AF4 revealed that SARs are spanning nearly the complete breakpoint cluster region of the AF4 gene. By using short DNA fragments in "scaffold reassociation experiments" (to define MARs), similar results were obtained for both genes. However, Distamycin A competition experiments in combination with "scaffold reassociation experiments" revealed specific differences in the affinity of each tested DNA fragment to bind the isolated nuclear matrix proteins. When the latter data were compared with the known location of chromosomal breakpoints for both genes, an unexpected correlation was observed. DNA areas with strong MAR affinity contained fewer translocation breakpoints, while areas with weak or absent MAR affinity showed a higher density of chromosomal breakpoints.     

Physical mapping of 5S rDNA reveals a new locus on 3R and unexpected complexity in a rye translocation used in chromosome mapping

Using fluorescence in situ hybridization (FISH) with probe pScT7, three different 5S rDNA loci were detected in the satellite of rye chromosome 1R (5SDna-R1) and in the short arms of chromosomes 3R (5SDna-R3) and 5R (5SDna-R2) respectively. All three loci showed polymorphism for the hybridization signal intensity. In order to determine the localization of these rye 5S rDNA multigene loci with higher precision within the corresponding chromosome arms, the probe pScT7 was physically mapped by FISH in relation to the following five translocations (Wageningen Tester Set): T850W (1RS/4RL), T248W (1RS/6RS), T273W (1RS/5RL), T305W (2RS/5RS) and T240W (3RS/5RL). Accurate physical maps of the translocation breakpoints had previously been made using electron microscope analysis of spread pachytene synaptonemal complexes of heterozygotes for the different translocations. The results indicate that locus 5SDna-R3 is located between the breakpoint of translocation T240W and the telomere, whereas locus 5SDna-R2 is located between the breakpoint of translocation T305W and the centromere, the hybridization of probe pScT7 on T305W translocated chromosomes demonstrating the complex nature of this translocation. On the other hand, the simultaneous detection of probes pScT7 and pTA71 (18S-5.8S-26S rDNA) with two different fluorochromes, indicated that the breakpoints of translocations T850W and T248W are located between loci Nor-R1 and 5SDna-R1.     

Localization of ribosomal DNA within the proximal X heterochromatin of Sciara coprophila (Diptera,Sciaridae)

By means of several reciprocal translocations in Sciara coprophila, each having a break-point in the proximal X heterochromatin, it has been possible in the salivary gland nucleus to bring about separation of specific regions of this heterochromatin and then, by means of in situ hybridization, to determine the relative number of ribosomal RNA cistrons in each. The three blocks of heterochromatin delineated by the translocation break-points have been designated H1, H2, and H3; H1 is the most proximal, lying immediately to the right of the X centromere, and H3 is the most distal, constituting the very end (right) of the chromosome. The distribution of ribosomal RNA cistrons is as follows: 10% are located in H1; 50% in H2; and 40% in H3. For the first time it has been possible to confirm by grain count our previous biochemical estimate of a 60% deletion of rRNA cistrons in the proximal heterochromatin of the X _W homologue. The grain count data also support the conclusion of our previously published cytological analysis, that the exchange points in the X heterochromatin are identical in translocations T29 and T32 (between H1 and H2), also in translocations T23 and T70 (between H2 and H3). The coincidence of break-points in the X heterochromatin is considered in relation to the chromomere make-up of the region. Also, the occurrence of ribosomal RNA cistrons in all three heterochromomeres is discussed in relation to the functional significance of chromomeres.     

X heterochromatin subdivision and cytogenetic analysis in Sciara coprophila (diptera,sciaridae)

The so-called controlling element (CE), which normally programs the curious behavior of the sex chromosome in this genus, has been localized in the short right arm of the polytene X in S. coprophila. The localization was accomplished by use of five X-autosome translocations whose break points define three blocks of heterochromatin (heterochromomeres) extending from the X centromere to the very end (right) of the chromosome. The behavior of the translocation chromosomes at the crucial second spermatocyte division was examined and the precocious chromosome identified in all five cases. Then, knowing the heterochromomere make-up of each chromosome, the position of the CE could be mapped; it is located in heterochromomere H2, the same block of heterochromatin that contains 50% of the ribosomal RNA cistrons. — The question of whether the CE can manipulate any centromere in the nucleus has been only partially answered. It can manipulate translocation chromosomes which possess the centromere of the metacentric autosome (salivary chromosome IV) or that of the shorter rod (salivary chromosome II); but the longer rod (salivary chromosome III) whose proximal end, as seen in the polytene nucleus, is heavily laden with heterochromatin of its own, has not been brought under CE control. — In one of the translocations, T23, the precocious chromosome is a very large metacentric chromosome which resembles the peculiar V-shaped X of S. pauciseta. This peculiarity is not observed in the J-shaped precocious chromosome of T29. These points are discussed.Dedicated to Professor Hans Bauer on the occasion of his 75th birthday.     

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.