Variant Bands Research Articles

Mutation screening of the BRCA1 gene in sporadic breast cancer in southern Chinese populations

The comparison of freshly collected breast cancer tissue DNA with that of the matched blood specimens of 144 patients was studied, and breast tissue from 30 unrelated normal women without cancer was selected as controls. The entire BRCA1 coding sequence was amplified by PCR with primers especially designed for comprehensive mutation screening by single-strand conformation polymorphism (SSCP) analysis. Amplification and electrophoresis were repeated for the confirmation of altered migration patterns. Variant bands were subsequently cut from gels, resuspended in distilled water, subjected again to PCR, and then sequenced. The amplified fragments of exon 2 and exon 3, which were not suitable for SSCP (>300 bp) were sequenced directly. A total of 20 nucleotide alterations were observed in the breast cancer tissue DNA, and all were single-base substitutions. Sixteen missense mutations (which change the coding from one amino acid to another) have been identified throughout the gene. Ten cases of single nucleotide changes in BRCA1 detected in the study without records in the BIC database consisted of two missense mutations in exon 5 (273C > G, 287A > T), one polymorphism in exon 11 (2630T > G), three missense mutations in exon 11 (2532T > G, 3191C > G, 3876C > A), one missense mutation in exon 12 (4285G > A), two missense mutations in exon 17 (5115T > C, 5116A > G), and one missense mutation in exon 18 (5206T > A). The same analysis was carried out on matched blood specimens for each of the 20 nucleotide alterations revealed to be present in the germline. No nucleotide alterations were detected in the controls. These results suggest that somatic mutations of BRCA1 are infrequent in sporadic breast cancer, and nucleotide alterations were more easily observed in the breast cancer tissue DNA. It is likely that there are other important susceptibility genes that remain to be identified in patients with sporadic breast cancer.

Calcium extrusion by plasma membrane calcium pump is impaired in caveolin-1 knockout mouse small intestine

Plasma membrane calcium ATPase (PMCA) is an important calcium extrusion mechanism in smooth muscle cells. PMCA4 is the predominant isoform operating in conditions of high intracellular calcium during contraction. PMCA appears to be localized in lipid rafts and caveolae. In this study we examined the effects of the PMCA4-selective inhibitor caloxin 1c2 (5 µM) in intestine of caveolin-1 knockout mice and in bovine tracheal smooth muscle after caveolae disruption on PMCA4 function. Small intestinal tissues from control mice treated with caloxin 1c2 showed a higher contractile response of the longitudinal smooth muscle to Carbachol (10 µM) when compared to control tissues treated with a similar concentration of a control peptide. This effect of caloxin 1c2 was not found in tissues from caveolin-1 knockout mice. Immunohistochemistry and Western blotting of membrane fractions showed that PMCA was co-localized with caveolin-1 in smooth muscle plasma membrane in control tissues. One of the PMCA4 splice variant bands was missing in the lipid raft-enriched fraction prepared from caveolin-1 knockout tissue. In bovine tracheal smooth muscle tissue, caveolae disruption by cholesterol depletion led to the diminution of caveolin-1 and PMCA4b immunoreactivities, previously co-localized in the smooth muscle plasma membrane, and to the loss of the increase in Carbachol-induced contraction by caloxin 1c2. Our results suggest that the calcium removal function of PMCA4 in smooth muscle cells is dependent on its presence in intact caveolae. We suggest that this is due to the close spatial arrangement that allows calcium extrusion from a privileged cytosolic space between caveolae and sarcoplasmic reticulum.

Genetic and epigenetic instabilities induced by tissue culture in wild barley (Hordeum brevisubulatum (Trin.) Link)

A simple tissue culture protocol was developed for efficient plant regeneration from young inflorescence-derived calli in wild barley, Hordeum brevisubulatum (Trin.) Link, an important pasturage grass. Genetic and epigenetic instabilities in the regenerated plants (regenerants) were assessed by three molecular markers AFLP, S-SAP and MSAP. Two pools of calli derived from young inflorescences of a single donor plant and 44 randomly chosen regenerants were subjected to AFLP analysis. Results showed that 74 out of 793 scored bands were polymorphic among the studied samples, giving rise to a genetic variation frequency of 9.3%. The number of variant bands as compared to the donor plant varied greatly among the regenerants, with a small number of regenerants accumulated a large number of variant bands (maximum 55), while the majority of regenerants showed only 2–3 variant bands. A subset of regenerants together with the two pools of calli were selected for S-SAP and MSAP analysis to detect possible retrotranspositional activity of a prominent retroelement family, BARE-1, in the genomes of Hordem species, and possible alterations in cytosine methylation. S-SAP analysis showed that of the 768 scored bands, 151 were polymorphic among the analyzed samples, giving rise to a genetic variation frequency of 19.7%, albeit no evidence for retrotranspositional event was obtained based on locus-specific PCR amplifications. MSAP analysis revealed that tissue culture has caused cytosine methylation alterations in both level and pattern compared with the donor plant. Sequencing of selected variant bands indicated that both protein-coding genes and transposon/retrotransposons were underlying the genetic and epigenetic variations. Correlation analysis of the genetic and epigenetic instabilities indicated that there existed a significant correlation between MSAP and S-SAP (r = 0.8118, 1,000 permutations, P < 0.05), whereas the correlation between MSAP and AFLP (r = 0.1048) is not statistically significant.

Analysis of Beta-Tubulin Gene Exon 4 Mutations in Advanced Stage III or IV Gastric Cancer

The mechanisms that cause chemoresistance of gastric cancer have yet to be elucidated. Taxanes and promising agents that were recently approved for treatment of advanced or recurrent gastric cancer. Mutations of beta-tubulin, which is a target of taxianes, have been shown to confer chemoresistance against these agents. The aim of the present study is to investigate the presence of mutations of the beta-tubulin in gastric cancer tissues. Sixty-six patients with advanced stage III or IV gastric cancer patients enrolled in this study. Paired samples of gastric cancer tissue and normal mucosa were obtained by endoscopy. The guanosine 5'-triphosphate (GTP)-binding site in exon 4 of the beta-tubulin gene was examined by polymerase chain reaction single-strand conformational polymorphism (PCR-SSCP) analysis, followed by sequencing of the products with abnormally shifted bands. SSCP analysis showed abnormal bands upstream of the GTP-binding site in 7 of the 66 patients, but sequence analysis found no nucleotide substitutions in these patients. Three variant bands were also detected down stream of the the GTP-binding site, but the sequences of the 3 products corresponded to those of two independent pseudogenes. Thus, none of the tumor samples showed mutation of the beta-tubulin exon 4 GTP-binding site. In conclusion, these findings suggest that mutations of the beta-tubulin gene are rare and are unlikely to be an important cause of taxane resistance to taxians.

Band 3 Courcouronne: Homozygous Mutation Ser667Phe Causes Severe Hereditary Spherocytosis and Incomplete Distal Renal Tubular Acidosis.

We describe the second case of a homozygous mutation in band 3 (anion exchanger 1 (AE1), SLC4A1) causing both hereditary spherocytosis (HS) and distal renal tubular acidosis (dRTA). This new variant differed from the previous homozygous variant (Band 3 Coimbra, Ribeiro et al, Blood 2000: 96, 1602) in that a significant amount of band 3 was present in the red cell membrane and the dRTA was incomplete. In the proband, an Algerian male baby, a severe hemolytic anemia rapidly developed following birth. Low hemoglobin (3.5 g Hb/dL) at D12 demanded a first transfusion. Hepato-splenomegaly, marked palor and jaundice were noted. Eight transfusions were administered in the following months. Subtotal splenectomy, performed at the age of 9 months, cancelled the transfusional needs. Veinous blood examined after splenectomy showed band 3 to be reduced to ~35% of normal, as shown by immunoblotting. The other known proteins of the band 3/Rh macrocomplex were also found to be reduced. The parents were first cousins. Both showed mild spherocytosis associated with a mild band 3 deficiency. DNA sequence analysis revealed a novel homozygous mutation: TCC to TTC at codon 667 in exon 16, leading to an amino acid substitution: Ser667Phe, located in proposed transmembrane helix 8. Both parents were heterozygous for the same mutation. Anion transport (sulphate uptake) in the patient's red cells was ~40% normal, showing that transport specific activity of the mutant band 3 was not affected. The mutant red cell band 3 and kidney band 3 were expressed in Xenopus oocytes, with and without co-expression of glycophorin A (GPA). There was very little chloride transport detected in oocytes expressing either mutant red cell or kidney protein alone, but transport was partially rescued by co-expression of GPA. After birth the child showed a temporary acidosis which spontaneously receded. No nephrocalcinosis has been noted to date. At 2 years of age, an ammonium chloride challenge suggested that the child has incomplete dRTA; over the seven hours of the test the blood bicarbonates decreased down to 15.6 mmoles/L, but urinary pH remained above 5.90. Stable expression of mutant kidney band 3 in non-polarised Madin-Darby canine kidney (MDCK) cells showed that the mutant protein was retained in the endoplasmic reticulum. We are currently investigating the effects of this mutant in polarized MDCK cells. Overall our results suggest that the Ser667Phe does not affect the anion transport function of band 3 but causes a trafficking defect in both red blood cells and kidney cells. The trafficking defect may be less severe in red blood cells where it is probably attenuated by the chaperone-like effect of GPA, which is not expressed in kidney cells. The fact that the hematological manifestations are far more conspicuous than their nephrologic counterpart will be discussed.

Patterns and variability in electrophoretic polypeptide profiles of human saliva in a healthy population

Electrophoretic polypeptide profiles of normal human saliva differ markedly between different reports. Since both methodological variations and polymorphism may explain these differences, in this study we aimed to establish whether or not the salivary electrophoretic polypeptide profiles of subjects from a healthy population share discrete molecular features. To this end, parotid, submandibular/sublingual and whole salivas were collected separately from each of 40 young and 34 elderly clinically healthy adults and processed for SDS-polyacrylamide gel electrophoresis and Coomassie blue staining. Each type of glandular saliva displayed a different group of invariant (i.e. present in every subject) electrophoretic polypeptide bands while whole saliva showed a profile that reflected mostly the combined contribution of the major salivary glands. Some minor variant (i.e. absent in some subjects) bands were identified in each type of saliva. Regarding those interindividual variations, no age- or sex-dependence was appreciated. Altogether, these results demonstrate the occurrence of distinctive electrophoretic polypeptide patterns, in addition to some minor variations, for each type of normal saliva, thus providing a background for further populational studies on salivary polypeptide profiles.

Search for genetic variants in the p66Shc longevity gene by PCR-single strand conformational polymorphism in patients with early-onset cardiovascular disease

BackgroundAmong the possible candidate genes for atherosclerosis experimental data point towards the longevity gene p66Shc. The p66Shc gene determines an increase of intracellular reactive oxygen species (ROS), affecting the rate of oxidative damage to nucleic acids. Knock-out p66Shc-/- mice show reduction of systemic oxidative stress, as well as of plasma LDL oxidation, and reduced atherogenic lesions. Thus, p66Shc may play a pivotal role in controlling oxidative stress and vascular dysfunction in vivo.MethodsWe searched for sequence variations in the p66Shc specific region of the Shc gene and its upstream promoter by PCR-SSCP in a selected group of early onset coronary artery disease (CAD) subjects (n. 78, mean age 48.5 ± 6 years) and in 93 long-living control subjects (mean age 89 ± 6 years).ResultsThe analysis revealed two variant bands. Sequencing of these variants showed two SNPs: -354T>C in the regulatory region of p66Shc locus and 92C>T in the p66 specific region (CH2). Both these variants have never been described before. The first substitution partially modifies the binding consensus sequence of the Sp1 transcription factor, and was detected only in two heterozygous carriers (1 CAD subjects and 1 control subject). The 92C>T substitution in the CH2 region consists in an amino acid substitution at codon 31 (proline to leucine, P31L), and was detected in heterozygous status only in one CAD subject. No subjects homozygous for the two newly described SNPs were found.ConclusionOnly two sequence variations in the p66Shc gene were observed in a total of 171 subjects, and only in heterozygotes. Our observations, in accordance to other studies, suggest that important variations in the p66Shc gene may be extremely rare and probably this gene is not involved in the genetic susceptibility to CAD.

Biochemistry and Genetic Analysis of Bioeffects of Low Energy N+ Implantation and Y-Radiation on Arabidopsis thaliana

We analyzed the activities of specific enzymes, esterase and peroxidase, and the electrophoresis profiling of isozymes of both the second-generation (M1) and the third-generation (M2) individuals of Arabidopsis thaliana after the parental generation was treated with low-energy N+ implantation and γ-radiation. Results indicated that the mutagenic effects of both treatments were inheritable. The obvious difference between N+ implantation and γ-radial on the relationship of doses and effects was also observed. For the correlation between effects and doses, the former presented a particular saddle-like pattern, while the latter showed a pattern of linearity. Random amplification of polymorphic DNA analysis showed that the variant DNA bands were consistent in M1 and M2 generations treated with both γ-radiation and ion implantation. However, the ratio of individual plant variation between the two treatments was remarkably different.

Clinical follow up of Mexican women with early onset of breast cancer and mutations in the BRCA1 and BRCA2 genes

This study describes the presence of mutations in BRCA1 and BRCA2 genes in a group of Mexican women and the clinical evolution of early onset breast cancer (EOBC). A prospective hospital-based study was performed in a sample of 22 women with EOBC (7 in clinical stage IIA, 8 in IIB, and 7 in IIIA). The patients attended a tertiary care hospital in northeastern Mexico in 1997 and were followed up over a 5-year period. Molecular analysis included: 1) a mutation screening by heteroduplex analysis (HA) of BRCA1 and BRCA2 genes and 2) a sequence analysis. Of 22 patients, 14 (63.6%) showed a variant band detected by heteroduplex analysis of the BRCA1 and BRCA2 genes: 8 polymorphisms, 4 mutations of uncertain significance, and 2 novel truncated protein mutations, one in BRCAI (exon 11, 3587delT) and the other in the BRCA2 gene (exon 11, 2664InsA). These findings support future studies to determine the significance and impact of the genetic factor in this Mexican women population.

Increased Susceptibility of Mice Lacking Clara Cell 10-kDa Protein to Lung Tumorigenesis by 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone, a Potent Carcinogen in Cigarette Smoke

Ninety percent of all human lung cancers are related to cigarette smoking. Both tobacco smoke and lung tumorigenesis are associated with drastically reduced levels of Clara cell 10-kDa protein (CC10), a multifunctional secreted protein, naturally produced by the airway epithelia of virtually all mammals. We previously reported that the expression of CC10 is markedly reduced in animals exposed to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK, a potent carcinogen in tobacco smoke. Furthermore, it has been reported that CC10 expression, induced in certain tumor cells, reverses the transformed phenotype. We demonstrate here that NNK exposure of CC10-knock-out (CC10-KO) mice causes a significantly higher incidence of airway epithelial hyperplasia and lung adenomas compared with wild type (WT) littermates (30% CC10-KO versus 5% WT, p = 0.041). We also found that compared with NNK-treated WT mice, CC10-KO mice manifest increased frequency of K-ras mutation, elevated level of Fas ligand (FasL) expression, and increased MAPK/Erk phosphorylation, all of which are considered predisposing events in NNK-induced lung tumorigenesis. We propose that CC10 has a protective role against NNK-induced lung tumorigenesis mediated via down-regulation of the above-mentioned predisposing events.

Search for genetic variants in the retinoid X receptor-gamma-gene by polymerase chain reaction-single-strand conformation polymorphism in patients with resistance to thyroid hormone without mutations in thyroid hormone receptor beta gene.

Resistance to thyroid hormone (RTH) is an inherited disease characterized by reduced tissue sensitivity to thyroid hormone. Approximately 90% of subjects with RTH have mutation in the thyroid hormone receptor beta (TRbeta) gene. Approximately 10% of subjects diagnosed as having RTH do not carry mutation in the TRbeta gene. A possible linkage was reported with the retinoid X receptor-gamma (RXR-gamma) gene in two families. The aim of this study is to search for mutation within the RXR-gamma gene in unrelated subjects with diagnosed RTH without mutations in the TRbeta gene. Four subjects with RTH were studied, and sequence variants in the RXR-gamma gene were searched by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP). Analysis of all the 10 exons of the RXR-gamma gene, including intron-exon boundaries, promoter region and 3' untranslated region (UTR) reveled two variant bands in subjects II and III. Sequencing of these variants showed two single nucleotide polymorphisms (SNPs): 447C > T in exon 3 for patients II and IVS9 + 6A > G for patient III. Both SNPs were also present at high frequency in a group of normal subjects and in nonaffected relatives of subject III. In conclusion, in patients with RTH we have found two SNPs in the RXR-gamma gene; these SNPS are common in the general population, thus excluding a role for the RXR-gamma gene in these patients.

Association between the CCK-A receptor gene and panic disorder.

Cholecystokinin (CCK) is one of the most abundant neurotransmitter peptides in the brain. CCK appears to play an important role in the neurobiology of anxiety and panic disorders (PD) in both humans and animals. Recently, we reported that lack of CCKAR had a significant anxiogenic-like effect in rats. In this study, to investigate the role of CCKAR in PD, we compared the CCKAR gene in PD patients and normal controls. Subjects who fulfilled the DSM-IV criteria for PD were 17 males and 26 females. The sequence containing the Pst I polymorphic site in the boundary between intron 1 and exon 2 of the CCKAR gene was studied. Pst I digestion of the PCR products gave two individual alleles: A1 and A2. The A1 allele was the undigested fragment and the A2 allele was the digested one with two variant bands at 264 and 180 bp. Genotypic frequencies were 20.9% (A1-A1), 55.8% (A1-A2), and 41.7% (A2-A2) in patients, and 20.5% (A1-A1), 46.2% (A1-A2), and 33.3% (A2-A2) in controls. Allelic frequencies were 48.8% (A1) and 51.2% (A2) in patients, and 43.6% (A1) and 56.4% (A2) in controls. The chi-square test did not show a significant difference in either genotypic or allelic frequencies between patients and control subjects. The Pst polymorphism of CCKAR may not be associated with PD.

BRCA1 germline mutations in Indian familial breast cancer.

Germline mutation analysis of BRCA1 gene has demonstrated significant allelic heterogeneity. These differences represent historical influences of migration, population structure and geographic or cultural isolation. To date, there have been no reports of Indian families with mutations in BRCA1. We have screened for mutations in selected coding exons of BRCA1 and their flanking intron regions in three breast or breast and ovarian cancer families with family history of three or more cases of breast cancer under age 45 and/or ovarian cancer at any age. We have also analyzed 10 female patients with sporadic breast cancer regardless of age and family history, as well as 50 unrelated normal individuals as controls. Thus a total of 90 samples were analyzed for BRCA1 mutations using polymerase chain reaction-mediated site directed mutagenesis (PSM) and single stranded conformation polymorphism (SSCP) analysis for various selected exons followed by sequencing of variant bands. Eight point mutations were identified. Two deleterious pathogenic, protein truncating non-sense mutations were detected in exon 11 (E1250X) and exon 20 (E1754X) and six novel and unique amino acid substitutions (F1734S, D1739Y, V1741G, Q1747H, P1749A, R1753K). One complex missense mutation of exon 20 [V1741G; P1749A] was seen in two out of three families and another complex combination of missense and non-sense mutations of the same exon [V1741G; E1754X] was observed in only one family. These complex mutations exist only in breast cancer families but not in control populations of women. Three splice site variants (IVS20+3A>C, IVS20+4A>T, IVS20+5A>T) and two intronic variants (IVS20+21_22insG, IVS20+21T>G) were also detected. In the group of 10 sporadic female patients no mutations were found.

The prevalence of gastrointestinal symptoms in patients with amyotrophic lateral sclerosis

We report on an Austrian pedigree with autosomal dominant amyotrophic lateral sclerosis (ALS), diagnosed in six patients from two generations. The only surviving clinically affected family member was examined in our ALS clinic. Historical information on other affected individuals was obtained from knowledgeable family members. The mean ± S.D. age of onset of the disease was 54 ± 6.9 years, with a range of 43–66 years. The duration of the index patient's disease until death was 8 months. Using single strand conformational polymorphism (SSCP) analysis, we studied the index patient's exons 1,2 and 4 of the Cu/Zn superoxide dismutase gene (SOD1) on chromosome 21. A variant banding pattern was observed for exon 1. Sequencing studies showed a previously underscribed T to A missense mutation at position 8 in exon 1 of the SOD1 gene. This mutation results in the elimination of an Eco571 restriction site. Whereas the index patient was heterozygous for this restriction site, 50 unrelated healthy controls and an unaffected brother were not. The mutation lies in a region involved in dimer contact in the three-dimensional structure of the SOD1 protein. This region comprises other known sites for ALS-causing mutations.

Restriction endonuclease fingerprinting enhanced conformation sensitive gel electrophoresis (REF-CSGE) in the analysis of BRCA1 exon 11 mutations in a high-risk breast cancer cohort.

Efficient genetic analysis of large exonic regions containing heterozygous mutations and common polymorphisms can be difficult. We have analyzed 30 patients for inherited susceptibility mutations (ISM) within exon 11 of the BRCA1 gene as part of an ongoing genetic epidemiological study of high-risk breast cancer (HRBC). A novel combination of restriction endonuclease fingerprinting (REF) and conformation sensitive gel electrophoresis (CSGE) was developed for rapid and efficient screening of mutations. This method (REF-CSGE) was compared side-by-side with standard CSGE and evaluated for both efficiency and sensitivity of detection. REF-CSGE detected 100% of the alterations found by CSGE. However, one variant was only detectable by REF-CSGE. All samples with variant bands were sequenced to confirm the nature of the alteration. In total, two small deletions (frameshifts) and 62 point mutations (60 known polymorphisms and two variants of unknown significance) were found in our cohort. The majority of the exon 11 polymorphisms detected are inherited as a linked haplotype. Point mutations that comprise these haplotypes could be simultaneously detected on a single gel by REF-CSGE, thereby decreasing the number of sequencing reactions necessary to elucidate heteroduplex patterns seen on CSGE gels. An analysis of the overall efficiency of both techniques revealed that REF-CSGE required 67% fewer confirmatory sequencing reactions, resulting in savings in both reagents and technician time.

Mutational analysis of the Sonic Hedgehog gene in 220 newborns with oral clefts in a South American (ECLAMC) population.

Oral clefts generally have a multifactorial etiology. A number of genes contribute to the formation of the face and palate. Cleft lip and/or palate can occur in pedigrees with autosomal dominant holoprosencephaly due to mutations in Sonic Hedgehog (SHH). In addition, animal models have shown that SHH is involved in face development. We thus examined the human SHH gene in 220 newborn infants with nonsyndromic oral clefts registered by the Estudio Colaborativo Latinoamericano de Malformaciones Congenitas: ECLAMC (Latin American Collaborative Study of Congenital Malformations). We found 15 variant bands in 13 patients with oral clefts, representing five different base changes, all of which were found by sequencing to represent silent polymorphisms. Four occurred in introns. The alteration occurring in an exon, Ser190Ser, may create a consensus sequence for the 3'splice site 6 bp downstream of the original consensus sequence. Thus, we did not identify any clearly disease-causing mutation in SHH in these patients, and conclude that SHH mutations are not a frequent cause of isolated oral clefts in humans.

Report of 16 kindreds and one kindred with hMLH1 germline mutation.

To analyze the diagnosis and treatment of 16 hereditary nonpolyposis colorectal cancer (HNPCC) kindreds, and to report the first kindred with hMLH1 germline mutation in Mainland China. The diagnosis, treatment and follow-up study of 16 HNPCC kindreds were retrospectively reviewed. Data concerning site of the malignant tumor, age at the diagnosis, history of synchronous and/or metachronous cancer, and histopathology of tumors were recorded. All treatments had won formal consent. PCR and SSCP were used to screen the coding region of hMLH1 and hMSH2 genes. Variant bands were sequenced by a 377 DNA sequencer. Among sixteen kindreds, sixty-eight patients had a mean age of 50.8 years, including twenty-one multiple cancer patients and forty-six colorectal cancer patients (metachronous colorectal cancers in sixteen). A total of one hundred and one malignant neoplasms were found in these sixty-eight patients, including 50 colonic, 17 rectal, 11 gastric, 7 endometrial, and 4 esophageal cancers. 39.5% colorectal patients had metachronous cancers within ten years who needed reoperations. A germline G265T nonsense mutation was found in the third exon of hMLH1, resulting in a stop codon and truncated protein. Three phenotypically normal family members were also found to carry the mutated gene. HNPCC is a typical auto-dominant hereditary disease, the main characteristics include early onset and frequency of cancers; predominance of colorectal, especially right-sided colon cancers; frequency of multiple primary cancers (especially colorectal cancers). Segmental resection for colorectal cancers is not eligible for colorectal cancer patient in HNPCC kindreds. Intensive follow-up is essential for all patients and possible gene carriers. The first HNPCC kindred with hMLH1 gene germline mutation was identified in Mainland China, and three phenotypically normal family members were found to be carriers of the mutated gene. The G265T germline (nonsense) mutation in the third exon of hMLH1 found here had not been reported previously in the literature.

Comparative mapping in equids: the asine X chromosome is rearranged compared to horse and Hartmann’s mountain zebra

The X chromosomes of the extant equids, in general, share morphology and banding pattern similarities. However, the donkey X is, in part, an exception because of significantly different centromeric index and variant banding patterns in the pericentromeric region. To verify the underlying molecular basis of this difference, twelve equine BAC clones were FISH mapped to donkey (EAS) and Hartmann’s mountain zebra (EZH) metaphase spreads. Loci from the terminal region of Xp and distal to terminal regions of the Xq showed the same order and relative position in all three species, implying cross-species conservation of these chromosomal segments. However, loci from the proximal/pericentromeric regions of either arms showed similar FISH locations in horse and zebra but a slightly deviant location and relative position in the donkey. Three of the markers (tel–OTC, TRAP170 and ^psALDH2– cen) located on the short arm of ECAX and EZHX were found inverted on the long arm of EASX, along with the transposition of the centromere. This molecular evidence of a pericentromeric inversion helps define the likely evolutionary breakpoints causing the rearrangement. The breakpoints most likely correspond to the region between Xp16→q12 in the horse and Xp12→q13 in the donkey. The findings coupled with the highly conserved X-chromosome gene order between horse and outgroup species, human and cat, suggest that the equine type X is ancestral while the asine type X arose as a result of an independent inversion event. The study adds two new markers to horse, 11 to donkey and 12 to Hartmann’s zebra gene maps, thus contributing to the expansion of comparative maps in the equids.

Endoscopic Mucosal Resection of the Esophagus

Endoscopic mucosal resection (EMR) is a minimally invasive endoscopic technique used in treating superficial cancers of premalignant lesions in the gastrointestinal tract. An attraction of this technique is that it can be curative with low morbidity, often providing the entire lesion for pathologic evaluation. The band ligation technique of EMR uses the existing technology of variceal band ligation to endoscopically place a band on flat mucosal lesions of the gastrointestinal tract to create a "pseudopolyp" before resection with an electrocautery snare. The band ligating technique and two variant band ligating devices are described.

The chromosome 9q genes TGFBR1, TSC1, and ZNF189 are rarely mutated in bladder cancer.

This study assessed a series of bladder tumours and bladder tumour cell lines for sequence variation in the Krüppel-like zinc finger gene ZNF189, the tuberous sclerosis complex gene 1 (TSC1), and the TGF beta receptor type I (TGFBR1). All three genes have been mapped to 9q regions commonly deleted in transitional cell carcinoma of the bladder. Mutation analysis of the coding sequence of these genes revealed several variant bands that were shown to represent polymorphisms. Mutation analysis of the ZNF189 gene in bladder cancer cell lines identified one amino acid substitution (lysine-->isoleucine) at position 323 in exon 4. For the TSC1 gene, two mutations were identified in two out of 27 independent cell lines. Both mutations result in a truncated protein. Furthermore, one out of 36 bladder tumours had a frameshift mutation in exon 7 of the TSC1 gene. No tumour-specific mutations were found in the TGFBR1 gene. The length of the polyalanine tract present in exon 1 of the TGFBR1 gene was also investigated. It has been suggested that the allele with six alanines (6A) is more frequent in patients with bladder and other cancers, so bladder cancer patients were compared with normal controls. In both groups, the percentage of heterozygotes was 17%. These data do not support a role for the 6A allele in bladder cancer susceptibility.