Premutation allele pool in myotonic dystrophy type 2.

BACKGROUND: The myotonic dystrophies (DM1, DM2) are the most common adult muscle diseases and are characterized by multisystem involvement. DM1 has been described in diverse populations, whereas DM2 seems to occur primarily in European Caucasians. Both are caused by the expression of expanded microsatellite repeats. In DM1, there is a reservoir of premutation alleles; however, there have been no reported premutation alleles for DM2. The (CCTG)(DM2) expansion is part of a complex polymorphic repeat tract of the form (TG)(n)(TCTG)(n)(CCTG)(n)(NCTG)(n)(CCTG)(n). Expansions are as large as 40 kb, with the expanded (CCTG)(n) motif uninterrupted. Reported normal alleles have up to (CCTG)(26) with one or more interruptions. METHODS: To identify and characterize potential DM2 premutation alleles, we cloned and sequenced 43 alleles from 23 individuals. Uninterrupted alleles were identified, and their instability was confirmed by small-pool PCR. We determined the genotype of a nearby single nucleotide polymorphism (rs1871922) known to be in linkage disequilibrium with the DM2 mutation. RESULTS: We identified three classes of large non-DM2 repeat alleles: 1) up to (CCTG)(24) with two interruptions, 2) up to (CCTG)(32) with up to four interruptions, and 3) uninterrupted (CCTG)(22-33). Large non-DM2 alleles were more common in African Americans than in European Caucasians. Uninterrupted alleles were significantly more unstable than interrupted alleles (p = 10(-4) to 10(-7)). Genotypes at rs1871922 were consistent with the hypothesis that all large alleles occur on the same haplotype as the DM2 expansion. CONCLUSIONS: We conclude that unstable uninterrupted (CCTG)(22-33) alleles represent a premutation allele pool for DM2 full mutations.

Expression of bcr-abl mRNA in individual chronic myelogenous leukaemia cells as determined by in situ amplification.

We present the results of a novel method developed for evaluation of in situ amplification, a molecular genetic method at the cellular level. Reverse transcription polymerase chain reaction (RT-PCR) was used to study bcr-abl transcript levels in individual cells from patients with chronic myelogenous leukaemia (CML). After hybridizing a fluorochrome-labelled probe to the cell-bound RT-PCR product, bcr-abl mRNA-positive cells were determined using image analysis. A dilution series of bcr-abl-positive BV173 into normal cells showed a good correlation between expected and actual values. In 25 CML samples, the percentage of in situ PCR-positive cells showed an excellent correlation with cytogenetic results (r = 0.94, P < 0.0001), interphase fluorescence in situ hybridization (FISH) (r = 0.95, P = 0.001) and hypermetaphase FISH (r = 0.81, P < 0.001). The fluorescence intensity was higher in residual CML cells after interferon (IFN) treatment than in newly diagnosed patients (P = 0.004), and was highest in late-stage CML resistant to IFN therapy and lowest in CML blast crisis (P = 0.001). Mean fluorescence values correlated with bcr-abl protein levels, as determined by Western blot analysis (r = 0.62). Laser scanning cytometry allowing automated analysis of large numbers of cells confirmed the results. Thus, fluorescence in situ PCR provides a novel and quantitative approach for monitoring tumour load and bcr-abl transcript levels in CML.

Frequency of minisatellite repeat number changes at the MS205 locus in human sperm before and after cancer chemotherapy.

To determine whether the measurement of repeat number mutations at a minisatellite locus could detect human germline mutations induced by chemotherapy, we performed a longitudinal study of the mutation frequencies in sperm from 10 patients treated for Hodgkin's disease. Polymerase chain reaction on small pools of DNA equivalent to 100 sperm and Southern blotting were used to screen at least 7900 sperm in each sample to quantify the mutation frequency at the minisatellite MS205 locus. Pretreatment and posttreatment semen samples were obtained at least 2 months after completion of therapy from 4 patients treated with a regimen (Novantrone, Oncovin, vinblastine and prednisone [NOVP]) that lacks alkylating agents and from three patients treated with regimens (Cytoxan, vinblastine, procarbazine and prednisone/Adriamycin, bleomycin, dacarbazine, lomustine, and prednisone [CVPP/ABDIC] or mechlorethamine, Oncovin, procarbazine and prednisone [MOPP]) containing alkylating agents. There were no effects of NOVP or CVPP/ABDIC on the mutation frequencies. In the 1 patient treated with MOPP, the treatment with the highest dose of gonadotoxic alkylating agents, there was a statistically significant increase in mutation frequency from 0.79% pretreatment to 1.14% posttreatment, indicating induction of mutations in stem spermatogonia. During-treatment semen samples obtained from 2 patients treated with ABVD, which does not contain gonadotoxic alkylating agents, and 1 with NOVP also did not show any increases above the baseline mutation frequencies, indicating no increase in the minisatellite mutation frequency in spermatocytes. Thus, measurement of repeat number changes at minisatellite MS205 appears to be able to detect induced germline mutations in human sperm. However, most chemotherapy regimens do not significantly increase this class of mutations.

Early detection of relapse by hypermetaphase fluorescence in situ hybridization after allogeneic bone marrow transplantation for chronic myeloid leukemia.

PURPOSE: Standard G-band cytogenetic analysis (CG) provides information on approximately 25 metaphases for monitoring the presence of Philadelphia chromosome positive (Ph+) cells in chronic myelogenous leukemia (CML) patients, making the detection of a low frequency of Ph+ cells problematic. The purpose of this study was to improve the detection of a low frequency of Ph+ cells. PATIENTS AND METHODS: We combined fluorescence in situ hybridization (FISH) with long-term colcemid exposure, capturing several hundred metaphases in bone marrow cultures (hypermetaphase FISH [HMF]). Using probes that identify Ph+ cells, HMF was compared with CG analysis in the follow-up evaluations of 51 patients with CML at various time points after allogeneic bone marrow transplant (BMT). RESULTS: Thirty-five patients never showed the presence of Ph+ cells by either method. In four patients, high frequencies of Ph+ cells were detected by both methods. In the remaining 12 patients, Ph+ cells were detected by HMF at time points after BMT when they were not detected by CG. In seven of the 12 patients, low but statistically significant frequencies of Ph+ cells (0.37% to 5.20%) were detected 3 months or later after BMT, and when no intervention was initiated, all seven patients later relapsed. Based on those data, an eighth patient with mixed chimerism and a similar HMF-detected Ph+ frequency (1.8% at 27 months after BMT) was reinfused with donor lymphocytes and achieved remission with 0% Ph+ cells studied by HMF (up to 50 months after BMT). Ph+ cells detected by HMF but not by CG less than 3 months after BMT disappeared on later examination in two of four patients. After detection of Ph+ cells by HMF only, the median time to cytogenetic progression (detection of Ph+ cells by CG) was 101 days. CONCLUSION: The results demonstrate the ability of HMF to detect low but clinically relevant levels of leukemic cells not detected by CG in transplant patients. The data indicate that HMF can detect low levels of Ph+ cells before standard cytogenetics at a time that may be useful in monitoring disease status and planning clinical interventions.

Characterization of human retinal fascin gene (FSCN2) at 17q25: close physical linkage of fascin and cytoplasmic actin genes.

Retinal fascin is a newly identified photoreceptor-specific paralog of the actin-bundling protein fascin. Fascins crosslink f-actin into highly ordered bundles within dynamic cell extensions such as neuronal growth cone filopodia. We have isolated cDNA and genomic clones of human retinal fascin and characterized the structure of the human retinal fascin gene (FSCN2). The cDNA predicts a protein of 492 amino acids and molecular mass 55,057 that shows 94% identity to bovine retinal fascin and 56% identity to human fascin. Promoter analysis reveals a consensus retinoic acid response element and several potential binding sites for transcription factors Crx and Nrl, which correlates with the retina-specific expression of FSCN2 mRNA. Fluorescence in situ hybridization analysis and genomic clone sequencing indicate that the FSCN2 gene lies within 200 kb of the actin gene ACTG1 at 17q25. Database searches revealed that the human fascin gene FSCN1 and actin gene ACTB at 7p22 also coexist within a 200-kb genomic clone. The close physical linkage of these fascin/actin gene pairs suggests that they derive from a common gene duplication event and allows comparison of fascin and actin phylogenetic analyses. Finally, a possible link to the retinitis pigmentosa 17 allele (RP17) at distal 17q was excluded by demonstration of multiple independent segregation events in two RP17 kindreds. Informative FSCN2 polymorphisms were identified and will serve as useful markers in future linkage studies. The likely function of retinal fascin, in light of known fascin roles in other cell types, is to assemble actin microfilaments in support of photoreceptor disk morphogenesis.

Human sulfotransferases SULT1C1 and SULT1C2: cDNA characterization, gene cloning, and chromosomal localization.

Sulfate conjugation catalyzed by sulfotransferase (SULT) enzymes is an important pathway in the biotransformation of many drugs, other xenobiotics, neurotransmitters, and hormones. We previously described a human cDNA, SULT1C1, that encoded a protein similar in sequence to that of rat ST1C1. Subsequently, a related human cDNA, SULT1C2, was reported. In the present study, we set out to characterize further the human SULT1C1 cDNA and then to clone, structurally characterize, and map its gene. As an initial step, we performed 5'- and 3'-RACE with SULT1C1 cDNA. Those experiments demonstrated that a small number of SULT1C1 transcripts contained an "insert," which we later showed resulted from alternative splicing that involved an Alu sequence in intron 3 of SULT1C1. We then cloned and structurally characterized the SULT1C1 gene from a human genomic BAC library. Because the sequence of SULT1C2 was closely related to that of SULT1C1 and because the genes for other human SULT paralogues occur in clusters, we screened the BAC clones that had been positive for SULT1C1 to search for SULT1C2 and discovered a clone that contained both genes. That BAC was used to sequence and structurally characterize SULT1C2. SULT1C1 and SULT1C2 were approximately 21 and 10 kb in length, respectively. Both genes contained seven exons that encoded protein, and both had structures that were similar to those of other genes that encode members of the SULT1 family. Finally, human SULT1C1 and SULT1C2 mapped to 2q11.2 by fluorescence in situ hybridization. The cloning and structural characterization of SULT1C1 and SULT1C2 will now make it possible to perform molecular genetic and pharmacogenomic studies of these sulfate-conjugating enzymes in humans.

Human 3'-phosphoadenosine 5'-phosphosulfate synthetase 1 (PAPSS1) and PAPSS2: gene cloning, characterization and chromosomal localization.

Sulfae conjugation is an important pathway in the metabolism of a large number of exogenous and endogenous compounds. These reactions are catalyzed by sulfotransferase (SULT) enzymes that utilize 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as a sulfate donor. PAPS is synthesized from ATP and inorganic sulfate by PAPS synthetase (PAPSS). Two separate PAPSS cDNAs, PAPSS1 and PAPSS2, have been identified in human tissues. We have cloned and characterized the genes for human PAPSS1 and PAPSS2 to make it possible to study the pharmacogenomics of these enzymes. Both genes consisted of 12 exons with virtually identical exon-intron splice junction locations. All splice junctions conformed to the "GT-AG" rule. The total length of PAPSS1 was approximately 108 kb, while that of PAPSS2 was greater than 37 kb. The 5'-flanking region of PAPSS1 did not include a TATA box sequence near the site of transcription initiation, but PAPSS2 had a TATA motif located 21 bp upstream from the site of transcription initiation. Northern blot analysis showed that the major PAPSS1 and PAPSS2 transcripts were approximately 2.7 and 4.2 kb in length, respectively. PAPSS1 mapped to human chromosome band 4q24 while PAPSS2 mapped to 10q22-23 by fluorescence in situ hybridization analysis. Cloning and structural characterization of PAPSS1 and PAPSS2 will make it possible to perform molecular genetic and pharmacogenomic studies of these important enzymes in humans.

Dramatic, expansion-biased, age-dependent, tissue-specific somatic mosaicism in a transgenic mouse model of triplet repeat instability.

Myotonic dystrophy type 1 (DM1) is one of a growing number of inherited human diseases whose molecular basis has been implicated as the expansion of a trinucleotide DNA repeat. Expanded disease-associated alleles of >50 CTG repeats are unstable in both the germline and soma. Expansion of the unstable alleles over time and variation of the level of mutation between the somatic tissues of an individual are thought to account at least partially for the tissue specificity and progressive nature of the symptoms. We previously generated a number of transgenic mouse lines containing a large expanded CTG repeat tract that replicated a number of the features of unstable DNA in humans, including frequent sex-specific changes in allele length during intergenerational transmission. Small length change mutations were apparent in the somatic tissues of young mice in all of the lines generated, but the gross instability observed in human DM1 patients was not replicated. We now show that in one of the lines, Dmt -D, spectacular, expansion-biased, tissue-specific instability is observed in older mice. The highest levels of instability were detected in kidney with gains of >500 repeats, representing a tripling of allele length, in some cells. Mosaicism accumulated in an age-dependent manner, but the tissue specificity did not obviously correlate with cell turnover. Such gross somatic mosaicism was not observed in three other lines examined, further emphasizing a role for flanking DNA in modulating repeat stability.

Identification and characterization of a novel gene (C4orf5) located on human chromosome 4q with specific expression in cardiac and skeletal muscle.

The loci of several genes responsible for arrhythmogenic right ventricular dysplasia (ARVD) have been mapped. Since ARVD involves the right ventricle, we sought candidate genes preferentially expressed in the right ventricle utilizing differential display polymerase chain reaction (PCR) on mRNA from the chambers of an adult human heart. PCR products were cloned, sequenced, and used to screen an adult heart cDNA library. A novel 1.3-kb cDNA (HGMW-approved symbol C4orf5) with an open reading frame of 795 bp was identified. A probe designed from the 3' untranslated region of the 1.3-kb cDNA was hybridized to the 1.3-kb transcript and an alternatively spliced 2.5-kb transcript in the heart and skeletal muscle RNA lanes on a multitissue Northern blot. Analysis of a 39-kb partial genomic sequence identified three intronic splice sites in the 1.3-kb transcript. The gene was mapped to human chromosome 4q26-q27. Computer-based analysis indicated that this gene is novel with no known function.

Human indolethylamine N-methyltransferase: cDNA cloning and expression, gene cloning, and chromosomal localization.

Indolethylamine N-methyltransferase (INMT) catalyzes the N-methylation of tryptamine and structurally related compounds. We recently cloned and characterized the rabbit INMT cDNA and gene as a step toward cloning the cDNA and gene for this enzyme in humans. We have now used a PCR-based approach to clone a human INMT cDNA that had a 792-bp open reading frame that encoded a 263-amino-acid protein 88% identical in sequence to rabbit INMT. Northern blot analysis of 35 tissues showed that a 2.7-kb INMT mRNA species was expressed in most tissues. When the cDNA was expressed in COS-1 cells, the recombinant enzyme catalyzed the methylation of tryptamine with an apparent K(m) value of 2.9 mM. The human cDNA was then used to clone the human INMT gene from a human genomic BAC library. The gene was 5471 bp in length, consisted of three exons, and was structurally similar to the rabbit INMT gene as well as genes for nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase in several species. All INMT exon-intron splice junctions conformed to the "GT-AG" rule, and no canonical TATA or CAAT sequences were present within the 5'-flanking region of the gene. Human INMT mapped to chromosome 7p15.2-p15.3 on the basis of both PCR analysis and fluorescence in situ hybridization. Finally, two possible single nucleotide polymorphisms were identified within exon 3, both of which altered the encoded amino acid. The cloning and expression of a human INMT cDNA, as well as the cloning, structural characterization, and mapping of its gene represent steps toward future studies of the function and regulation of this methyltransferase enzyme in humans.

hRAD17, a structural homolog of the Schizosaccharomyces pombe RAD17 cell cycle checkpoint gene, stimulates p53 accumulation.

The RAD17 gene product of S. Pombe is an essential component of the checkpoint control pathway which responds to both DNA damage and disruption of replication. We have identified a human cDNA that encodes a polypeptide which is structurally conserved with the S. Pombe Rad17 protein. The human gene, designated hRAD17, predicts an encoded protein of 590 amino acids and a molecular weight of 69 kD. Amino acid sequence alignment revealed that hRadl7 has 28.3% and 52.5% similarity with the S. Pombe Rad17 protein, and 21.8% identity and 45.8% similarity to the budding yeast cell cycle checkpoint protein, Rad 24. When introduced into the S. Pombe rad17 mutant, hRAD17 was able to partially revert its hydroxyurea and ionizing radiation hypersensitivity, but not its UV hypersensitivity. Permanent overexpression of the hRAD17 gene in human fibrosarcoma cells resulted in p53 activation and a significant reduction of S- and G2/M-phase cells accompanied by an accumulation of the G1-phase population, suggesting that hRAD17 may have a role in cell cycle checkpoint control. Immunostaining of HT-1080 cells transiently transfected with a hRAD17 construct confirmed the nuclear accumulation of p53, which mimics the induction caused by DNA damage. Using FISH analysis, we have mapped the hRAD17 locus to human chromosome 5q11.2.

Human hydroxysteroid sulfotransferase SULT2B1: two enzymes encoded by a single chromosome 19 gene.

We have cloned and characterized cDNAs that encode two human hydroxysteroid sulfotransferase (SULT) enzymes, SULT2B1a and SULT2B1b, as well as the single gene that encodes both of these enzymes. The two cDNAs differed at their 5'-termini and had 1050- and 1095-bp open reading frames that encoded 350 and 365 amino acids, respectively. The amino acid sequences encoded by these cDNAs included "signature sequences" that are conserved in all known cytosolic SULTs. Both cDNAs appeared, on the basis of amino acid sequence analysis, to be members of the hydroxysteroid SULT "family, " SULT2, but they were only 48% identical in amino acid sequence with the single known member of that family in humans, SULT2A1 (also referred to as DHEA ST). Northern blot analysis demonstrated the presence of SULT2B1 mRNA species approximately 1.4 kb in length in human placenta, prostate, and trachea and-faintly-in small intestine and lung. Expression of the two human SULT2B1 cDNAs in COS-1 cells showed that both of the encoded proteins catalyzed sulfation of the prototypic hydroxysteroid SULT substrate, dehydroepiandrosterone, but both failed to catalyze the sulfate conjugation of 4-nitrophenol or 17beta-estradiol, prototypic substrates for the phenol and estrogen SULT subfamilies. Both of these cDNAs were encoded by a single gene, SULT2B1. The locations of most exon-intron splice junctions in SULT2B1 were identical to those of the only other known human hydroxysteroid SULT gene SULT2A1 (previously STD). The divergence in 5'-terminal sequences of the two SULT2B1 cDNAs resulted from alternative transcription initiation prior to different 5' exons, combined with alternative splicing. SULT2B1 mapped to human chromosome band 19q13.3, approximately 500 kb telomeric to the location of SULT2A1.

RAD1, a human structural homolog of the Schizosaccharomyces pombe RAD1 cell cycle checkpoint gene.

Cell cycle checkpoints are gating mechanisms that govern cell cycle progression in the presence of DNA damage and incomplete DNA replication. The Schizosaccharomyces pombe Rad1 protein is an essential component of cell cycle checkpoints activated by both types of genomic stress. In this study, we report the isolation of a human homolog of the S. pombe RAD1 gene. The hRAD1 protein is also similar to the Saccharomyces cerevisiae cell cycle checkpoint protein Rad17 and the Ustilago maydis 3' --> 5' exonuclease, Rec1. We show that human RAD1 partially complements the hydroxyurea and ionizing radiation hypersensitivities of a S. pombe rad1 mutant, suggesting phylogenetic conservation of the DNA damage and replication checkpoints. The human RAD1 locus was mapped to human chromosome 5p13.2, a locus frequently altered in non-small-cell lung cancer and bladder cancer.

Chromosome 5 aberrations and genetic predisposition to lung cancer.

In this study, we aimed to confirm the finding that chromosome 5 aberrations are predisposing factors for lung cancer. The study population consisted of 118 previously untreated lung cancer patients and 101 healthy controls. Lymphocytes were treated with bleomycin for 5 hr and then allowed to recover in a drug-free medium for 48 hr. The mean number of cells with chromosome 5 abnormalities among 100 cells examined was significantly higher in patients (9.12) than in controls (4.69) (p < 0.001). The most frequent aberration was a 5q deletion and the breakpoints clustered at the 5q13-5q31 region. We then dichotomized the number of induced chromosome 5 abnormalities in peripheral blood lymphocytes by the 75th percentile in that of the controls. 103 (87.3%), of the 118 patients, but only 31 (30.7%) of the 101 controls, exhibited induced breaks above this point. After adjustment for age, sex, ethnicity and smoking status, we found that the sensitive group was at 14.4-fold increased risk for lung cancer. There was also a significant (p < 0.01) gradient of increased risk for lung cancer with an increasing number of chromosome 5 lesions. Therefore, chromosome 5 lesions, especially those at 5q, may be a molecular target of carcinogens in the development of lung cancer.

XRCC2 and XRCC3, new human Rad51-family members, promote chromosome stability and protect against DNA cross-links and other damages.

The phenotypically similar hamster mutants irs1 and irs1SF exhibit high spontaneous chromosome instability and broad-spectrum mutagen sensitivity, including extreme sensitivity to DNA cross-linking agents. The human XRCC2 and XRCC3 genes, which functionally complement irs1 and irs1SF, respectively, were previously mapped in somatic cell hybrids. Characterization of these genes and sequence alignments reveal that XRCC2 and XRCC3 are members of an emerging family of Rad51-related proteins that likely participate in homologous recombination to maintain chromosome stability and repair DNA damage. XRCC3 is shown to interact directly with HsRad51, and like Rad55 and Rad57 in yeast, may cooperate with HsRad51 during recombinational repair. Analysis of the XRCC2 mutation in irs1 implies that XRCC2's function is not essential for viability in cultured hamster cells.

Philadelphia chromosome-positive myeloid cells in the peripheral blood of chronic myelogenous leukemia patients: comparison with the frequency detected in cycling cells of the bone marrow.

Monitoring the frequency of the Philadelphia (Ph) chromosome in chronic myelogenous leukemia (CML) is important in determining the effectiveness of treatment for patients during therapy. This can be done with high resolution by subjecting short-term bone marrow cultures (48 h) to 24 h of mitotic arrest before harvest and detecting Ph-positive (Ph+) metaphases by fluorescence in situ hybridization (FISH) in a procedure termed hypermetaphase FISH or HMF. Here, we compared procedures for detecting Ph+ interphase cells (interphase FISH or I-FISH) in peripheral blood polymorphonucleocytes (PMNs) with HMF results on the bone marrow of the same 26 CML patients in different stages of remission. The probes for I-FISH in these experiments were relatively large (200-300 kb) and sufficiently resolved in PMNs so that 97.5% of the cells were scorable. The correlation between the frequencies of Ph+ cells from the two different cell sources was excellent (r = 0.983, P < 0.0001); however, there was a consistently higher level of Ph+ cells observed in the cycling marrow cells than in the peripheral blood PMNs. This was discussed in terms of current theories of apoptosis in CML cells. The large number of PMNs analyzable by I-FISH (approximately 500/patient in this study) provided sufficiently narrow 99% confidence intervals to suggest the procedure as an effective and efficient method for monitoring the frequency of Ph+ cells in CML patients undergoing therapy. However, for detection and quantification of minimal residual disease, HMF is preferable to I-FISH because of the much lower frequency of false-positive readings with the former procedure.

Cytogenetics of chronic myelogenous leukaemia.

The Philadelphia (Ph) chromosome is present in the leukaemic cells of most patients with chronic myelogenous leukaemia. Variant translocations occur in 10% of patients but breakpoints on chromosomes 9 and 22 remain the same, so prognosis of these patients is unchanged. Clonal evolution is infrequent in chronic phase and its significance depends on the specific chromosome involved, the number of metaphases affected and the timing in the chronic phase. The majority of patients in blastic phase demonstrate clonal evolution; three specific abnormalities (+Ph, +8 and isochromosome 17q) are present in 70% of patients. Loss of the Ph chromosome on therapy is associated with prolonged survival. For monitoring these events conventional G-band cytogenetics (CG) is essential at presentation to characterize the Disease cytogenetically, while fluorescence in situ hybridization (FISH) on hypermetaphase preparations (hypermetaphase FISH (HMF)) is important for establishing the specific frequency of Ph+ cells. During treatment FISH on interphase cells (I-FISH) can monitor the level of Ph+ cells in circulation, while CG may be used to identify any suspected clonal evolution. Where I-FISH is negative, HMF is essential to evaluate minimal residual disease.

Hypermutable myotonic dystrophy CTG repeats in transgenic mice.

Myotonic dystrophy (DM) is one of a growing number of inherited human disorders associated with the expansion of triplet repeat DNA sequences. Expanded alleles are highly unstable in both the germline and soma, accounting in large part for the unusual genetics of this disorder, its phenotypic variability and probably, the progressive nature of the symptoms. However, the molecular mechanisms and the genetic factors modulating repeat stability in DM and the other human disorders associated with expanded repeats are not well understood. To provide a model system in which the turnover of triplet repeats could be studied throughout mammalian development, we have generated five transgenic mouse lines incorporating expanded CTG/CAG arrays derived from the human DM locus. Transgene analysis has revealed germline hypermutability, including expansions, deletions and parent-of-origin effects, somatic and early embryonic instability and segregation distortion. Mutational differences between lines and sexes demonstrate that stability, as in humans, is modulated by as yet unidentified cis and trans acting genetic elements.

An 80 Kb P1 clone from chromosome 3p21.3 suppresses tumor growth in vivo.

High frequencies of allelic loss on the short arm of chromosome 3 in small cell lung cancer (SCLC) and a number of other tumors suggest the existence of a tumor suppressor gene(s) within the deleted regions. Two small cell lung cancer lines, NCI H740 and GLC20, have been described which have homozygous deletions in the region 3p21.3. The deleted region overlaps with a 2 Mb fragment of human DNA present in the interspecies hybrid HA(3)BB9F, that suppresses tumor formation by mouse A9 fibrosarcoma cells. Human sequences from this cell hybrid were isolated using inter Alu PCR. From this starting point, a P1 contig was developed for the region of 450 Kb that is common to the homozygous deletions seen in the SCLC lines NCI H740 and GLC20 and is also present in HA(3)BB9F, the suppressed A9 hybrid. Individual P1 clones were assayed for their ability to suppress the tumorigenicity of the mouse fibrosarcoma cell line A9 as assayed by injection of transfected A9 cells into athymic nude mice. The introduction of one of the P1 clones into A9 cells resulted in suppression of tumor growth whereas two other P1 clones from the contig failed to suppress tumor formation in athymic nude mice. These data functionally delimit a tumor suppressor locus to a region of 80 kb within a P1 clone at 3p21.3.

Myotonic dystrophy phenotype without expansion of (CTG)n repeat: an entity distinct from proximal myotonic myopathy (PROMM)?

Myotonic dystrophy (DM) is associated with an expansion of an unstable (CTG)n repeat in the 3' untranslated region of the DM protein kinase (DMPK) gene on chromosome 19q13.3. We studied six patients from two families who showed no expansions of the repeat, in spite of their clinical diagnosis of DM. These patients had multi-systemic manifestations that were distinguishable from those seen in other myotonic disorders, including proximal myotonic myopathy (PROMM). In one additional family, two symptomatic members showed no expanded (CTG)n repeats, while their affected relatives had the expanded repeats. DM haplotype analysis failed to exclude the DMPK locus as a possible site of mutation in each family; however, DMPK mRNA levels were normal. We conclude that a mutation(s) other than the expanded (CTG)n repeat can cause the DM phenotype. The mutation(s) in these families remain(s) to be mapped and characterized.