Somatic cell hybrid studies of fragile (X) expression in a carrier female and transmitting male.

We have extended our previous studies of fra(X) expression in somatic cell hybrids to the analysis of a carrier female with a low level of expression and to an unaffected, transmitting male who shows no expression in lymphocytes or lymphoblasts. Optimum conditions for fra(X) expression was treatment with 10(-8) M FUdR for 16 hours. In recent experiments, addition of 2.2 mM caffeine 2 hours before harvest was found to increase expression consistently. Two clones from the carrier female containing the fra(X) chromosome but not the normal X showed expression of 2-4%, indicating that expression in heterozygous females is not influenced by the presence or absence of the normal X. Expression rate was increased to 20% by exposure to FUdR plus caffeine. Analysis of hybrids containing only the fra(X) in an inactive state, and after reactivation by 5-azacytidine, showed no change in the frequency of expression. A hybrid clone from the nonexpressing, transmitting male containing only the X and chromosome 13, showed expression ranging from 2% without caffeine to 12% with caffeine in three different experiments. The ability to induce fra(X) expression in hybrid from this nonexpressing male may be explained in one of several ways: 1) a second mutation has occurred, 2) an autosomal suppressor locus was lost, or 3) the hamster genome or unusually short cell cycle lowers the threshold for expression, particularly in the presence of caffeine.

Caffeine enhances fragile (X) expression in somatic cell hybrids.

Yunis and Soreng recently demonstrated enhanced expression of common fragile sites as and of fra(X) when 2.2 mM caffeine is added to FUdR treated lymphocyte cultures 6 hours before harvest. We failed to replicate this finding for fra(X) expression in lymphocytes. However, we do find a consistent increase in expression levels in somatic cell hybrids between a Chinese hamster cell line and 3 unrelated individuals with the fra(X) mutation when caffeine is present for the last 2 hours before harvest. This was particularly true for "low-expressing" cell lines, in which a 4-6 fold increase was observed. Using a thymidylate synthase deficient hybrid which could be blocked in the S phase by thymidine deprivation, we found that caffeine significantly reduced the recovery time from thymidine release to mitosis. This produced the highest level of fra(X) expression (48%) only one hour after release from thymidine deprivation. These results show that caffeine does enhance fra(X) expression in at least some cell types. The effect is probably indirect, inhibiting the mitotic delay usually associated with DNA damage.

A rodent-human hybrid containing Xq24-qter translocated to a hamster chromosome expresses the Xq27 folate-sensitive fragile site.

A somatic cell hybrid containing a single human X chromosome bearing the Xq27 fragile site was lethally irradiated and re-hybridized to its HPRT- Chinese hamster parent. One of 24 colonies surviving selection for HPRT was found to have retained human G6PD but not PGK. This line, X3000-11, which shows Xq24-qter translocated to a hamster chromosome by trypsin G-banding and a single human chromatin fragment corresponding to this segment of the X by G-11 staining, expresses the fragile site on exposure to 5-fluorodeoxyuridine. Dot blots using total human DNA suggest that X3000-11 retains approximately 0.2% of the human genome. By Southern blotting, X3000-11 retains Factor IX, DXS11 and DXS42 but lacks DXYS1, DXS3 and DXS17. This hybrid is being used to construct a cosmid library in the vector pCOS2 from which a sub-library of 500-1000 clones of human origin will be isolated using in vivo recombination with cloned Alu and Kpn family repeats. Such a sub-library will greatly facilitate chromosome walking to the fragile site as well as the testing of individual clones for their ability to create a folate-sensitive fragile site by DNA transfer into permissive Chinese hamster recipient cells.

Recombination and amplification of pyrimidine-rich sequences may be responsible for initiation and progression of the Xq27 fragile site: an hypothesis.

HYPOTHESIS: A pyrimidine-rich sequence (PRS) of DNA is present as a normal sequence in the q27 band of the human X chromosome. Under conditions of pyrimidine nucleotide triphosphate deprivation during S phase, deoxyuridine monophosphate is misincorporated and has to be excised during G2 by DNA repair mechanisms. When a simple PRS is present on both homologous X chromosomes during oogenesis, PRS may undergo amplification through non-homologous crossing-over to produce the initial lesion of the fragile (X). Carriers of such initial lesions will be unaffected transmitting females or males. When an X chromosome bearing such an initial lesion is itself paired with a homologous X carrying a simple PRS during oogenesis, a much higher rate of non-homologous crossing-over may occur resulting in progression to an even longer stretch of pyrimidine rich DNA in this region; the increased length of PRS through amplification makes the region too long to be repaired during G2 and allows it to be seen as a fragile site in metaphase chromosome preparations. Furthermore, this amplified lesion may interfere with transcription of one or more genes in this region and produce the phenotype of the Martin-Bell syndrome.

Thymidylate synthase-deficient Chinese hamster cells: a selection system for human chromosome 18 and experimental system for the study of thymidylate synthase regulation and fragile X expression.

Chinese hamster lung (CHL) V79 cells already deficient in hypoxanthine phosphoribosyltransferase were exposed to uv light and selected for mutations causing deficiency of thymidylate synthase (TS) by their resistance to aminopterin in the presence of thymidine and limiting amounts of methyl tetrahydrofolate. Three of seven colonies chosen for initial study were shown to be thymidylate synthase deficient (TS-) by enzyme assay, thymidine auxotrophy, and their inability to incorporate labeled deoxyuridine into their DNA in vivo. Complementation analysis of human X TS- hamster hybrids revealed that TS activity segregated with human chromosome 18. Southern analysis of a panel of 14 human X hamster hybrids probed with complementary DNA from mouse TS confirmed the chromosome assignment of TS to human chromosome 18; quantitative Southern blotting using unbalanced human cell lines further localized the gene to 18q21.31----qter. Another hybrid was generated that contained a human X chromosome with the Xq28 folate-dependent fragile site as its only human chromosome in a hamster TS- background. The fragile site could be easily and reproducibly expressed in this hybrid without the use of antimetabolites simply by removing exogenous thymidine from the medium. These TS-deficient cells are useful for: somatic cell genetics as a unique selectable marker for human chromosome 18, studies on regulation of the TS gene, and analysis of the fragile (X) chromosome and other folate-dependent fragile sites.

Fragile (X) expression induced by FUdR is transient and inversely related to levels of thymidylate synthase activity.

Thymidylate synthase (TS) activity was monitored in fluorodeoxyuridine (FUdR)-treated lymphoblasts from individuals carrying the fragile (X) [fra(X)] chromosome. Fra(X) expression and levels of TS activity were measured over a 72-hr period at different cell densities. TS activity was 80%-90% inhibited immediately after exposure to FUdR and remained suppressed for the first 24 hrs. Fra(X) expression was not found until 6-8 hrs after FUdR treatment, and at 24 hrs, reached a maximum expression of approximately 50%. At 48 and 72 hrs, however, increasing levels of TS activity paralleled a dramatic drop in fra(X) expression. High fra(X) expression at 48 and 72 hrs could be maintained by rechallenging cultures with increasing doses of FUdR. At low cell densities, fra(X) expression was maintained at high levels for a much longer period of time. In two lymphoblastoid cell lines from obligate carriers, which either expressed at very low levels or did not express the fra(X) in routine cultures, TS activity was also 90% inhibited but with no corresponding fra(X) expression 12 or 24 hrs after FUdR treatment. We conclude that: FUdR inhibits TS activity immediately and induces fra(X) expression 6-8 hrs later, FUdR-induced fra(X) expression and TS activity are inversely related, the FUdR effect on fra(X) expression and TS activity is time and cell-density dependent, and inhibition of TS activity is a necessary but not sufficient condition for fra(X) expression.

New chromosomal syndrome: Miller-Dieker syndrome and monosomy 17p13.

The Miller-Dieker Syndrome (MDS) consists of lissencephaly, characteristic facies, pre- and postnatal growth retardation, plus various other birth defects. Autosomal recessive inheritance has been presumed based on four reported families with two or more affected siblings. We present substantial evidence that monosomy 17p13.3 causes the MDS phenotype. This includes two patients with ring chromosome 17, one patient with a de novo 17p13 deletion, and one patient with monosomy 17p due to an unbalanced 7p; 17p translocation. We report the first prenatal diagnosis of MDS in a 20-week fetus from this latter family. Additionally, we report a balanced translocation between chromosome 17 and different autosomes (8, 12, and 15) in three of the four familial cases of lissencephaly. The finding of a chromosomal basis for this presumed autosomal recessive disorder significantly alters genetic counseling and makes prenatal diagnosis possible in some families.

Expression of the fragile (X) chromosome in an interspecific somatic cell hybrid.

Interspecific somatic cell hybrids were constructed between a Chinese hamster lung cell line deficient in hypoxanthine phosphoribosyltransferase and two lymphoblastoid cultures (GM 4025 and GM 3200) from unrelated males affected with the fragile (X) syndrome. Thirteen independent colonies survived selection in hypoxanthine-azaserine, while only one colony survived selection in hypoxanthine-aminopterin-thymidine. One hybrid formed from GM 4025 was found to contain a human X chromosome as the only detectable human chromosome in the majority of cells analyzed. Induction of fragile (X) expression in this hybrid at frequencies up to 20% was achieved by treatments with 5-fluoro-2'-deoxyuridine (5 X 10(-8) M or 1 X 10(-7) M) or methotrexate (5 X 10(-6) or 1 X 10(-5) for 12 h. Use of the somatic cell hybrid system may allow study of the fragile (X) from different patients on a homogeneous xenogeneic background and may provide a better system for characterization of the fragile (X) at the biochemical and molecular level.

Chromosome 15 abnormalities and the Prader-Willi syndrome: a follow-up report of 40 cases.

High-resolution chromosome analysis and multiple banding techniques were performed on blood samples from 40 patients with Prader-Willi syndrome (PWS) as a follow-up to our recent report in which we found interstitial deletions of 15q in four of five patients with this syndrome. Of the 40 new patients, 19 had interstitial del(15q), one had an apparently balanced 15;15 translocation, and one was mos46,XX/47,XX+idic(15) (pter leads to q11::q11 leads to pter). These data confirm our previous report and demonstrate that half of all patients with the clinical diagnosis of PWS have chromosome abnormalities involving chromosome 15 detectable by high-resolution methods. Although the majority of these involve a specific deletion of bands 15q11-q12, other alterations of chromosome 15 may be present.