Strategy for constructing somatic hybrids isolating the two derivative chromosomes in X;autosome translocations. Application to a female patient t(X;5) with Hunter syndrome

X; autosomal translocations are excellent tools for genetic analysis because of the easy selection of clones isolating the derivative bearing the HPRT gene in somatic cell hybrids. We have developed a strategy to select clones isolating the other derivative avoiding fastidious and time consuming technics, mainly based on immunofluorescent screening using MIC 2 and MIC 5 antigenic markers and we have succeeded in isolating in a rodent context the two X;5 translocated derivative chromosomes of a female patient with Hunter syndrome. The location of MIC 5 gene was specified between the IDS and G6PD DXS369 (RN1), DXS296 (VK21c), and DXS304 (U62), DXS52 and F8c (F814) are proximal and distal from the breakpoint disrupting the IDS gene respectively.     

Physical mapping of new DNA probes near the fragile X mutation (FRAXA) by using a panel of cell lines

The fragile X syndrome is a very common disorder, but there has been little progress toward isolating the fragile X mutation (FRAXA). We describe a panel of 14 somatic cell hybrid lines, lymphoblastoid cell lines, and peripheral lymphocytes with X-chromosome translocation or deletion breakpoints near FRAXA. The locations of the breakpoints were defined with 16 established probes between pX45d (DXS100) and St14-1 (DXS52). Seven of the cell lines had breakpoints between the probes RN1 (DXS369) and U6.2 (DXS304), which flank FRAXA at distances of 3-5 centimorgans. The panel of cell lines was used to localize 16 new DNA probes in this region. Six of the probes-VK16, VK18, VK23, VK24, VK37, and VK47--detected loci near FRAXA, and it was possible to order both the X-chromosome breakpoints and the probes in relation to FRAXA. The order of probes and loci near FRAXA is cen-RN1,VK24-VK47-VK23-VK16,FRAXA-++ +VK21A-VK18-IDS-VK37-U6.2-qter. The breakpoints near FRAXA are sufficiently close together that probes localized with this panel can be linked on a large-scale restriction map by pulsed-field gel electrophoresis. This panel of cell lines will be valuable in rapidly localizing other probes near FRAXA.     

Genetic mapping of new RFLPs at Xq27-q28.

The development of the human gene map in the region of the fragile X mutation (FRAXA) at Xq27 has been hampered by a lack of closely linked polymorphic loci. The polymorphic loci DXS369 (detected by probe RN1), DXS296 (VK21A, VK21C), and DXS304 (U6.2) have recently been mapped to within 5 cM of FRAXA. The order of loci near FRAXA has been defined on the basis of physical mapping studies as cen-F9-DXS105-DXS98-DXS369-DXS297-FRAXA-++ +DXS296-IDS-DXS304-DXS52-qter. The probe VK23B detected HindIII and XmnI restriction fragment length polymorphisms (RFLPs) at DXS297 with heterozygote frequencies of 0.34 and 0.49, respectively. An IDS cDNA probe, pc2S15, detected StuI and TaqI RFLPs at IDS with heterozygote frequencies of 0.50 and 0.08, respectively. Multipoint linkage analysis of these polymorphic loci in normal pedigrees indicated that the locus order was F9-(DXS105, DXS98)-(DXS369, DXS297)-(DXS293,IDS)-DXS304-DXS52. The recombination fractions between adjacent loci were F9-(0.058)-DXS105-(0.039)-DXS98-(0.123)-DXS369-(0.00)- DXS297-(0.057)-DXS296- (0.00)-IDS-(0.012)-DXS304-(0.120)-DXS52. This genetic map will provide the basis for further linkage studies of both the fragile X syndrome and other disorders mapped to Xq27-q28.     

Chromosome localization of the human renin gene (REN) by in situ hybridization

Previous studies by Southern blot analysis of human X mouse somatic cell hybrids localized the renin gene to region p21----qter of human chromosome 1. Using a DNA insert encoding exons 2-5, the renin gene was mapped to human chromosome bands 1q25----q32 by in situ hybridization. The sublocalization of the renin gene will facilitate subsequent detailed linkage analysis of human chromosome 1.     

Assignment of cathepsin E (CTSE) to human chromosome region 1q31 by in situ hybridization and analysis of somatic cell hybrids

A full length cathepsin E (CTSE) cDNA clone was used to assign the corresponding gene to human chromosome region 1q31 by in situ hybridization. Southern blot analysis of DNA from three independent human x rodent somatic cell hybrids containing X;1 translocations confirmed the assignment of the CTSE gene to the distal region of the long arm of chromosome 1.     

Localization of X chromosome short arm markers relative to synovial sarcoma- and renal adenocarcinoma-associated translocation breakpoints

A series of thirteen different DNA markers was mapped relative to papillary renal cell carcinoma- and synovial sarcoma-associated translocation breakpoints in Xp11.2 using a panel of tumor-derived somatic cell hybrids in conjunction with Southern blot analysis. Our results indicate that the two translocation breakpoints differ from each other and that the chromosomal break in t(X; 1)-positive papillary renal cell carcinoma is located between the markers PFC-TIMP-OATL1-SYP-TFE3 and DXS226-DXS146-DXS255-OATL2-DXS14. In addition, our current breakpoint analysis has resulted in a revision of the regional localization of the proximal Xp marker DXS226.     

Hunter disease (mucopolysaccharidosis type II) associated with unbalanced inactivation of the X chromosomes in a karyotypically normal girl.

The mechanism of profound generalized iduronate sulfatase (IDS) deficiency in a developmentally delayed female with clinical Hunter syndrome was studied. Methylation-sensitive RFLP analysis of DNA from peripheral blood lymphocytes from the patient, using MspI/HpaII digestion and probing with M27 beta, showed that the paternal allele was resistant to HpaII digestion (i.e., was methylated) while the maternal allele was digested (i.e., was hypomethylated), indicating marked imbalance of X-chromosome inactivation in peripheral blood lymphocytes of the patient. Similar studies on DNA from maternal lymphocytes showed random X-chromosome inactivation. Among a total of 40 independent maternal fibroblast clones isolated by dilution plating and analyzed for IDS activity, no IDS- clone was found. Somatic cell hybrid clones containing at least one active human X chromosome were produced by fusion of patient fibroblasts with Hprt- hamster fibroblasts (RJK88) and grown in HAT-ouabain medium. Methylation-sensitive RFLP analysis of DNA from the hybrids showed that of the 22 clones that retained the DXS255 locus (M27 beta), all contained the paternal allele in the methylated (active) form. No clone was isolated containing only the maternal X chromosome, and in no case was the maternal allele hypermethylated. We postulate from these studies that the patient has MPS II as a result of a mutation resulting in both the disruption of the IDS locus on her paternal X chromosome and unbalanced inactivation of the nonmutant maternal X chromosome.     

Mapping of the Emery-Dreifuss gene through reconstruction of crossover points in two Italian pedigrees

Summary Two unrelated pedigrees, which show recurrence of Emery-Dreifuss muscular dystrophy (EDMD) in three generations, have been studied using 13 X-linked DNA polymorphisms and somatic cell hybrids to establish the phase of the corresponding alleles in some obligate carriers. The reconstruction of cross-over points on the X chromosomes carrying the EDMD gene excludes from mapping most regions of the X chromosome except for the terminal portion of Xq. Pooled linkage data from the two pedigrees confirm the linkage previously reported with locus DXS15. A cross-over in a carrier female suggests that the EDMD gene is probably located distally to DXS15. In addition the recombinant meioses from one of the two pedigrees suggest the following order for some Xq polymorphic loci: DXS1 (DXYS1-DXS178) DXS42 (F9-DXS15).     

An Xp22 microdeletion associated with ocular albinism and ichthyosis: approximation of breakpoints and estimation of deletion size by using cloned DNA probes and flow cytometry.

Ocular albinism of the Nettleship-Falls type (OA1) and X-linked ichthyosis (XI) due to steroid sulfatase (STS) deficiency are cosegregating in three cytogenetically normal half-brothers. The mother has patchy fundal hypopigmentation consistent with random X inactivation in an OA1 carrier. Additional phenotypic abnormalities that have been observed in other STS "deletion syndromes" are not present in this family. STS is entirely deleted on Southern blot in the affected males, but the loci MIC2X, DXS31, DXS143, DXS85, DXS43, DXS9, and DXS41 are not deleted. At least part of DXS278 is retained. Flow cytometric analysis of cultured lymphoblasts from one of the XI/OA1 males and his mother detected a deletion of about 3.5 million bp or about 2% of the X chromosome. Southern blot and RFLP analysis in the XI/OA1 family support the order tel-[STS-OA1-DXS278]-DXS9-DXS41-cen. An unrelated patient with the karyotype 46,X,t(X;Y) (p22;q11) retains the DXS143 locus on the derivative X chromosome but loses DXS278, suggesting that DXS278 is the more distal locus and is close to an XI/OA1 deletion boundary. If a contiguous gene deletion is responsible for the observed XI/OA1 phenotype, it localizes OA1 to the Xp22.3 region.     

Localization of the ornithine aminotransferase gene and related sequences on two human chromosomes

Summary We have used a full length cDNA clone to determine the chromosomal location ofthegene encoding human ornithine aminotransferase (OAT), a mitochondrial matrix enzyme. Southern blot analysis of ScaI-digested DNA from 34 human-mouse somatic cell hybrids revealed 11 human fragments. Three fragments mapped to chromosome 10q23-10qter, confirming the previous provisional assignment of the functional gene to this autosome by analysis of OAT expression in somatic cell hybrids (O'Donnell et al. 1985). The remaining eight fragments were assigned to the X chromosome, and regionally assigned to Xp21-Xp11 by use of an X-chromosome mapping panel. These X chromosome sequences could represent pseudogenes, or related members of a multigene family. Two of the X chromosome fragments are alternate alleles of a restriction fragment length polymorphism (RFLP) making this OAT-related locus an excellent genetic marker. The RFLP may now be used to determine any possible relationship between this locus and several X-linked eye defects.     

Regional localization of polymorphic DNA loci on the proximal long arm of the X chromosome using deletions associated with choroideremia

Summary In two unrelated families, males have been identified who suffer from choroideremia and at the same time have an interstitial deletion on the proximal long arm of the X chromosome. By high-resolution banding we have characterized the deletion chromosomes as del(X)(q21.1-q21.33) and del(X)(q21.2-q21.31) respectively. By Southern blot analysis we have mapped ten different polymorphic DNA loci relative to the position of the deletion and the choroideremia locus TCD. One probe, p31, was shown to cover one of the breakpoints of the smallest deletion. The following order of the loci was suggested by deletion mapping: cen-DXS106-DXS72-TCD-(DXYS1/DXYS23/DXYS5)-DXYS2-(DXYS12/DXS3)-(DXS17/DXS101)-Xqter.     

Linkage studies do not confirm the cytogenetic location of incontinentia pigmenti on Xp11

Summary Linkage studies have been performed in 5 incontinentia pigmenti (IP) families totaling 29 potentially informative meioses. Ten probes of the Xp arm were used, six of them were precisely localized on the X chromosome, using hamster x human somatic cell hybrids containing a broken X chromosome derived from an incontinentia pigmenti patient carrying an X;9 translocation [46,XX,t(X;9)(p11.21;q34)]. The following order for probes is proposed: pter-(DXS7, DXS146, DXS255)-IP1-(DXS14, DXS90)-DXS106-qter. The negative lod scores obtained exclude the possibility that in the families studied, the gene for IP is located in Xp11 or in the major part of the Xp arm.     

Localization of the gene encoding human Factor V to chromosome 1q21–25

The gene encoding human coagulation Factor V (FV), one of the cofactors in the blood clotting process, has been mapped to chromosome 1 by both Southern hybridization to DNA from human-hamster somatic cell hybrids and in situ hybridization. The whole plasmid pUC3A containing a 1.5-kb cDNA sequence for FV was 32P-labeled for Southern analysis and 3H-labeled for in situ hybridization to metaphase chromosomes. The results localized the FV gene to the region of 1q21-25.     

Structural gene aberrations in mucopolysaccharidosis II (Hunter)

Summary A total of 14 unrelated German patients with X-linked iduronate-2-sulfatase (IDS) deficiency (Hunter syndrome, MPS II) showing variable clinical manifestations was screened for structural gene aberrations by Southern analysis. Using the IDS cDNA clone c2S15 as a probe, no Southern fragments could be detected in blots in the severely affected patient G-65 with respect to DNA digested by HindIII, PstI and TaqI, suggesting a total loss of the IDS structural gene. In this patient, the flanking loci DXS 297, DXS 296 and DXS 466 were tested. The locus DXS 466 is involved in the deletion, whereas both of the other loci are present. A normal 9.0-kb fragment disappeared and an aberrant fragment of 3.5 kb occurred in the HindIII blot of patient G-117. A normal Southern pattern was found in PstI and TaqI blots of this patient. This result can be interpreted as the generation of an additional HindIII restriction site by point mutation in an IDS gene intron.     

Isolation and characterization of a highly polymorphic human locus (DXS455) in proximal Xq28

Human Xq28 is highly gene dense with over 27 loci. Because most of these genes have been mapped by linkage to polymorphic loci, only one of which (DXS52) is informative in most families, a search was conducted for new, highly polymorphic Xq28 markers. From a cosmid library constructed using a somatic cell hybrid containing human Xq27.3----qter as the sole human DNA, a human-insert cosmid (c346) was identified and found to reveal variation on Southern blot analyses with female DNA digested with any of several different restriction endonucleases. Two subclones of c346, p346.8 and p346.T, that respectively identify a multiallelic VNTR locus and a frequent two-allele TaqI polymorphism were isolated. Examination of 21 unrelated females showed heterozygosity of 76 and 57%, respectively. These two markers appeared to be in linkage equilibrium, and a combined analysis revealed heterozygosity in 91% of unrelated females. Families segregating the fragile X syndrome with key Xq28 crossovers position this locus (designated DXS455) between the proximal Xq28 locus DXS296 (VK21) and the more distal locus DXS374 (1A1), which is proximal to DXS52. DXS455 is therefore the most polymorphic locus identified in Xq28 and will be useful in the genetic analysis of this gene dense region, including the diagnosis of nearby genetic disease loci by linkage.     

The terminal deoxynucleotidyltransferase gene is located on human chromosome 10 (10q23----q24) and on mouse chromosome 19

Terminal deoxynucleotidyltransferase (TdT) is a DNA polymerase expressed in immature lymphocytes of the thymus and bone marrow, as well as certain leukemic cells. Chromosomal assignment of the gene coding for human TdT was accomplished by in situ hybridization of a 3H-labeled cDNA probe to human chromosome preparations and by Southern blot analysis of somatic cell hybrid DNAs. The human TdT gene was mapped to the region q23----q24 of chromosome 10. Breaks at this site have been reported in different translocations in human leukemias. The mouse TdT gene was assigned to chromosome 19 by Southern blot analysis of mouse X Chinese hamster somatic cell hybrids. This result adds a fourth locus to the conserved syntenic group on mouse chromosome 19 and human chromosome 10.     

Human monoamine oxidase gene (MAOA): chromosome position (Xp21-p11) and DNA polymorphism

An essentially full-length cDNA clone for the human enzyme monoamine oxidase type A (MAO-A) has been used to determine the chromosomal location of a gene encoding it. This enzyme is important in the degradative metabolism of biogenic amines throughout the body and is located in the outer mitochondrial membrane of many cell types. Southern blot analysis of PstI-digested human DNA revealed multiple fragments that hybridized to this probe. Using rodent-human somatic cell hybrids containing all or part of the human X chromosome, we have mapped these fragments to the region Xp21-p11. A restriction fragment length polymorphism (RFLP) for this MAOA gene was identified and used to evaluate linkage distances between this locus and several other loci on Xp. The MAOA locus lies between DXS14 and OTC, about 29 cM from the former.     

Localization of the murine lambda 5 gene on chromosome 16

The chromosomal location of the murine lambda 5 gene was analyzed by Southern hybridization using restriction enzyme-digested DNA from a panel of 15 mouse X hamster somatic cell hybrids. Sequences homologous with those of lambda 5 DNA were detected in DNA of 5 hybrids. In all 5 hybrids lambda 5 was contained in restriction fragments of equal sizes, the lengths of which indicated that the germline configuration of lambda 5 with three exons and the restriction sites expected from its genomic structure were present. Southern hybridization with the murine lambda 1 gene as a probe detected the same 5 hybrids as positive. The only mouse chromosome present on all of the positive hybrids, and absent from negative ones, was number 16. We conclude that lambda 5 is situated on the same chromosome as lambda 1, i.e., on the murine chromosome 16.     

Human gastric intrinsic factor: characterization of cDNA and genomic clones and localization to human chromosome 11

A human gastric intrinsic factor (IF) cDNA clone was isolated using a rat cDNA clone as a probe. Comparison of the predicted amino acid sequence revealed 80% identity of human IF with rat IF. These cDNA clones were used to isolate and map two overlapping clones encoding the human IF gene. The first exon of the cloned region (exon 2) contains 30 bp of the 5' untranslated region, the signal peptide, and the first 8 amino acids of the mature protein. Exons 3-10 encode the remainder of the coding and 3' noncoding regions. Southern analysis of genomic DNA indicated the presence of a single human IF gene and also revealed the presence of strong hybridizing sequences in genomic DNA from monkey, rat, mouse, cow, and human, suggesting that the IF gene is well conserved. The IF gene was localized to human chromosome 11 by concurrent cytogenetic and cDNA probe analysis of a panel of human X mouse somatic cell hybrids. Southern analysis of genomic DNA from patients with congenital pernicious anemia (lacking intrinsic factor) revealed normal restriction fragment patterns, suggesting that a sizable gene deletion was not responsible for the deficiency.     

The pronatriodilatin gene is located on the distal short arm of human chromosome 1 and on mouse chromosome 4.

Atrial natriuretic factors (ANF) are polypeptides having natriuretic, diuretic, and smooth muscle-relaxing activities that are synthesized from a single larger precursor: pronatriodilatin. Chromosomal assignment of the gene coding for human pronatriodilatin was accomplished by in situ hybridization of a [3H]-labeled pronatriodilatin probe to human chromosome preparations and by Southern blot analysis of somatic cell hybrid DNAs with normal and rearranged chromosomes 1. The human pronatriodilatin gene was mapped to the distal short arm of chromosome 1, in band 1p36. Southern blot analysis of mouse X Chinese hamster somatic cell hybrids was used to assign the mouse pronatriodilatin gene to chromosome 4. This assignment adds another locus to the conserved syntenic group of homologous genes located on the distal half of the short arm of human chromosome 1 and on mouse chromosome 4.