Effect of Polypurine Reverse Hoogsteen Hairpins on Relevant Cancer Target Genes in Different Human Cell Lines.

We studied the ability of polypurine reverse Hoogsteen hairpins (PPRHs) to silence a variety of relevant cancer-related genes in several human cell lines. PPRHs are hairpins formed by two antiparallel polypurine strands bound by intramolecular Hoogsteen bonds linked by a pentathymidine loop. These hairpins are able to bind to their target DNA sequence through Watson-Crick bonds producing specific silencing of gene expression. We designed PPRHs against the following genes: BCL2, TOP1, mTOR, MDM2, and MYC and tested them for mRNA levels, cytotoxicity, and apoptosis in prostate, pancreas, colon, and breast cancer cell lines. Even though all PPRHs were effective, the most remarkable results were obtained with those against BCL2 and mammalian target of rapamycin (mTOR) in decreasing cell survival and mRNA levels and increasing apoptosis in prostate, colon, and pancreatic cancer cells. In the case of TOP1, MDM2, and MYC, their corresponding PPRHs produced a strong effect in decreasing cell viability and mRNA levels and increasing apoptosis in breast cancer cells. Thus, we confirm that the PPRH technology is broadly useful to silence the expression of cancer-related genes as demonstrated using target genes involved in metabolism (DHFR), proliferation (mTOR), DNA topology (TOP1), lifespan and senescence (telomerase), apoptosis (survivin, BCL2), transcription factors (MYC), and proto-oncogenes (MDM2).

Frequency and distribution of simple and compound microsatellites in forty-eight Human papillomavirus (HPV) genomes.

Simple sequence repeats (SSRs) are tandem-repeated sequences ubiquitously present but differentially distributed across genomes. Present study is a systematic analysis for incidence, composition and complexity of different microsatellites in 48 representative Human papillomavirus (HPV) genomes. The analysis revealed a total of 1868 SSRs and 120 cSSRs. However, four genomes (HPV-60, HPV-92, HPV-112 and HPV-136) lacked any cSSR content; while HPV-31 accounted for a maximum of 10 cSSRs. An overall increase in cSSR% with higher dMAX was observed. The SSRs and cSSRs were prevalent in coding regions. Poly(A/T) repeats were significantly more abundant than poly(G/C) repeats possibly due to high (A/T) content of the HPV genomes. Further, higher prevalence of di-nucleotide repeats over tri-nucleotide repeats may be attributed to instability of former because of higher slippage rate. An in-depth study of the satellite sequences would provide an insight into the imperfections and evolution of microsatellites.

Poly (AT) polymorphism in the XPC gene and smoking enhance the risk of prostate cancer in a low-risk Chinese population.

We investigated two polymorphisms of xeroderma pigmentosum complementary group C (XPC) in 202 subjects with prostate cancer (PCa) and 221 healthy controls in a Chinese Han population. Genotyping was performed using a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) technique. Our results indicated that smoking is associated with an increased risk for PCa (odds ratio [OR]: 1.51; 95% confidence interval [CI]: 1.02-2.22). Subjects carrying the XPC-PAT+/+ genotype exhibited a significantly increased risk for PCa (OR: 2.11; 95% CI: 1.12-3.99). The combined subjects with either the PAT+/+ or PAT+/- genotype also exhibited a 1.54-fold increased risk associated with PCa (OR: 1.54; 95% CI: 1.04-2.26). Moreover, smokers with PAT+/- or PAT+/+ had a higher risk for PCa (OR: 1.98; 95% CI: 1.08-3.64; P = 0.026 and OR: 3.56; 95% CI: 1.45-8.76; P = 0.004, respectively) compared with never smokers with the PAT-/- genotype. Analyses of the XPC Lys939Gln polymorphism did not show an association with PCa risk. Our findings support the hypothesis that XPC-PAT polymorphisms may contribute to the risk of developing PCa. More important, an elevated risk of PCa associated with a gene-environment (smoking) interaction was determined in a Chinese population.

Replication error deficient and proficient colorectal cancer gene expression differences caused by 3'UTR polyT sequence deletions.

Replication error deficient (RER+) colorectal cancers are a distinct subset of colorectal cancers, characterized by inactivation of the DNA mismatch repair system. These cancers are typically pseudodiploid, accumulate mutations in repetitive sequences as a result of their mismatch repair deficiency, and have distinct pathologies. Regulatory sequences controlling all aspects of mRNA processing, especially including message stability, are found in the 3'UTR sequence of most genes. The relevant sequences are typically A/U-rich elements or U repeats. Microarray analysis of 14 RER+ (deficient) and 16 RER- (proficient) colorectal cancer cell lines confirms a striking difference in expression profiles. Analysis of the incidence of mononucleotide repeat sequences in the 3'UTRs, 5'UTRs, and coding sequences of those genes most differentially expressed in RER+ versus RER- cell lines has shown that much of this differential expression can be explained by the occurrence of a massive enrichment of genes with 3'UTR T repeats longer than 11 base pairs in the most differentially expressed genes. This enrichment was confirmed by analysis of two published consensus sets of RER differentially expressed probesets for a large number of primary colorectal cancers. Sequence analysis of the 3'UTRs of a selection of the most differentially expressed genes shows that they all contain deletions in these repeats in all RER+ cell lines studied. These data strongly imply that deregulation of mRNA stability through accumulation of mutations in repetitive regulatory 3'UTR sequences underlies the striking difference in expression profiles between RER+ and RER- colorectal cancers.

Disease-causing mutations improving the branch site and polypyrimidine tract: pseudoexon activation of LINE-2 and antisense Alu lacking the poly(T)-tail.

Cryptic exons or pseudoexons are typically activated by point mutations that create GT or AG dinucleotides of new 5' or 3' splice sites in introns, often in repetitive elements. Here we describe two cases of tetrahydrobiopterin deficiency caused by mutations improving the branch point sequence and polypyrimidine tracts of repeat-containing pseudoexons in the PTS gene. In the first case, we demonstrate a novel pathway of antisense Alu exonization, resulting from an intronic deletion that removed the poly(T)-tail of antisense AluSq. The deletion brought a favorable branch point sequence within proximity of the pseudoexon 3' splice site and removed an upstream AG dinucleotide required for the 3' splice site repression on normal alleles. New Alu exons can thus arise in the absence of poly(T)-tails that facilitated inclusion of most transposed elements in mRNAs by serving as polypyrimidine tracts, highlighting extraordinary flexibility of Alu repeats in shaping intron-exon structure. In the other case, a PTS pseudoexon was activated by an A>T substitution 9 nt upstream of its 3' splice site in a LINE-2 sequence, providing the first example of a disease-causing exonization of the most ancient interspersed repeat. These observations expand the spectrum of mutational mechanisms that introduce repetitive sequences in mature transcripts and illustrate the importance of intronic mutations in alternative splicing and phenotypic variability of hereditary disorders.

Poly(T) variation within mitochondrial protein-coding genes in Globodera (Nematoda: Heteroderidae).

We sequenced a mitochondrial subgenome from the nematode Globodera rostochiensis, in two overlapping pieces. The subgenome was 9210 bp and contained four protein-coding genes (ND4, COIII, ND3, Cytb) and two tRNA genes (tRNA(Thr), tRNA(Gln)). Genome organization was similar to that of Globodera pallida, which is multipartite. Together with the small number of genes on this subgenome, this suggests that the mitochondrial genome of G. rostochiensis is also multipartite. In the initial clones sequenced, COIII and ND3 were full-length, while ND4 and Cytb were interrupted by premature stop codons and contained point indels that disrupted the reading frame. However, sequencing of multiple clones, from DNA extracted both from multiple individuals and from single cysts, revealed a predominant source of variation-in the length of polythymidine tracts. Comparison of our genomic sequences with ESTs similarly revealed variation in the length of polythymidine tracts. We subsequently sequenced both genomic DNA and mRNA from populations of G. pallida. In each case, variation in the length of polythymidine tracts was observed. The levels of expression of mitochondrial genes in G. pallida were representative of the subgenomes present: little evidence of differential expression was observed. These observations are consistent with the operation of posttranscriptional editing in Globodera mitochondria, although this is difficult to show conclusively in the presence of intraindividual gene sequence variation. Further, alternative explanations cannot be discounted; these include the operation of slippage during translation or that genomic copies of most genes are pseudogenes with a small proportion of full-length sequences able to maintain mitochondrial function.

Compound genetic abnormalities in patients with cystic fibrosis transmembrane regulator gene mutation.

OBJECTIVE: To determine the prevalence of compound genetic abnormalities in patients who are carriers of cystic fibrosis mutations. DESIGN: Case report. SETTING: Tertiary referral center for male infertility. PATIENT(S): Between 2000 and 2005, 65 patients were identified to be carriers of cystic fibrosis transmembrane regulator gene (CFTR) mutations or have a polymorphism of the polythymidine tract of intron 8. INTERVENTION(S): Patients were evaluated for male factor infertility. Additional genetic testing for karyotype abnormalities or Y chromosome microdeletions was performed when indicated because of evidence of impaired spermatogenesis during surgical sperm retrieval or on semen analysis. A comparison of similar patients is in the published literature. MAIN OUTCOME MEASURE(S): Characteristics of patients with compound genetic abnormalities presenting to an academic male fertility practice. Comparison to similar patients reported in the literature. RESULT(S): Two patients (3.1%) out of 65 were identified in our database to have compound genetic abnormalities. One patient had a W1282X mutation while the other had an I148T mutation. Both patients had deletions of AZF b + c regions. There were no karyotype abnormalities identified in our database. An additional two patients with compound CFTR mutations and Y chromosome microdeletions were identified in the literature. Three patients in the literature had compound CFTR mutations and karyotype abnormalities. CONCLUSION: Compound genetic abnormalities in CFTR mutation patients can be a contributing factor when abnormal spermatogenesis is encountered. A secondary genetic etiology should be considered in these types of patients.

Mutations in the MYB intron I regulatory sequence increase transcription in colon cancers.

Although MYB overexpression in colorectal cancer (CRC) is known to be a prognostic indicator for poor survival, the basis for this overexpression is unclear. Among multiple levels of MYB regulation, the most dynamic is the control of transcriptional elongation by sequences within intron 1. The authors have proposed that this regulatory sequence is transcribed into an RNA stem-loop and 19-residue polyuridine tract, and is subject to mutation in CRC. When this region was examined in colorectal and breast carcinoma cell lines and tissues, the authors found frequent mutations only in CRC. It was determined that these mutations allowed increased transcription compared with the wild type sequence. These data suggest that this MYB regulatory region within intron 1 is subject to mutations in CRC but not breast cancer, perhaps consistent with the mutagenic insult that occurs within the colon and not mammary tissue. In CRC, these mutations may contribute to MYB overexpression, highlighting the importance of noncoding sequences in the regulation of key cancer genes.

Identification of an 11T allele in the polypyrimidine tract of intron 8 of the CFTR gene.

PURPOSE: Most of the kits or reagents available for testing for mutations in the cystic fibrosis transmembrane conductance regulator gene include testing for the 5/7/9T polypyrimidine tract, but these methods only screen for three variants in this region: 5T, 7T, and 9T. Although such commercial products may not have been designed to screen for rare alleles of the polypyrimidine tract, we demonstrate that at least one of them (Tag-It Cystic Fibrosis Kit, Tm Bioscience, Toronto, Ontario, Canada) has enough sensitivity to differentiate samples with rare alleles by describing how this product allowed us to detect a previously uncharacterized 11T allele. METHODS: A total of 139 banked and anonymized clinical samples from carrier adults and children with cystic fibrosis (The Hospital for Sick Children, Toronto, Canada) were tested and analyzed using the Tag-It Cystic Fibrosis Kit. RESULTS: Two samples displayed allelic ratios for the polypyrimidine tract that were significantly different from the other samples and did not correspond to values expected to be seen for samples with 5T, 7T, or 9T alleles. Further tests with sequencing and an extended Tag-It assay confirmed the presence of an 11T allele. CONCLUSION: Although commercial products used in cystic fibrosis testing may not have been designed to screen for rare alleles of the polypyrimidine tract, we demonstrated that at least the Tag-It assay may have enough sensitivity to differentiate samples with such rare alleles, which can then be further analyzed for clarification.

Direct molecular haplotyping of the IVS-8 poly(TG) and polyT repeat tracts in the cystic fibrosis gene by melting curve analysis of hybridization probes.

BACKGROUND: Molecular haplotyping is a developing technology with great potential for use in clinical diagnostics. We describe a haplotyping method that uses PCR combined with hybridization probes. METHODS: We designed a LightCycler assay that uses fluorescence resonance energy transfer hybridization probes to haplotype the poly(TG) and polyT (TG-T) tract in the IVS-8 region of the CFTR gene. The reporter probe was designed as a perfect match to the TG12-5T allele. RESULTS: Analysis of 132 samples revealed 9 unique derivative melting temperatures (Tms); the lowest was 42.4 degrees C and the highest was 63.6 degrees C. The lowest Tms were in the TGn-9T group, the intermediate Tms in the TGn-7T group, and the highest Tms in the TGn-5T group. Haplotype frequencies were highest (39%) for TG11-7T and lowest (0.4%) for TG13-5T. CONCLUSIONS: Different combinations of polymorphisms under the reporter hybridization probe had unique and characteristic Tms. This property enables genotyping as well as determination of the phase of multiple variants under the probe, a principle we demonstrated by haplotyping the TG-T repeat tract in the IVS-8 region of the CFTR gene.

Variations of the CFTR gene in the Hanoi-Vietnamese.

In order to investigate polymorphic backgrounds of the cystic fibrosis transmembrane conductance regulator gene (CFTR) in the Vietnamese, we analyzed 495 blood samples of randomly selected healthy individuals in Hanoi for the delta F508 mutation and TG-repeats, poly-T, and M470V polymorphisms. We compared their distributions with those of Caucasians and other Asian populations. No delta F508 mutation was found, being consistent with the extremely low incidence of cystic fibrosis (CF) in Vietnam. Allele frequency of the T5 allele promoting exon 9 skipping was 0.037. Greater number of TG-repeats, which is known to facilitate this aberrant splicing, was a predominant trend in the Vietnamese and other Asians. A "T5-TG12-V470" haplotype was most common (29/37) among T5-bearing haplotypes. Three major haplotypes, T7-TG12-M470, T7-TG11-V470, and T7-TG12-V470, estimated by PHASE program, related to 92% of the population. This is the first study of the CFTR gene among the Vietnamese.

Cystic fibrosis prenatal screening in genetic counseling practice: recommendations of the National Society of Genetic Counselors.

For over a decade, prenatal screening for cystic fibrosis (CF) has been considered a model for the integration of genetic testing into routine medical practice. Data from pilot studies and public policy discourse have led to recommendations by some professional organizations that CF screening should be offered or made available to pregnant women and their partners, and to couples planning a pregnancy. It is crucial that genetic counselors gain thorough understanding of the complexities of CF and the implications of positive test results, so that they may serve as a reliable, educated referral base and resource for health care providers and their patients. While not all pregnant women will be referred for genetic counseling prior to CF carrier testing, genetic counselors often will be asked to counsel clients after they have a positive test result, or who are found to be at increased risk. Genetic counselors can play an important role in providing accurate and current information as well as support for patients' informed decisions. These recommendations were created by a multicenter working group of genetic counselors with expertise in CF and are based on personal clinical experience, review of pertinent English language medical articles, and reports of expert committees. The recommendations should not be construed as dictating an exclusive course of management, nor does the use of such recommendations guarantee a particular outcome. These recommendations do not displace a health care provider's professional judgment based on the clinical circumstances of a particular client.

Analysis of 3208 cystic fibrosis prenatal diagnoses: impact of carrier screening guidelines on distribution of indications for CFTR mutation and IVS-8 poly(T) analyses.

PURPOSE: To evaluate and quantify indications for CFTR mutation analysis of prenatal specimens, and to determine if a significant portion of tests are performed only for the identification of 5T alleles, we surveyed our laboratory data over a 3-year time period that spanned the issuance of the cystic fibrosis (CF) carrier screening guidelines. METHODS: Referral indications for 3208 prenatal specimens were compared for an 18-month period before (April 2000 to September 2001) and after (October 2001 to April 2003) publication of the ACMG/ACOG statement regarding prenatal and preconception testing for CF. RESULTS: The frequency of cases received for testing when one or both parents were CF mutation carriers did not change significantly after publication of the guidelines. The most frequent indication during the entire 3-year period was fetal ultrasound abnormality, yet in the post-ACMG/ACOG period the percentage decreased significantly due to an increase in the number of prenatal screening cases. Testing indications related to parental 5T status also increased significantly in the post-ACMG/ACOG period and accounted for 2.9% of testing over the 3-year period. A small subset (1.6%) of prenatal specimens were tested for poly(T) even though the parents did not carry 5T allele(s). However, more than 40% of these cases could be attributed to parental R117H mutations. CONCLUSION: These data indicate that although indications for prenatal testing shifted after the issuance of carrier screening guidelines, prenatal testing related to parental 5T alleles comprised < 3% of the total referral indications.

Germline mutations in CFTR and PSTI genes in chronic pancreatitis patients.

Mutations in the cationic trypsinogen, cystic fibrosis transmembrane conductance regulator (CFTR) and pancreatic secretory trypsinogen inhibitor (PSTI) genes have recently been associated with chronic pancreatitis. This paper investigates the frequency of CFTR and PSTI gene mutation in patients with idiopathic and alcoholic chronic pancreatitis, the clinical course of patients with these two kinds of disease, and examines the clinical differences between carriers and noncarriers of mutation. In idiopathic pancreatitis a significant increase was found in mutation frequency both in the CFTR gene (13%) and N34S mutation in the PSTI gene (3.9%), as well as an increase in familial disposition to pancreatic disorders. In alcohol-induced pancreatitis an increase in calcification, exocrine insufficiency, and diabetes mellitus was observed. In conclusions, mutations in the genes investigated are involved in causing idiopathic pancreatitis. Such mutations have no connection either with the age at onset or the clinical course of the disease.

DNA variants of BAT-25 in Japanese, a locus frequently used for analysis of microsatellite instability.

BAT-25 is a DNA marker, intragenic to the c-kit protooncogene, assigned to 4q12, containing a polythymine tract, mostly repeats of 25 poly(T) (T25). The BAT-25 locus is frequently used in the analysis of microsatellite instability (MSI) in cancer tissues. The number of poly(T) repeats at BAT-25 is reported to be quasi-monomorphic and this property permits the easy identification of the MSI status. We report DNA variants of BAT-25, in one patient with hereditary nonpolyposis colorectal cancer presenting T21 and T25 alleles and another carrying T18 and T25 alleles in the analysis of 100 normal Japanese donors. Observed allelic frequencies were 0.5% for both T21 and T18 alleles. So far, DNA variants of BAT-25 locus have been reported in African Americans with relatively high frequencies, but not in Japanese.

An intronic poly (AT) polymorphism of the DNA repair gene XPC and risk of squamous cell carcinoma of the head and neck: a case-control study.

Inherited polymorphisms of DNA repair genes may contribute to variations in DNA repair capacity and genetic susceptibility to cancer. In a hospital-based case-control study of 287 non-Hispanic white patients with newly diagnosed SCCHN and 311 control subjects matched on age, sex, ethnicity, and smoking status, we investigated the role of a newly identified variant allele of XPC, XPC-PAT+. We found that the frequency of the XPC-PAT+ allele was higher in the cases (0.409) than in the controls (0.333; P = 0.007). Fifty cases (17.4%) and 37 controls (11.9%) were XPC-PAT+/+, and 135 (47.0%) cases and 133 controls (42.8%) were XPC-PAT+/-. XPC-PAT+/- and XPC-PAT+/+ subjects were at significantly increased risk for SCCHN [adjusted odds ratios = 1.44 and 1.85, respectively (95% confidence intervals, 1.01-2.05 and 1.12-3.05, respectively; trend test, P = 0.007)]. We did not find ethnic difference in the frequency of XPC-PAT+ allele among four groups aged between 19 and 75 years: non-Hispanic whites, 294; African-Americans, 178; Hispanic-Americans, 103; and native Chinese, 119 (0.333, 0.281, 0.296, and 0.353, respectively). The case-control findings support the hypothesis that the XPC-PAT+ allele may contribute to the risk of developing SCCHN.

Genomic structure of human alpha-pix, and variable deletions in a poly (T) tract in gastric cancer tissue.

PAK-interacting exchange factor (PIX) has been reported to mediate the recruitment of PAK into focal adhesions and activate Rac, thus creating a feedback loop that stimulate PAK and other targets. This pathway is thought to be related to cellular changes, such as transformation and migration, that are often encountered in cancer cells. Here, we report the genomic structure of alpha-PIX, one of the PAK- interacting exchange factors, including the identification of the promoter region, which consisted 772 amino acids in 22 exons, spanning about 100 kb on genome of X chromosome. All splice sites conformed to the GT-AT rule. To investigate the role of alpha-PIX in carcinogenesis, we screened 60 cases of gastric cancer for mutations and polymorphisms using an intron-primer that covered all the exons, but no mutations or polymorphisms were found in the coding region. However an 18 bp repeat of thymidine tract was present in 50 bp downstream from exon 12 and the deletion of variable numbers of mononucleotide repeats was observed in seven out of the 60 gastric cancer tissue specimens that were examined. These seven cases all exhibited a mutator phenotype, suggesting that the deletions are passenger mutations. Thus our results revealed that alpha-PIX probably does not play any primary role in human gastric carcinogenesis.