PCR-single Strand Conformation Polymorphism Analysis Research Articles

Tongue Carcinogenic Susceptibility of p53 Deficient Mice to Methyl-<i>n</i>-amylnitrosamine

Mutation of the p53 tumor suppressor gene is a common genetic alteration in human squamous cell carcinoma of the tongue. Mice deficient in p53 have recently attracted attention for their potential to identify chemical genotoxins. In this study we investigated the susceptibility of p53 nullizygous (-/-), heterozygous (+/-), and wild type (+/+) mice to methyl-n-amylnitrosamine (MNAN) induced squamous cell carcinoma (SCC) of the tongue. The p53 (+/-), and (+/+) mice were treated with 5 p.p.m. MNAN in drinking water for 8 weeks then held without further treatment for an additional 7 or 17 weeks, and killed at 15 or 25 experimental weeks. A separate group of the p53 (-/-) mice were given 5 p.p.m. MNAN for 8 weeks and were killed at 15 weeks. At 15 weeks, SCCs and papillomas were observed in 5/12 (41.7%) and 2/12 (16.7%) of p53 (-/-) mice, respectively, but not in p53 (+/-) and (+/+) mice. At 25 weeks, carcinomas in situ (CIS) were detected in 1/16 (6.3%) of p53 (+/-) and 1/13 (7.7%) of p53 (+/+) mice, and a papilloma was observed in the p53 (+/-) mouse which had CIS. PCR-single strand conformation polymorphism analysis of exons 5-8 of the p53 gene demonstrated a missense mutation in the CIS from p53 (+/+) mouse. These results suggest that a lack of p53 gene function predisposes the tongue to the development of SCCs in mice treated with MNAN, and show that p53 (-/-) mouse was a useful model for demonstrating carcinogenicity of MNAN to tongue.

TP53 mutations in human meningiomas.

Overexpression of p53 has been reported to play a role in the development of neoplasms of the central nervous system. Meningiomas are generally benign intracranial tumors originating from the meninges. Overexpression of the p53 protein in meningiomas and an association with histological type and recurrence has been reported. Mutation of the TP53 gene leads to a more stable p53 protein in quantities high enough for detection by immunohistochemistry. In the search for these mutations the core domain of the TP53 gene of meningiomas has been analyzed. Only a very low incidence of mutations was reported. The apparent discordance between overexpression of p53 protein and TP53 gene mutations may be explained by mutations located outside the core domain. This issue was addressed in the present study. All 11 exons of 17 meningiomas were analyzed for DNA alterations by PCR single-strand conformation polymorphism (PCR-SSCP) analysis with subsequent sequencing. PCR-SSCP analysis showed a various number of band shifts and nucleotide alterations, caused either by alterations in the flanking introns or common polymorphisms (codon 36 and 72). The allele frequencies of the polymorphisms found in this small population of tumors resemble the frequencies reported in the literature. In addition, three nucleotide changes located in introns 2, 3 and 7 were found in 11, 3 and 4, respectively, of 17 specimens. Based on this study and on reports by others we conclude that it is not very likely that TP53 mutations are involved in the etiology of meningiomas.

FHIT alterations in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine in rats.

Alteration of the FHIT gene was investigated in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl) amine (BHP) in male Wistar rats. Animals at 6 weeks of age were given 2000 p.p.m. of BHP in drinking water for 12 weeks, then maintained without further treatment until killed at the end of week 25. A total of 25 lung adenocarcinomas were obtained and total RNAs were extracted from each for assessment of aberrant transcription of the FHIT gene by reverse transcription (RT)-polymerase chain reaction (PCR) analysis. Aberrant transcripts were detected in 15 adenocarcinomas (60%) as absence in the regions of nucleotides (nt) -9 to 279, -98 to 279, -98 to 348 or -98 to 447. Genomic DNAs were also extracted from all 25 adenocarcinomas and exons 5-9 were examined for mutations, using PCR-single strand conformation polymorphism (SSCP) analysis and sequencing. A mutation was detected in only one adenocarcinoma (4%), an ACC to ATC (Thr to IIe) transition at codon 76. Southern blot analysis of eight tumors did not show any evidence of gross rearrangement or deletion of the FHIT gene. Western blot analysis revealed reduced expression of Fhit protein in six out of 10 adenocarcinomas (60%). These results suggest that alteration of the FHIT gene may be involved in the development of lung adenocarcinomas induced by BHP in rats.

Genetic alterrations of microsatellite markers at chromosome 17 in non-small cell lung cancer

Objective: To analyse the microsatellite instability (MI) and loss of heterozygosity (LOH) in non-small cell lung cancer (NSCLC). Methods: MI and LOH at chromosome 17 were checked in 35 cases of NSCLC tumor-normal paired tissues using four microsatellite markers TP53 (17pl3.1), THRA1 (17qll.2–12), D17S579 (17ql2–21) and D17S855 (17q21) by PCR based analysis. Mutations of P53 exons 5–8 were also tested using PCR-single strand conformation polymorphism (PCR-SSCP) and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis. Results: 22 of 35 tumors (62.8%) displayed MI or LOH. 14 tumors (40.0%) exhibited MI, 11 tumors (31.4%) exhibited LOH, while 3 tumors (8.6%) exhibited MI and LOH concurrently. 23 tumors (65.7%) exhibited P53 gene mutations. The frequency of MI or LOH was obviously higher in the early-stage (stages I and II, 78.9%) than in the advanced-stage (stage III, 43.8%). However, the frequency of MI or LOH had no difference either between high-grade (75.0%) and low-grade (52.6%) differentiated NSCLC or between the tumors with P53 mutations (59.1%) and those without P53 mutations (69.2%). No relationship was observed between the presence of MI or LOH and the histologic subtype of NSCLC. Conclusion: The results suggest that MI and LOH at chromosome 17 may play a significant role in the development of NSCLC. The high frequency of MI or LOH in the early-stage tumors indicates that these genetic alterations could occur early during NSCLC development. Our data also show that the genetic alterations of microsatellite at chromosome 17 was not associated with P53 gene mutations.

Alterations of the insulin-like growth factor system in patients with polycythemia vera

The molecular etiology of Polycythemia vera (PV) is still undetermined. Recently, enhanced tyrosine phosphorylation of the insulin-like growth factor-I receptor (IGF-IR) has been shown in PV bone marrow progenitors and peripheral blood mononuclear cells (PBMNC), and elevated levels of IGF binding protein-1 (IGFBP-1) in the serum of PV patients have been reported. To identify further alterations of circulating IGFBPs, the IGFBP profile in the serum of 12 PV patients was compared with age- and sex-matched controls by Western ligand blot (WLB), two-dimensional WLB, IGFBP-3 immunoblot and specific RIA for IGFBP-1, -2, -3 and IGFBP-4. To elucidate a role for the IGF-IR in the pathogenesis of PV, basal and IGF-I stimulated tyrosine phosphorylation of the IGF-IR β-subunit in PBMNC of PV patients or controls was determined by WLB. Furthermore, exons 2, 3 and 15-21 of the IGF-IR were screened for mutations by PCR-single strand conformation polymorphism analysis (PCR-SSCP). We found alterations of the IGFBP profile in the serum of eight out of 12 examined patients including elevated levels of IGFBP-1, -2 and -4, decreased levels of IGFBP-3 and an increase in IGFBP-3 fragment. However, no differences in tyrosine phosphorylation of the IGF-IR in PV patients, neither basal nor IGF-I induced, were detected. Furthermore, no mutations within the screened exons of the IGF-IR could be identified by PCR-SSCP. We conclude that there is no direct impairment of IGF-IR structure or function, but an altered IGFBP profile in a significant portion of PV patients which might contribute to the pathogenesis of PV in these patients.

Structure of the human acyl-CoA:cholesterol acyltransferase-2 (ACAT-2) gene and its relation to dyslipidemia

Acyl-CoA:cholesterol acyltransferase (ACAT) catalyzes cholesterol esterification in mammalian cells. Two isoforms of ACAT have been reported to date (ACAT-1 and ACAT-2). ACAT-1 is ubiquitously expressed in tissues except the intestine. In contrast, ACAT-2 is expressed mainly in the intestine in humans. To investigate the relationship between ACAT-2 and dyslipidemia, we determined the structure of the human ACAT-2 gene and then studied the relationship between mutations of the ACAT-2 gene and dyslipidemia. To isolate human ACAT-2 genomic DNA, we designed primers based on the human ACAT-2 cDNA sequence: forward primer 5′-ACACCTCGATCTTGGTCCTGCCATA-3′ and reverse primer 5′-GGAATGCAGACAGGGAGTCCT-3′. Using these primers, a human P1-derived artificial chromosome (PAC) library was screened by PCR-based procedures. Isolated PAC clones were completely digested with BamHI and subcloned into plasmid vector. Subclones that contained exons were screened by dot-blot hybridization using partial ACAT-2 cDNA fragments. The coding region of the ACAT-2 gene was encoded in 15 exons from 51 to 265 base pairs on a 21 kilobase span of genomic DNA. The exonic sequences coincided completely with that of ACAT-2 cDNA, and each exon-intron junction conserved splicing consensus sequences. Next, 187 (91 dyslipidemic and 96 normolipidemic) subjects were screened by PCR single-strand conformational polymorphism analysis of the ACAT-2 gene. Three mutations were identified by DNA sequencing: two missense mutations (E14G in exon 1 and T254I in exon 7) and a point mutation in intron 7 (−35G→A). Mutations in exon 1 and intron 7 were not associated with plasma concentrations of lipids and apolipoproteins (apo). However, plasma apoC-III levels in T254I heterozygotes were significantly higher than those in subjects without mutation. Plasma triglyceride (TG) levels in T254I heterozygotes were similar to those in subjects without mutation. Although further studies are needed, our data suggest that ACAT-2 may contribute to apoC-III gene expression and the assembly of apoC-III and TG, possibly in the intestine.

Genetic diversity of Pneumocystis carinii in HIV-positive and -negative patients as revealed by PCR–SSCP typing

To describe the epidemiology of severe Pneumocystis carinii pneumonia (PCP) in HIV-infected and non HIV-infected patients. Bronchoalveolar lavage specimens from 212 European patients with PCP were typed using PCR--single strand conformation polymorphism analysis of four genomic regions of P. carinii f. sp. hominis. Demographic and clinical information was obtained from all patients. Twenty-three per cent of the patients were presumably infected with a single P. c. hominis type. The other patients presented with two (50%) or more (27%) types. Thirty-five genetically stable and ubiquitous P. c. hominis types were found. Their frequency ranged from 0.4% to 10% of all isolates, and up to 15% of those from a given hospital. There was no significant association between the P. c. hominis type or number of co-infecting types per patient and geographical location, year of collection, sex, age, or HIV status. No more than three patients infected with the same type were observed in the same hospital within the same 6 month period, and no epidemiological link between the cases was found. The broad diversity of types observed seems to indicate that multiple sources of the pathogen co-exist. There was no evidence that in our study population inter-human transmission played a significant role in the epidemiology of P. carinii.

Early-onset type 2 diabetes: metabolic and genetic characterization in the mexican population.

The objective of this study was to investigate possible defects in the insulin sensitivity and/or the acute insulin response in a group of Mexican patients displaying early-onset type 2 diabetes and to evaluate the contribution of mutations in three of the genes linked to maturity-onset diabetes of the young. We studied 40 Mexican patients with an age of diagnosis between 20 and 40 yr in which the insulin sensitivity as well as the insulin secretory response were measured using the minimal model approach. A partial screening for possible mutations in 3 of the 5 genes linked to maturity-onset diabetes of the young was carried out by PCR-single strand conformation polymorphism analysis. A low insulin secretory capacity (AIRg = 68.5 +/- 5 muU/mL.min) and a near-normal insulin sensitivity (3.43 +/- 0.2 min/muU.mL x 10(4)) were found in these patients. Among this group we found two individuals carrying missense mutations in exon 4 of the hepatocyte nuclear factor-1alpha (HNF-4alpha) gene (Asp(126)-->His/Tyr and Arg(154)-->Gln, respectively) and one carrying a nonsense mutation in exon 7 of the HNF-1alpha gene (Gln(486)-->stop codon); 7.5% had positive titers for glutamic acid decarboxylase antibodies. Thirty-five percent of cases had insulin resistance; these subjects had the lipid abnormalities seen in the metabolic syndrome. A defect in insulin secretion is the hallmark in Mexican diabetic patients diagnosed between 20 and 40 yr of age. Mutations in either the HNF-1alpha or the HNF-4alpha genes are present among the individuals who develop early-onset diabetes in our population. These particular sequence changes have not been previously reported and therefore represent putative new mutations. Even in the absence of endogenous hyperinsulinemia, insulin resistance is associated with an adverse lipid profile.

Incidence of human papillomavirus 16 and 18 infection and p53 mutation in patients with oral squamous cell carcinoma in Japan

The purpose of this study was to investigate the prevalence of human papillomaviruses (HPVs) 16 and 18 infection, and p53 mutation in oral squamous cell carcinomas (SCCs) in Japanese patients. Our results showed a higher incidence of HPV16 and 18 infections than previous studies because we combined the findings of a consensus polymerase chain reaction (PCR), restriction fragment length polymorphism by using the restriction enzyme digestion of the PCR products and Southern blot hybridization. Each HPV16 and 18 E6/E7 DNA was detected in 9 (20%) and 25 (54%) of 46 samples. The p53 mutation in the exons from 5 to 8 were detected in 20 out of 46 samples (43%) by a PCR-single strand conformation polymorphism analysis. There was a significant relationship between HPV16 and the p53 mutation (P =0.02) suggesting that HPV16 infection has a mutagenic effect in oral SCC. However, neither HPV infection nor p53 mutation influenced survival.

Typing of Pneumocystis carinii f. sp. hominis by PCR-SSCP to indicate a high frequency of co-infections.

Broncho-alveolar lavage specimens from patients with Pneumocystis carinii pneumonia were investigated by PCR-single strand conformation polymorphism (SSCP) analysis of four genomic regions for P. carinii f. sp. hominis. In all, 32% of specimens produced two bands (one allele) for each of the four genomic regions, suggesting an infection with a single P.c. hominis type. The other specimens displayed more than two bands for at least one of the four genomic regions, suggesting several theoretical possibilities: co-infections, heterozygosity of diploid or polyploid organisms, or multicopy genes. Quantification of the different alleles and analysis of mixtures of specimens showed that different alleles of a genomic region were most often present in different proportions in a given specimen. In contrast, experiments with plasmid insertion of two alleles resulted in the detection of identical proportions of the two alleles. This suggests that neither heterozygosity of diploid organisms nor multicopy genes are responsible for the presence of two alleles. Unequal proportions are most likely explained by the occurrence of co-infections. The putative co-infecting types could be identified in the majority of specimens.

Characterisation of extended-spectrum beta-lactamases of the SHV family using a combination of PCR-single strand conformational polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP).

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) has been developed to extend the identification of SHV beta-lactamases previously characterised by PCR-single strand conformational polymorphism (PCR-SSCP) analysis alone. Eight bacteria, each producing a different SHV beta-lactamase, were used in this study. These bacteria harbour bla(SHV-1), bla(SHV-2a), bla(SHV-3), bla(SHV-4), bla(SHV-5) (two strains), bla(SHV-11) and bla(SHV-12). All isolates were characterised by PCR-SSCP and PCR-RFLP with DdeI and NheI digestion. By a combination of these techniques, the genes encoding these beta-lactamases could be differentiated from each other. In addition, the PCR-RFLP technique theoretically can be applied to distinguish the genes encoding SHV-7, SHV-9, SHV-10, SHV-15, SHV-17 and SHV-24 from those encoding other SHV variants. We report a simple PCR-RFLP technique that can be used in epidemiological studies to enable the rapid characterisation of known SHV beta-lactamases in a combination with the previously published PCR-SSCP analysis.

Characterisation of extended-spectrum β-lactamases of the SHV family using a combination of PCR-single strand conformational polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP)

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) has been developed to extend the identification of SHV β-lactamases previously characterised by PCR-single strand conformational polymorphism (PCR-SSCP) analysis alone. Eight bacteria, each producing a different SHV β-lactamase, were used in this study. These bacteria harbour bla SHV-1, bla SHV-2a, bla SHV-3, bla SHV-4, bla SHV-5 (two strains), bla SHV-11 and bla SHV-12. All isolates were characterised by PCR-SSCP and PCR-RFLP with DdeI and NheI digestion. By a combination of these techniques, the genes encoding these β-lactamases could be differentiated from each other. In addition, the PCR-RFLP technique theoretically can be applied to distinguish the genes encoding SHV-7, SHV-9, SHV-10, SHV-15, SHV-17 and SHV-24 from those encoding other SHV variants. We report a simple PCR-RFLP technique that can be used in epidemiological studies to enable the rapid characterisation of known SHV β-lactamases in a combination with the previously published PCR-SSCP analysis.

Transgenic rats carrying human c-Ha-ras proto-oncogenes are highly susceptible to N-methyl-N-nitrosourea mammary carcinogenesis.

A rat line carrying three copies of the human c-Ha-ras proto-oncogene, including its own promoter region, was established and designated Hras128. Expression of the transgene was detected in all organs examined from Hras128 rats by northern blot analysis. To examine its influence on susceptibility to N-methyl-N-nitrosourea (MNU)-induced mammary carcinogenesis, female rats were treated with 50 mg/kg MNU i.v. at 50 days of age. All 22 Hras128 transgenic rats rapidly developed multiple and large mammary carcinomas within as little as 8 weeks after MNU treatment (14.1 tumors/rat, average diameter 16.4 mm). In contrast, 24 non-transgenic littermates developed no or only small tumors (0.46 tumors/rat, average diameter 7.4 mm) within this period. PCR-restriction fragment length polymorphism (RFLP) analysis and direct sequencing for the transduced human c-Ha-ras proto-oncogene indicated that 38 out of 44 tumors (86.4%) contained cells with mutations at codon 12 in exon 1. However, the signal densities of the mutated bands observed in the RFLP analyses revealed the presence of mixed populations of mutated and non-mutated cells in the tumors, the latter being in the majority. PCR-single strand conformation polymorphism analysis detected no mutations in codons 12 or 61 of the endogenous rat c-Ha-ras gene of Hras128 rat tumors. The results thus indicate that rats carrying the transduced human c-Ha-ras proto-oncogene are highly susceptible to MNU-induced mammary carcinogenesis and that this is not primarily due to mutations of the transgene or endogenous c-Ha-ras gene.

Rapid identification of bacteria from positive blood cultures by fluorescence-based PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene.

Bacteremia continues to result in significant morbidity and mortality, particularly in patients who are immunocompromised. Currently, patients with suspected bacteremia are empirically administered broad-spectrum antibiotics, as definitive diagnosis relies upon the use of blood cultures, which impose significant delays in and limitations to pathogen identification. To address the limitations of growth-based identification, the sequence variability of the 16S rRNA gene of bacteria was targeted for rapid identification of bacterial pathogens isolated directly from blood cultures using a fluorescence-based PCR-single-strand conformation polymorphism (SSCP) protocol. Species-specific SSCP patterns were determined for 25 of the most common bacterial species isolated from blood cultures; these isolates subsequently served as a reference collection for bacterial identification for new cases of bacteremia. A total of 272 blood-culture-positive patient specimens containing bacteria were tested. A previously determined SSCP pattern was observed for 251 (92%) specimens, with 21 (8%) specimens demonstrating SSCP patterns distinct from those in the reference collection. Time to identification from blood culture positivity ranged from 1 to 8 days with biochemical testing, whereas identification by fluorescence-based capillary electrophoresis was obtained as early as 7 h at a calculated cost of $10 (U.S. currency) per specimen when tested in batches of 10. Limitations encountered included the inability to consistently detect mixed cultures as well as some species demonstrating identical SSCP patterns. This method can be applied directly to blood cultures or whole-blood specimens, where early pathogen identification would result in a timely diagnosis with possible implications for patient management costs and the mortality and morbidity of infections.

CDKN2 Mutation Is Infrequent in German Hepatocellular Carcinoma

For hepatocellular carcinoma, only scarce and controversial data on CDKN2 alterations are available. A high rate of mutations in a Chinese study contrasts with a low rate found in Japanese tumors and a CDKN2 germline mutation in 4/26 Swiss tumors examined. We analyzed 23 hepatocellular carcinomas from German patients for homozygous deletions of CDKN2 by coamplification with the human tyrosine hydroxylase (TH) gene and for CDKN2 mutations by PCR-single strand conformation polymorphism analysis and direct DNA sequencing. Our results indicate the lack of homozygous deletions. In one tumor, DNA sequencing showed a GCG-ACG (alanine-threonine) substitution at codon 148, a polymorphism in exon 2 of CDKN2. We conclude that the alteration of CDKN2 by deletion or mutation appears not to be a frequent event in hepatocarcinogenesis in German patients.

High resolution HLA-DQB1 typing by combination of PCR-RFLP and PCR-SSCP

A reliable method for high-resolution HLA-DQB1 typing using a combination of PCR- restriction fragment length polymorphism (RFLP) and PCR-single strand conformation polymorphism (SSCP) analysis is described. The second exon of the DQB1 gene was subjected to PCR using generic primers and digested with two restriction enzymes, MspA1I and HaeIII, and the DQB1 alleles were divided into seven groups. According to the RFLP patterns, appropriate group specific primers for DQ5, 6 and DQ2, 3, 4 groups were used to selectively amplify the alleles and the SSCP technique was used to distinguish the individual alleles. A total of 88 quality control samples of various ethnic groups distributed in the International Cell Exchange and HLA DNA Exchange programs and the ASHI/CAP Proficiency Tests were investigated by the PCR-RFLP/SSCP method. The concordance between our typing results and the consensus results of the surveys were 100%, and a total of 14 DQB1 alleles in 49 homozygous and heterozygous combinations were all correctly identified by the method described. This method is accurate, economical and relatively easy to interpret and well suited for routine clinical and research uses.

Establishment and Characterization of a Fas-Resistant T Cell Line

Fas is a cell surface receptor that controls a signal transduction pathway leading to apoptosis. We established an antihuman Fas monoclonal antibody (mAb)-resistant variant, kit-225-FR, from the human T cell line, kit-225. Flow cytometric analysis revealed that the expression of Fas molecules on kit-225-FR was preserved. The defect in Fas molecule was not detected either by reverse transcription polymerase chain reaction (PCR) analysis of the Fas transcript or by PCR-single strand conformation polymorphism analysis of the Fas gene in kit-225-FR. Although kit-225-FR was resistant to a high concentration of anti-Fas mAb, apoptosis could be induced, as with the wild type, by exogenous C2-ceramide exposure. MORT1/FADD was expressed at wild-type level in kit-225-FR, as determined by Western blot analysis. It therefore appears that the apoptotic signal transduction in kit-225-FR is defective between FADD and the sphingomyelin-ceramide pathway. By comparing the differences from the wild-type kit-225, kit-225-FR would serve as a useful cell line for analyzing Fas-specific signal transduction pathways in detail.

Alteration of p16 (CDKN2) gene is associated with interleukin-2–induced tumor cell growth in adult T-cell leukemia

Because tumorigenesis frequently involves the dysfunction of cell cycle-related proteins, we examined the effect of mutations in CDK inhibitor p16 and its linked genomic loci p15, cl.B, and 1063.7 on the growth of primary adult T-cell leukemia (ATL) cells. Southern blot analysis of primary ATL cells showed a significantly higher incidence of p16 gene alteration in acute ATL than in chronic ATL [67.7% (23/34) vs 26.1% (6/23), respectively; p < 0.003]. Similarly, polymerase chain reaction (PCR) analysis of p16 exon 2 revealed a higher incidence of alteration in acute ATL than in chronic ATL [52.9% (18/34) vs 26.1% (6/23), respectively; p < 0.05]. PCR-single strand conformation polymorphism analysis of exons 1 and 2 of p16 showed no mutations in the patients, with normal pattern by Southern blotting or PCR analysis. Notably five of six chronic ATL patients with abnormal p16 genes progressed to acute crisis within 4 months. PCR analysis of the p16 linked loci 1063.7, p15 exon 2, and cl.B found homozygous deletion in 55.9%, 20.6%, and 2.9% of acute ATL cells and 39.1%, 13.0%, and 0% of chronic ATL cells, respectively, showing no relationship of homozygous deletion in either loci with disease subtypes. In most cases, deletions were seen in multiple genes, including p16. Acute ATL cells had a higher frequency of multigene deletions than chronic ATL cells [44.1% vs 17.4%; p < 0.05]. When leukemic cells were analyzed for interleukin 2 (IL-2) responsive growth, only p16 gene alteration was directly associated with leukemic cell growth activity. Among leukemic cells showing high IL-2 responsiveness, 73.1% (19/26) had p16 gene alteration vs 27.8% (5/18) of leukemic cells that showed low IL-2 responsiveness ( p < 0.005). p16 gene alteration was found in 73.3% (14/19) of leukemic cells showing high autonomous growth rates but in only 40.0% (10/25) of those leukemic cells showing low autonomous growth ( p < 0.03). These results suggest the following: alteration of p16-related genomic regions in ATL is usually a wide rearrangement including the p16 gene; within this region, only p16 gene alteration is associated with disease aggressiveness; and p16 gene deletion may be a proximate event in leukemogenesis.

Homozygous Cys542→Arg substitution in GPIIIa in a Swiss patient with type I Glanzmann's thrombasthenia

Glanzmann's thrombasthenia (GT) arises from a qualitative or quantitative defect in the GPIIb-IIIa complex (integrin alphaIIbbeta3), the mediator of platelet aggregation. We describe a patient in whom clinical and laboratory findings typical of type I GT were found together with a second pathology involving neurological and other complications symptomatic of tuberous sclerosis. Analysis of platelet proteins by Western blotting revealed trace amounts of normally migrating GPIIb and equally small amounts of GPIIIa of slightly slower than normal migration. Flow cytometry confirmed a much decreased binding to platelets of monoclonal antibodies to GPIIb, GPIIIa or GPIIb-IIIa, and an antibody to the alphav subunit also showed decreased binding. Nonradioactive PCR single-strand conformation polymorphism analysis followed by direct sequencing of PCR-amplified DNA fragments showed a homozygous point mutation (T to C) at nucleotide 1722 of GPIIIa cDNA and which led to a Cys542-->Arg substitution in the GPIIIa protein. The mutation gave rise to a HinP1 I restriction site in exon 11 of the GPIIIa gene and allele-specific restriction enzyme analysis of family members confirmed that a single mutated allele was inherited from each parent. This amino acid substitution presumably changes the capacity for disulphide bond formation within the cysteine-rich core region of GPIIIa and its study will provide new information on GPIIb-IIIa and alphavbeta3 structure and biosynthesis.

Clonal Expansion of CD4+ TCRββ+ T Cells in TCR α-Chain- Deficient Mice by Gut-Derived Antigens

AbstractA population of CD4+ α−β+ T cells increases in the mucosal and peripheral lymphoid tissues of TCRα-chain-deficient mice with inflammatory bowel disease. The α−β+ T cells, which produce predominantly IL-4, mediate the proliferation of colonic epithelial crypts and the infiltration of large numbers of IgA-producing plasma cells into the lamina propria of the colon. To examine whether enteric Ags were recognized by a population of monoclonal α−β+ T cells leading to the intestinal inflammation, we examined the usage and clonotypes of TCR expressed by the α−β+ T cells in TCRα-chain-deficient mice with inflammatory bowel disease. Analyses of immunoprecipitates by two dimensional electrophoresis and single-cell RT-PCR revealed that TCR of the α−β+ T cells was a homodimer of β-chains that was capable of recognizing luminal bacterial Ags. PCR single-strand conformation polymorphism analysis of TCR Vβ transcripts revealed monoclonal accumulation of the α−β+ T cells in the colonic lamina propria of the diseased mice. DNA sequencing revealed the accumulation of the α−β+ T cells with the same CDR3 sequences in the colon. These findings suggest that the pathogenic CD4+ α−β+ T cells expressing a homodimeric form of the TCRβ-chains can be clonally expanded upon the stimulation with gut-derived Ags.