Regional mapping of the human No. 1 and X chromosome in interspecific cell hybrids using an X/1 translocation.

Fibroblasts from a carrier of an X/1 translocation, 46,XY,t(X;1)(q28;q31), were fused with Chinese hamster cells. The resulting hybrids were analyzed for human No. 1 and X-chromosome markers. The data indicate that the loci for PGM1, PGD, PPH, and GuK1 are situated either in the long arm proximal to a break point in band 1q31 or in the short arm. The loci for Pep-C, FH, and GuK1 are located distal to the break point. HPRT and G6PD are probably situated distal to a break point in band q28 of the X chromosome; alpha-Gal A is situated proximal to the break point, either on the long or short arm of the chromosome.     

CD40-CD40 ligand interaction is central to cell-mediated immunity against Toxoplasma gondii: patients with hyper IgM syndrome have a defective type 1 immune response that can be restored by soluble CD40 ligand trimer.

Cell-mediated immunity that results in IL-12/IFN-gamma production is essential to control infections by intracellular organisms. Studies in animal models revealed contrasting results in regard to the importance of CD40-CD40 ligand (CD40L) signaling for induction of a type 1 cytokine response against these pathogens. We demonstrate that CD40-CD40L interaction in humans is critical for generation of the IL-12/IFN-gamma immune response against Toxoplasma gondii. Infection of monocytes with T. gondii resulted in up-regulation of CD40. CD40-CD40L signaling was required for optimal T cell production of IFN-gamma in response to T. gondii. Moreover, patients with hyper IgM (HIGM) syndrome exhibited a defect in IFN-gamma secretion in response to the parasite and evidence compatible with impaired in vivo T cell priming after T. gondii infection. Not only was IL-12 production in response to T. gondii dependent on CD40-CD40L signaling, but also, patients with HIGM syndrome exhibited deficient in vitro secretion of this cytokine in response to the parasite. Finally, in vitro incubation with agonistic soluble CD40L trimer enhanced T. gondii-triggered production of IFN-gamma and, through induction of IL-12 secretion, corrected the defect in IFN-gamma production observed in HIGM patients. Our results are likely to explain the susceptibility of patients with HIGM syndrome to infections by opportunistic pathogens.     

Trans-splicing repair of CD40 ligand deficiency results in naturally regulated correction of a mouse model of hyper-IgM X-linked immunodeficiency

X-linked immunodeficiency with hyper-IgM (HIGM1), characterized by failure of immunoglobulin isotype switching, is caused by mutations of the CD40 ligand (CD40L), which is normally expressed on activated CD4(+) T cells. As constitutive expression of CD40L induces lymphomas, we corrected the mutation while preserving the natural regulation of CD40L using pre-mRNA trans-splicing. Bone marrow from mice lacking CD40L was modified with a lentivirus trans-splicer encoding the normal CD40L exons 2-5 and was administered to syngenic CD40L-knockout mice. Recipient mice had corrected CD40L mRNA, antigen-specific IgG1 responses to keyhole limpet hemocyanin immunization, regulated CD4(+) T-cell CD40L expression after CD3 stimulation in primary and secondary transplanted mice, attenuation of Pneumocystis carinii pneumonia, and no evidence of lymphoproliferative disease over 1 year. Thus, HIGM1 can be corrected by CD40L trans-splicing, leading to functional correction of the genetic defect without the adverse consequences of unregulated expression of the CD40L gene.     

Demethylation of CD40LG on the inactive X in T cells from women with lupus

Why systemic lupus erythematosus primarily affects women is unknown. Recent evidence indicates that human lupus is an epigenetic disease characterized by impaired T cell DNA methylation. Women have two X chromosomes; one is inactivated by mechanisms including DNA methylation. We hypothesized that demethylation of sequences on the inactive X may cause gene overexpression uniquely in women, predisposing them to lupus. We therefore compared expression and methylation of CD40LG, a B cell costimulatory molecule encoded on the X chromosome, in experimentally demethylated T cells from men and women and in men and women with lupus. Controls included TNFSF7, a methylation-sensitive autosomal B cell costimulatory molecule known to be demethylated and overexpressed in lupus. Bisulfite sequencing revealed that CD40LG is unmethylated in men, while women have one methylated and one unmethylated gene. 5-Azacytidine, a DNA methyltransferase inhibitor, demethylated CD40LG and doubled its expression on CD4(+) T cells from women but not men, while increasing TNFSF7 expression equally between sexes. Similar studies demonstrated that CD40LG demethylates in CD4(+) T cells from women with lupus, and that women but not men with lupus overexpress CD40LG on CD4(+) T cells, while both overexpress TNFSF7. These studies demonstrate that regulatory sequences on the inactive X chromosome demethylate in T cells from women with lupus, contributing to CD40LG overexpression uniquely in women. Demethylation of CD40LG and perhaps other genes on the inactive X may contribute to the striking female predilection of this disease.     

X chromosome inactivation in carriers of Barth syndrome.

Barth syndrome (BTHS) is a rare X-linked recessive disorder characterized by cardiac and skeletal myopathy, neutropenia, and short stature. A gene for BTHS, G4.5, was recently cloned and encodes several novel proteins, named "tafazzins." Unique mutations have been found. No correlation between the location or type of mutation and the phenotype of BTHS has been found. Female carriers of BTHS seem to be healthy. This could be due to a selection against cells that have the mutant allele on the active X chromosome. We therefore analyzed X chromosome inactivation in 16 obligate carriers of BTHS, from six families, using PCR in the androgen-receptor locus. An extremely skewed X-inactivation pattern (>=95:5), not found in 148 female controls, was found in six carriers. The skewed pattern in two carriers from one family was confirmed in DNA from cultured fibroblasts. Five carriers from two families had a skewed pattern (80:20-<95:5), a pattern that was found in only 11 of 148 female controls. Of the 11 carriers with a skewed pattern, the parental origin of the inactive X chromosome was maternal in all seven cases for which this could be determined. In two families, carriers with an extremely skewed pattern and carriers with a random pattern were found. The skewed X inactivation in 11 of 16 carriers is probably the result of a selection against cells with the mutated gene on the active X chromosome. Since BTHS also shows great clinical variation within families, additional factors are likely to influence the expression of the phenotype. Such factors may also influence the selection mechanism in carriers.     

Establishment and characterization of a clonal human T-cell line, MKB-1 derived from a patient with acute myeloblastic leukemia

A human T-cell line, designated as MKB-1, was established by cloning procedures in a suspension culture from a peripheral blood of a 17-year-old female patient with acute myeloblastic leukemia. The immunological marker profile of MKB-1 indicated that unlike a myeloid phenotype of the original leukemic cells, the cells were positive for CD3 (both cell surface and cytoplasm), T cell receptor (TcR) alpha/beta heterodimer, CD4, CD5, CD7, CD10, CD57 (Leu7), SN-1 and the cytoplasmic TcR beta chain. These findings indicate the T cell nature of the established cells. Terminal deoxynucleotidyl transferase (TdT) was also detected in 60%. We did not detect markers of human myeloid and B cell associated antigens, HLA-class II or immunoglobulin chains. Cytogenetic study revealed that the MKB-1 cells had a female hypo-tetraploid karyotype with chromosomal abnormalities including a translocation between chromosomes 10 and 14. The breakpoint of chromosome 14 of this translocation, 14q11.2, is known to be the location of TcR alpha and delta genes; t(10; 14) (q26; q11.2) is a variant type of a T cell neoplasm-associated translocation, t (10; 14) (q24; q11.2). The MKB-1 cell line is unusual in that its T cell characteristics are phenotypically and cytogenetically distinct from the original myeloid leukemia cells.     

Unusual X chromosome inactivation in a mentally retarded girl with an interstitial deletion Xq27: implications for the fragile X syndrome

Summary A de novo interstitial deletion (X)(q27.1q27.3), between the loci DXS 105 and F8, has been found in a mentally retarded female. The deleted X chromosome is preferentially early replicating in fibroblasts, B cells and T cells, suggesting that the missing region plays a role in inactivation of the X chromosome. None of the available DNA probes except DXS 98 maps to the deleted region of about 10000kb. The locus FRAXA is either included in the deletion, or located close to the distal break point.     

Fragile chromosome 16(q22) cause a balanced translocation at the same point

A father with a fragile 16(q22) has a son with a de novo balanced translocation 1;16. Both the fragile site and the break point at chromosome 16 are similar (q22). The question of whether the fragile site can cause a structural chromosome abnormality at the same point is discussed.     

Localization of human variable and constant region immunoglobulin heavy chain genes on subtelomeric band q32 of chromosome 14

Analysis of a group of human/rodent somatic cell hybrids with nucleic acid probes prepared from cloned human variable region (VH), junctional (JH), and constant region (C epsilon) heavy chain immunoglobulin genes indicates that all of these IgH genes are localized on the subtelomeric (q32) band of chromosome 14. Somatic cell hybrids were isolated in selective medium after fusing human fibroblasts with hprt- Chinese hamster cells. The human parental cells contained two translocation chromosomes representing a reciprocal translocation between chromosomes X and 14. Only those hybrid cell lines retaining a complete human autosome 14 or the X/14 translocation chromosome (i.e. containing band 14q32) retained the human IgH genes. Retention of these genes did not correlate with the presence of the other translocation chromosome, 14/X. These results indicate that all human IgH genes (VH, JH, and CH) map to the same chromosomal band (14q32) which is commonly involved in reciprocal translocations with human chromosome 8 (8q24) in B-cell neoplasms.     

A family case of fertile human 45,X,psu dic(15;Y) males

We report on a familial case including four male probands from three generations with a 45,X,psu dic(15;Y)(p11.2;q12) karyotype. 45,X is usually associated with a female phenotype and only rarely with maleness, due to translocation of small Y chromosomal fragments to autosomes. These male patients are commonly infertile because of missing azoospermia factor regions from the Y long arm. In our familial case we found a pseudodicentric translocation chromosome, that contains almost the entire chromosomes 15 and Y. The translocation took place in an unknown male ancestor of our probands and has no apparent effect on fertility and phenotype of the carrier. FISH analysis demonstrated the deletion of the pseudoautosomal region 2 (PAR2) from the Y chromosome and the loss of the nucleolus organizing region (NOR) from chromosome 15. The formation of the psu dic(15;Y) chromosome is a reciprocal event to the formation of the satellited Y chromosome (Yqs). Statistically, the formation of 45,X,psu dic(15;Y) (p11.2;q12) is as likely as the formation of Yqs. Nevertheless, it has not been described yet. This can be explained by the dicentricity of this translocation chromosome that usually leads to mitotic instability and meiotic imbalances. A second event, a stable inactivation of one of the two centromeres is obligatory to enable the transmission of the translocation chromosome and thus a stably reduced chromosome number from father to every son in this family.     

Digeorge anomaly associated with partial deletion of chromosome 22. Report of a case with X/22 translocation and review of the literature

Partial monosomy of 22q, resulting from a de novo unbalanced translocation t(X;22)(q28;q11) was detected in a newborn female with manifestations of the DiGeorge anomaly including multiple anomalies, type I truncus arteriosus, T-cell abnormalities, thymic aplasia and parathyroid hypoplasia noted on postmortem examination. Although DiGeorge anomaly is causally heterogeneous, our patient, together with 18 previously known cases, confirm that partial monosomy of the proximal long arm of chromosome 22 is the single most common cause of this polytopic developmental field defect.     

The gene that encodes the human CD20 (B1) differentiation antigen is located on chromosome 11 near the t(11;14)(q13;q32) translocation site

The human CD20 gene (B1) encodes a B lymphocyte-specific, cell-surface molecule that is involved in B cell activation and differentiation. We report that the CD20 gene is located on human chromosome 11 at position q12-q13. The location of CD20 was determined by in situ hybridization and was further confirmed by Southern blot analysis of DNA from rodent/human hybrids that contained only portions of human chromosome 11. This localization places the CD20 gene near the site of the t(11;14)(q13;q32) translocation that is found in a subgroup of B cell-lineage malignancies. The site of this translocation has been previously identified by DNA cloning and termed bcl-1. The CD20 gene was found to lie on the centromeric side of bcl-1 on chromosome 11 and to be separated from bcl-1 by at least 50 kb of DNA. These results raise the possibility that alterations in the expression of the CD20 gene may result after the t(11;14) chromosomal alteration.     

Different patterns of X inactivation in MZ twins discordant for red-green color-vision deficiency.

Two female identical twins who were clinically normal were obligatory heterozygotes for X-linked deuteranomaly associated with a green-red fusion gene derived from their deuteranomalous father. On anomaloscopy, one of the twins was phenotypically deuteranomalous while the other had normal color vision. The color vision-defective twin had two sons with normal color vision and one deuteranomalous son. X-inactivation analysis was done with the highly informative probe M27 beta. This probe detects a locus (DXS255) which contains a VNTR and which is somewhat differentially methylated on the active and inactive X chromosomes. In skin cells of the color vision-defective twin, almost all paternal X chromosomes with the abnormal color-vision genes were active, thereby explaining her color-vision defect. In contrast, a different pattern was observed in skin cells from the woman with normal color vision; her maternal X chromosome was mostly active. However, in blood lymphocytes, both twins showed identical patterns with mixtures of inactivated maternal and paternal X chromosomes. Deuteranomaly in one of the twins is explained by extremely skewed X inactivation, as shown in skin cells. Failure to find this skewed pattern in blood cells is explained by the sharing of fetal circulation and exchange of hematopoietic precursor cells between twins. These data give evidence for X inactivation of the color-vision locus and add another MZ twin pair with markedly different X-inactivation patterns for X-linked traits.     

Transmission-ratio distortion of X chromosomes among male offspring of females with skewed X-inactivation

We have begun a search for heritable variation in X-chromosome inactivation pattern in normal females to determine whether there is a genetic effect on the imprinting of X-chromosome inactivation in humans. We have performed a quantitative analysis of X-chromosome inactivation in lymphocytes from mothers in normal, three-generation families. Eight mothers and 12 grandmothers exhibited evidence of highly skewed patterns of X-chromosome inactivation. We observed that the male offspring of females with skewed X-inactivation patterns were three times more likely to inherit alleles at loci that were located on the inactive X chromosome (Xi) than the active X chromosome (Xa). The region of the X chromosome for which this phenomenon was observed extends from XP11 to -Xq22. We have also examined X-chromosome inactivation patterns in 21 unaffected mothers of male bilateral sporadic retinoblastoma patients. Six of these mothers had skewed patterns of X-chromosome inactivation. In contrast to the tendency for male offspring of skewed mothers from nondisease families to inherit alleles from the inactive X chromosome, five of the six affected males inherited the androgen receptor alleles from the active X chromosome of their mother. © 1995 Wiley-Liss, Inc.     

Females with PDHA1 gene mutations: a diagnostic challenge

Biochemical analysis was performed in muscle tissue and in fibroblasts of four unrelated females consecutively diagnosed with a 'de novo' point mutation in the PDHA1 gene. Pyruvate dehydrogenase E1 subunit deficiency was confirmed in the muscle sample of all patients, however, in three out of four cases the activity of the pyruvate dehydrogenase complex in fibroblasts showed a normal activity. A skewed inactivation was confirmed of the maternal X chromosome in fibroblasts in all children. Due to the possibility of a skewed X inactivation pattern enzyme measurements in fibroblasts are not always reliable for the diagnosis of a PDHc defect in females. Based on the overlapping features of PDHc deficiency with those of the disorders of the oxidative phosphorylation we suggest performing a fresh muscle biopsy for detailed biochemical analysis in females with a suspected pyruvate dehydrogenase deficiency, followed by molecular genetic analysis of the PDHA1 gene.     

X chromosome inactivation in X autosome translocation carrier cows.

The pattern of X chromosome inactivation in X autosome translocation carries in a herd of Limousin-Jersey crossbred cattle was studied using the reverse banding technique consisting of 5-bromodeoxyuridine incorporation and acridine orange staining and autoradiography on cultures of solid tissues and blood samples exposed to tritiated thymidine. The late-replicating X chromosome was noted to be the normal X in strikingly high proportions of cells in cultures of different tissues from all translocation carriers. It is suggested that the predominance of cells in which the normal X is inactivated may be the result of a post-inactivation selection process. Such a selection process during the prenatal life favouring cells in which the genes of the normal X chromosome remain unexpressed in translocation carrier females may be the mechanism that helps these conceptuses escape the adverse effects of functional aneuploidy. Based on the observation that the translocation carriers of this line of cattle are exclusively females and that there is a higher than expected rate of pregnancy loss, it is also postulated that the altered X chromosome may be lethal to all male conceptuses and to some of their female counterparts.     

Cytogenetics of fleas (Siphonaptera: Pulicidae). 1. Rat fleas of the genus Xenopsylla

Five populations of Xenopsylla cheopis exhibit a chromosome complement of 2n = 17, X1X2Y (male), and 2n = 18, X1X1X2X2 (female). A detailed analysis of populations of X. astia from Bombay and Trivandrum led to the identification of two distinct cytotypes which hybridisation studies indicated were sibling species. These are referred to as X. astia with a diploid chromosome number of 2n = 18, X1X2X3Y (male), and 2n = 20, X1X1X2X2X3X3 (female) and X. prasadii with 2n = 10, X1X2Y1Y2 (male), and 2n = 10 X1X1X2X2 (female). It is proposed that X. prasadii is derived from X. astia through translocation/fusion events since the average total chromosome lengths are remarkably similar in all three species.     

Skewed X-Chromosome Inactivation Is Common in Fetuses or Newborns Associated with Confined Placental Mosaicism

The inactivation of one X chromosome in females is normally random with regard to which X is inactivated. However, exclusive or almost-exclusive inactivation of one X may be observed in association with some X-autosomal rearrangements, mutations of the XIST gene, certain X-linked diseases, and MZ twinning. In the present study, a methylation difference near a polymorphism in the X-linked androgen-receptor gene was used to investigate the possibility that nonrandom X inactivation is increases in fetuses and newborns that are associated with confined placental mosaicism (CPM) involving an autosomal trisomy. Extreme skewing was observed in 7 (58%) of 12 cases with a meiotic origin of the trisomy, but in none of 10 cases examined with a somatic origin of the trisomy, and in only 1 (4%) of 27 control adult females. In addition, an extremely skewed X-inactivation pattern was observed in 3 of 10 informative cases of female uniparental disomy (UPD) of chromosome 15. This may reflect the fact that a proportion of UPD cases arise by "rescue" of a chromosomally abnormal conceptus and are therefore associated with CPM. A skewed pattern of X inactivation in CPM cases is hypothesized to result from a reduction in the size of the early-embryonic cell pool, because of either poor early growth or subsequent selection against the trisomic cells. Since approximately 2% of pregnancies detected by chorionic villus sampling are associated with CPM, this is likely a significant contributor to both skewed X inactivation observed in the newborn population and the expression of recessive X-linked diseases in females.     

Targeted mutagenesis of Tsix leads to nonrandom X inactivation.

During X inactivation, mammalian female cells make the selection of one active and one inactive X chromosome. X chromosome choice occurs randomly and results in Xist upregulation on the inactive X. We have hypothesized that the antisense gene, Tsix, controls Xist expression. Here, we create a targeted deletion of Tsix in female and male mouse cells. Despite a deficiency of Tsix RNA, X chromosome counting remains intact: female cells still inactivate one X, while male cells block X inactivation. However, heterozygous female cells show skewed Xist expression and primary nonrandom inactivation of the mutant X. The ability of the mutant X to block Xist accumulation is compromised. We conclude that Tsix regulates Xist in cis and determines X chromosome choice without affecting silencing. Therefore, counting, choice, and silencing are genetically separable. Contrasting effects in XX and XY cells argue that negative and positive factors are involved in choosing active and inactive Xs.     

ABL/BCR gene variant with two-step mechanism: Unusual localization and rare/novel chromosomal rearrangements in CML patients

Aims: Variant translocations involving 9q, 22q and at least one additional genomic locus occur in 5-10% of the patients with chronic myeloid leukemia (CML). The mechanisms for the formation of these variant translocations are not fully characterized. Here we report CML cases presenting a variant translocation indicating two-step mechanism with rare/novel chromosomal rearrangement. Methods: Karyotype analysis was performed on metaphases obtained through short-term cultures of bone marrow and blood. Detection of BCR-ABL fusion gene was performed using dual-color dual-fusion (D-FISH) and extra signal (ES) translocation probes. BAC-FISH was also carried out. Results: In Patient 1, the third partner chromosome was der(11)(p15) with a 2F2G1R signal pattern, which is an unusual signal pattern with the two-step mechanism. Patients 2 and 3 showed typical positive (2F1G1R) signal pattern. In Patient 2, both the chromosome 22s were involved in variant formation. The second fusion was observed below the BCR gene of the second homologue. In Patient 3 the third chromosome was der(13)(q14). The fourth patient showed a variant pattern with BCR/ABL-ES probe involving der(X)(q13) region. Conclusion: The presence of different rearrangements of both 9q34 and 22q11 regions highlights the genetic heterogeneity of this subgroup of CML. In each case with variants, further studies with FISH, BAC-FISH or more advanced technique such as microarray should be performed. Future studies should be performed to confirm the presence of true breakpoint hot spots and assess their implications in CML with variant Ph.