Mosaicism and haemophilia.

Mosaicism may affect the haemophilia phenotype. Well-known instances include chromosomal mosaicism due to aneuploidy and pseudo-mosaicism due to varying patterns of X-chromosome inactivation. Chromosomal mosaicism in a chimera is a potential source of phenotypic variation. Gene mosaicism is commonplace. Its pattern and effect depend on the stage of development at which a mutation occurs. Proven or possible genetic mosaicism is an important consideration when predicting the likelihood of transmission of haemophilia to a future generation.

High frequency of de novo mutations in Li-Fraumeni syndrome.

BACKGROUND: Li-Fraumeni syndrome is an autosomal dominant cancer predisposition syndrome caused by germline mutations in the TP53 gene. The frequency of germline de novo TP53 mutations is largely unknown; few unequivocal de novo mutations have been reported. METHODS AND RESULTS: Of 341 patients with early onset cancer sent for clinical testing to a national reference laboratory, 75 patients had TP53 germline mutations. Five (7%) de novo mutations were identified, as well as an additional 10 TP53 germline mutations likely to be de novo by family history. The frequency of de novo TP53 mutations in this patient sample is at least 7% and may be as high as 20%. CONCLUSIONS: The possibility that de novo germline TP53 mutations are relatively common has implications for testing and the identification of potential Li-Fraumeni syndrome in patients with little or no family history of cancer.

Mosaics and haemophilia.

Some mosaic conditions may affect the haemophilia phenotype. Well-known instances include chromosomal mosaicism because of aneuploidy and pseudo-mosaicism because of varying patterns of X-chromosome inactivation. Chromosomal mosaicism in a chimera is a potential source of phenotypic variation. Gene mosaicism is commonplace. Its pattern and effect depend on the stage of development at which a mutation occurs. Proven or possible genetic mosaicism is an important consideration when predicting the likelihood of transmission of haemophilia to a future generation. A mosaic is an individual who has two or more cell lines, genetically different with regard to chromosomes or genes. As techniques improve and studies accumulate, mosaics are being found to be more common than hitherto believed. Some mosaic conditions may affect the phenotype of haemophilia in males and of the carrier state in females. Cells may be mosaic with regard to chromosomes, as in some instances of aneuploidy, and in chimeras, and in females owing to the pattern of X-chromosome inactivation. Cells may be mosaic with regard to new gene mutations. The pattern of mosaicism depends upon the stage in embryogenesis or in germ-cell formation in which the mutation arose.

Low frequency of p53 gene mutations in breast cancers of Japanese-American women.

Differences in frequencies and patterns of somatic p53 gene mutations among racially and geographically diverse populations presumably reflect exposure to different mutagens or different responses to certain mutagens. On emigration to the United States, Japanese women experience, over several generations, a four- to fivefold increase in the incidence of breast cancer. To determine whether this increased incidence is associated with a change in the frequency and/or type of p53 mutation in their tumors, we examined paraffin-embedded samples of primary breast cancers from Japanese-American women in Los Angeles County, CA. Mutations in exons 5-9 and adjacent intronic regions of the p53 gene were identified and confirmed by direct sequencing. Seven mutations, including 5 missense, were detected in 44 primary breast carcinomas, a frequency of 16%. There were six transitions and one transversion. As expected, overexpression of p53 protein, detected by immunohistochemistry, occurred in tumors with missense mutations; tumors with nonsense or splice junction mutations had no detectable p53 protein. The frequency of p53 gene mutations showed no increase over that previously found in breast cancers of native Japanese women. The increased incidence of breast cancer in Japanese-American women is likely to be multifactorial in nature and warrants further studies.

Diagnosis of Duchenne dystrophy by enhanced detection of small mutations.

OBJECTIVE: To determine whether detection of small mutations of the dystrophin gene can be increased using an enhanced method of single-strand conformation polymorphism analysis. BACKGROUND: Usual methods of DNA analysis for Duchenne dystrophy cannot identify mutations in one-third of cases. Muscle biopsy, with its inherent risks and added liability for patients with Duchenne dystrophy, becomes the sole method of diagnosis. Even with a tissue diagnosis of dystrophin deficiency, many families are excluded from carrier detection and prenatal diagnosis. METHODS: Genomic DNA from a cohort of 93 patients with Duchenne dystrophy without identifiable gene mutations was screened for mutations. In each case, 22 kilobases of genomic DNA were scanned, including all 79 exons of the dystrophin gene, adjacent intronic regions, and six alternative exons 1. RESULTS: Sixty-eight (73%) had small mutations, including 34 nonsense mutations, 27 microdeletions and insertions, and 7 splice site mutations. No missense mutations were found. One nonsense mutation in exon 59 was detected in four patients. Most mutations were new; 54 of 62 different small mutations have not been reported. Mutations were found throughout the gene: 24% in the first quartile, 31% in the second, 16% in the third, and 29% in the fourth. CONCLUSIONS: A highly sensitive single-strand conformation polymorphism method substantially increased detection of small dystrophin gene mutations and made it possible to diagnose approximately 90% of patients with Duchenne dystrophy by DNA analysis. These findings, combined with cost savings and safety issues, provide compelling reasons to consider DNA analysis as the initial diagnostic test for the suspected dystrophin-deficient patient.

Scanning by DOVAM-S detects all unique sequence changes in blinded analyses: evidence that the scanning conditions are generic.

The [detection of virtually all mutations]-SSCP (DOVAM-S) is a highly sensitive variant of single strand conformation polymorphism (SSCP). Mutations in the factor IX gene were used to find a set of five SSCP conditions that detects virtually all mutations. A blinded analysis of the factor IX gene in patients with hemophilia B detected 82 of 82 unique mutations. Since the method was developed and tested on the factor IX gene, it is possible that the conditions selected work more efficiently in the factor IX gene than in other genes. To test the general applicability of the conditions under which DOVAM-S detected all mutations in this gene, blinded analyses were performed in the human factor VIII and ataxia-telangiectasia (ATM) genes. Segments were amplified individually, combined into groups of 16 to 18 amplified segments and electrophoresed in five different nondenaturing conditions of varying matrices, buffers, temperatures and additives. Blinded analyses were performed in 92 samples from patients with hemophilia A (factor VIII gene) and 19 samples from A-T patients (ATM gene). Combined with an earlier blinded analysis in the factor IX gene, all of the 250 mutations and polymorphisms (180 of which are unique) were detected in both analyses. For two, three and four joint conditions, the average detection frequency ranged from 77%-97%, 91%-100% and 95%-100%, respectively. For each of the genes, one mutation may have been missed if only four conditions were used. With DOVAM-S, approximately 500 kb of autosomal sequence can be scanned in five gels with virtually 100% detection of mutations within the scanned region. The detection of 180 out of 180 unique sequence changes implies that DOVAM-S detects at least 96.5% (P = 0.03) of mutations. Blinded analyses that detect 400 unique sequence changes are required to determine that a scanning method detects at least 98.5% of mutations.

Highly sensitive mutation screening by REF with low concentrations of urea: A blinded analysis of a 2-kb region of the p53 gene reveals two common haplotypes.

Restriction endonuclease fingerprinting (REF), a hybrid modification of single-strand conformation polymorphism (SSCP) and restriction endonuclease digestion, has been used previously to detect mutations in 1- to 2-kb segments of DNA. This paper demonstrates that fragment resolution, and thus sensitivity of REF, can be markedly improved by electrophoresis under partially denaturing, rather than nondenaturing, conditions, for genes with a high G+C content. A 2. 1-kb segment of the p53 tumor suppressor gene (54.5% G+C) containing exons 5-9, including the intervening introns, was screened in a blinded analysis of 48 samples from human breast tumors containing known wild-type or mutant p53 genes. In gels containing 0.5 M urea, 97% of the mutant samples were detected correctly, and more than 80% of the mutations were localized within a 200-bp region. In the process of this methodological analysis, it was discovered that: (1) there are two common and four uncommon haplotypes; (2) the two common haplotypes occurred in the three races examined, suggesting an ancient origin; and (3) haplotype II is of substantially higher frequency in the Chinese relative to Japanese (P = 0.023) and Caucasians (P = 0.005). Two other improvements in the REF procedure included (1) the selection of an optimal set of restriction endonucleases by new software (REF Select) developed recently in our laboratory; and (2) the addition of an oligonucleotide "tail," containing two recognition sequences for restriction endonucleases, to the PCR primers to prevent coterminal fragments at the end of amplified products. These modifications facilitate the use of REF for efficient and sensitive mutation screening in p53 and other genes with a high G+C content.

Detection of virtually all mutations-SSCP (DOVAM-S): a rapid method for mutation scanning with virtually 100% sensitivity.

Dideoxy fingerprinting (ddF) was used as a tool to search for a generic set of conditions with sufficient power to detect virtually all mutations. For each condition tested, a very large sample of mutation-containing, single-stranded segments (about 1500) were analyzed with ddF. Correlation coefficients identified pairs of conditions in which single-strand conformation polymorphism (SSCP) mobilities were poorly correlated. The data strongly suggest that tertiary structure (e.g., base-sugar and sugar-sugar interactions) rather than secondary structure is the predominant determinant of mobility shifts by SSCP. Five conditions were selected with sufficient redundancy to detect all the mutations. The sensitivity of detection of virtually all mutations-SSCP (DOVAM-S) was determined by blinded analyses on samples containing additional mutations scattered throughout the eight exons and splice junctions in the factor IX gene. The factor IX gene sequence (2.5 kb) was scanned in one lane by 15 PCR-amplified segments (125 kb of sequence scanned per gel). All of the 84 single-base substitutions were detected in the blinded analyses, the first consisting of 50 hemizygous mutant and wild-type (WT) samples and the second consisting of 50 heterozygous mutant and WT samples. DOVAM-S is estimated to be five times faster than fluorescent DNA sequencing for the detection of virtually all mutations when the five conditions are applied.

Quantitative RT-PCR assays show Xist RNA levels are low in mouse female adult tissue, embryos and embryoid bodies.

We have investigated expression of the Xist gene in mouse female adult kidney, embryos and embryonic stem (ES) cells undergoing in vitro differentiation as embryoid bodies. Using the quantitative RT-PCR single nucleotide primer extension (SNuPE) assay, we found that the amount of Xist RNA in adult kidney of three mouse strains was less than approximately 2000 transcripts per cell, with only modest differences between strains carrying different Xce alleles. Female embryos 7.5 days post coitum had the same number of Xist transcripts per cell as isogenic adult tissue. Using quantitative oligonucleotide hybridization assays after RT-PCR, we investigated Xist expression in ES lines heterozygous at the Pgk-1 and Xist loci. We found that, while in most (XX) ES lines Xist RNA levels increased during embryoid body formation, the levels seen were less than 10% those found in adult female kidney. In addition, we found that the allelic ratio of Xist transcripts from reciprocal (XX) ES cell lines differentiating in vitro was identical to that of isogenic 10.5 to 11.5 day female embryos. These latter results suggest that there is no pattern of preferential paternal imprinting during days 1 to 9 of in vitro differentiation of ES cells. However, the influence of the Xce locus on the randomness of X-inactivation in embryos seems to operate also in ES cell lines. Our overall conclusion is that the low levels of Xist RNA in female kidney, embryos and differentiating (XX) ES cells are compatible only with models that do not require Xist RNA to cover the entire inactive X chromosome.

Differential expression of heat shock proteins in Drosophila embryonic cells following metal ion exposure.

Drosophila embryonic cells were exposed to a number of metal ions that have been previously reported to act as teratogens in mammalian systems, including some known to induce heat shock (stress) proteins in a variety of model systems. This study examined the effects of these ions both on differentiation of muscles and neurons and on the induction of heat shock proteins. Metals such as arsenate, cadmium, and mercury all inhibited neuron and/or muscle differentiation in Drosophila embryonic cultures, while they also induced the entire set of heat shock proteins. Two metal ions, nickel and zinc, were shown to induce only the 22- and 23-K proteins, a pattern similar to that seen in "classical" teratogens reported previously. None of the metals tested induced only the 26- and 27-K proteins. These results suggest that there exist different regulatory mechanisms responsible for the heat shock response.

Induction of heat shock gene expression without heat shock by hepatocarcinogens and during hepatic regeneration in rat liver.

We investigated the expression of the rat hepatic heat-shock protein (hsp) genes under the influence of hepatocarcinogens and during hepatic regeneration. This was undertaken because of the inducibility of the heat-shock response in rat liver and because heat-shock genes can be expressed with or without heat shock in various cell states in a developmentally regulated manner. We found that acute administration of hepatocarcinogens to rats induced an increased hsp gene transcription in a time- and dose-dependent manner. Chronic exposure of rats to complete hepatocarcinogens induced increased levels of mainly Mr 83,000 heat-shock protein gene transcription and, to a lesser extent, Mr 70,000 heat-shock protein. However, the tumor promoter phenobarbital did not induce increased hsp gene expression. Increased levels of both Mr 83,000 heat-shock protein and Mr 70,000 heat-shock protein gene transcription were found during hepatic regeneration. Thus, increased hsp gene transcription, which correlated with increased heat-shock protein synthesis, was observed under the acute and chronic influence of hepatocarcinogens and during normal hepatic proliferation. These results are similar to those observed for c-H-ras and c-myc expression in rat liver, and they suggest that a coordinate expression of these three genes may occur in hepatic regeneration and in the early stages of experimental chemical hepatocarcinogenesis.

Developmental effects of chemicals and the heat shock response in Drosophila cells.

Exposure of prokaryotic and eukaryotic cells to heat shock (hyperthermia) or to a number of diverse environmental stresses such as teratogens, anoxia, and inhibitors of oxidative phosphorylation results in the enhanced synthesis of a number of proteins which have been previously referred to as heat shock proteins (hsps). More recently, in view of the diverse types of agents that can induce these proteins, they have also been referred to as stress proteins. This phenomenon is one of the most basic regulatory mechanisms in living organisms. Exposure of Drosophila embryos, larvae, or pupae to these types of stresses also results in a variety of developmental abnormalities in the ensuing adult. Although the function(s) of these heat shock proteins has yet to be determined, they are widely thought to play an important role in cell survival and protection following some types of environmental stress. In our laboratory, we have developed an in vitro assay for detecting agents that act as teratogens, utilizing Drosophila embryonic cultures. Drosophila embryonic cells differentiate in vitro to a number of functional cell types including myotubes and ganglia. A number of drugs that have been shown to act as teratogens in mammals have also been found to inhibit muscle and/or neuron differentiation in Drosophila embryonic cultures. We have examined, by two-dimensional gel electrophoresis, the effects of such teratogens on protein synthesis in Drosophila embryonic cells. Inhibition of muscle and/or neuron differentiation correlates well with the induction of two proteins of about 20 kilodaltons. These are identical to two of the heat shock proteins (hsp 23, 22) as shown by electrophoretic mobilities and peptide mapping by partial proteolysis. Heat shock and other treatments such as exposure to some of the metal ions and ether induces the entire set of seven major heat shock proteins in the Drosophila embryonic cells. Dose-response studies of several teratogens show a correlation between the degree of inhibition of differentiation and the level of induction of hsps. Since heat shock proteins have been suggested as possibly serving a protecting role, our present studies are aimed at identifying the role of hsps in teratogenesis and investigating the differential regulation of heat shock genes in response to different external stimuli.

Self-degradation of heat shock proteins.

The 70-kDa heat shock protein of Drosophila decays in vivo at a much faster rate than other abundantly labeled proteins. Degradation also occurs in vitro, even during electrophoresis. It appears that this degradation is not mediated by a general protease and that the 70-kDa heat shock protein has a slow proteolytic action upon itself. Heat-induced proteins in CHO cells and a mouse cell line also degrade spontaneously in vitro, as do certain non-heat shock proteins from Drosophila tissues as well as the cell lines.

Teratogens induce a subset of small heat shock proteins in Drosophila primary embryonic cell cultures.

Drosophila embryonic cells placed into culture just after gastrulation differentiate in vitro over the next 24 hr. A number of drugs that are teratogenic in mammalian systems have been found to inhibit muscle or neuron differentiation (or both) in these developing cultures. We have examined, by two-dimensional gel electrophoresis, the effects of these drugs on protein synthesis in embryonic cells. For nine teratogens tested, cells treated for 20 hr with the drug show a dramatic induction of three proteins of about 20 kilodaltons, in addition to the normal proteins synthesized by untreated cells. Three teratogens as well as all eight nonteratogens tested did not show this induction. The induced proteins appear to be identical to three of the heat shock proteins (hsp 23, 22a, and 22b), as shown by electrophoretic mobilities and peptide mapping by partial proteolysis. A 37 degrees C heat shock of the embryonic cells produces the full complement of heat shock proteins, whereas drug-treated cells induce only the subset hsp 23, 22a, and 22b but not hsp 26 or 27. beta-Ecdysterone, the Drosophila molting hormone, also inhibits embryonic differentiation and induces hsp 23, 22a, and 22b, a partial subset of the heat shock proteins (hsp 22, 23, 26, and 27) induced by the hormone in imaginal discs and some Drosophila continuous cell lines. Dose-response studies of several drugs show a correlation between the degree of inhibition of differentiation and the level of induction of hsp 23, 22a, and 22b. The induction of heat shock proteins by drugs may reflect specific types of stress that can also give rise to teratogenesis.