Sensitive detection of human papillomavirus in cervical, head/neck, and schistosomiasis-associated bladder malignancies.

We assayed for the presence of human papilloma virus (HPV) DNA in serum and/or peripheral blood fraction (PBF) of individuals with cervical, head/neck, or bladder cancer due to schistosomiasis. Using mass spectroscopy coupled with competitive PCR, HPV DNA was detected at the individual molecule level by using "MassARRAY" assays. The resultant sensitivity was superior to real-time fluorescent PCR-based assays, while specificity was maintained. Our principal findings were: (i) Virtually all tested cervical cancers and schistosomiasis-associated bladder cancers, and a plurality of head/neck cancers, are associated with HPV DNA in the tumor. (ii) All 27 bladder cancers due to schistosomiasis were associated with the presence of HPV-16 DNA, which can be detected in tumor and serum but not in PBF. In contrast, no serum HPV-16 DNA signal was detected in seven individuals with schistosomiasis-associated bladder cancers after surgical removal of the tumor. (iii) Among the head/neck cancers we studied, anterior tumors were more often associated with HPV DNA in tumor, serum, and/or PBF than posterior tumors. (iv) In cervical cancer, where all tumors contain HPV DNA, viral DNA could be detected often in serum and/or PBF. Further, HPV-16 DNA was detected in serum and/or PBF of most patients with untreated high-grade cervical dysplasia but disappeared if the dysplasia was eliminated. The sensitive, specific, and quantitative MassARRAY technique should make it feasible to monitor cancer occurrence and treatment and recurrence of malignancies and dysplasias associated with HPV DNA.

Rap1, a small GTP-binding protein is upregulated during arrest of proliferation in human keratinocytes.

Rap1 is a small GTP-binding protein (SMG) that exists in two 95% homologous isoforms, rap1A and rap1B. The functions of the rap1 proteins are not well understood. In this report we examined expression and function of rap1 in primary (HOKs) and immortalized (IHOKs) human oral keratinocytes under different growth conditions. In HOKs, rap1 increased with passage number, suggesting a role in differentiation and arrest of proliferation. Similarly, when inhibition of proliferation and differentiation were induced in HOKs by 1.2 mM CaCl2, both rap1 and involucrin increased with increasing concentrations of CaCl2. However, when similar experiments were done with IHOKs, which continue to proliferate in the presence of 1.2 mM CaCl2, the increase in involucrin expression was similar to HOKs but there was no substantial increase in rap1, suggesting that increased expression of rap1 is linked to inhibition of proliferation rather than differentiation of keratinocytes. Upon transfection of immortalized keratinocytes with rapGAP, which inactivates both isoforms of endogenous active rap1, enhanced proliferation was observed. Thus, we conclude that rap1 inhibits proliferation in keratinocytes.

Matroshka and ectopic polymorphisms: Two new classes of DNA sequence variation identified at the Van der Woude syndrome locus on 1q32-q41.

Van der Woude syndrome (VWS) is an orofacial clefting disorder with an autosomal dominant pattern of inheritance. In our efforts to clone the VWS gene, 900 kb of genomic sequence from the VWS candidate region at chromosome 1q32-q41 was analyzed for new DNA sequence variants. We observed that in clone CTA-321i20 a 7922 bp sequence is absent relative to the sequence present in PAC clone RP4-782d21 at positions 1669-9590, suggesting the presence of a deletion/insertion (del/ins) polymorphism. Embedded in this 7922 bp region was a TTCC short tandem repeat (STR). Genotype analysis showed that both the internal STR and the (del/ins) mutation were true polymorphisms. This is a novel example of intraallelic variation, a polymorphism within a polymorphism, and we suggest that it be termed a "Matroshka" polymorphism. Further genetic and DNA sequence analysis indicated that the ancestral state of the 1669-9590 del/ins polymorphism was the insertion allele and that the original deletion mutation probably occurred only once. A second class of novel DNA sequence variation was discovered on chromosome 5 that shared a 328 bp identical sequence with this region on chromosome 1. A single nucleotide polymorphism (SNP) was detected by SSCP using a pair of primers derived from the chromosome 1 sequence. Surprisingly, these primers also amplified the identical locus on chromosome 5, and the SNP was only located on chromosome 5. Since the probe unexpectedly detected alleles from another locus, we suggest that this type of sequence variant be termed an "ectopic" polymorphism. These two novel classes of DNA sequence polymorphisms have the potential to confound genetic and DNA sequence analysis and may also contribute to variation in disease phenotypes.

Podocyte depletion and glomerulosclerosis have a direct relationship in the PAN-treated rat.

BACKGROUND: Podocytes are highly differentiated glomerular epithelial cells with limited potential to divide. They are responsible for maintaining and supporting the glomerular basement membrane so as to facilitate efficient filtration. The hypothesis tested was whether the development of glomerulosclerosis in the puromycin aminonucleoside (PAN)-treated rat could be attributed to podocyte depletion. METHODS: PAN was injected in Sprague-Dawley rats once, twice, or three times at 30-day intervals. Podocytes were counted in glomeruli using immunoperoxidase histochemistry and antibodies to both GLEPP1 (PTPRO) and WT-1. Podocytes were assayed in urine using reverse transcription-quantitative polymerase chain reaction (RT-QPCR). Glomerular areas were measured by computerized morphometry. RESULTS: In a preliminary experiment, a single injection of PAN caused a reduction in the glomerular podocyte count by 25%. Additional independent confirmation that podocytes were lost from glomeruli after PAN injection was obtained identifying detached podocytes in Bowman's space, measurement of nephrin and GLEPP1 mRNAs in urine, ultrastructural analysis of glomeruli, and identification of TUNEL-positive apoptotic podocytes in glomeruli. In a second experiment, sequential podocyte depletion by 15, 31, and 53% was achieved by the administration of one, two, or three injections of PAN at 30-day intervals. The region of the glomerulus devoid of podocytes developed glomerulosclerosis, and this area progressively increased as podocytes were progressively depleted. The correlation coefficient (r(2)) value for the relationship between percent podocyte depletion and glomerulosclerotic area was 0.99. The Y intercept of this plot showed that glomerulosclerosis was initiated when only 10 to 20% of podocytes were lost. CONCLUSION: This report supports the growing body of data linking glomerulosclerosis directly to a reduction in relative podocyte number [increased glomerular area per podocyte (GAPP)]. It raises important questions related to the mechanisms of podocyte loss, strategies for prevention of podocyte depletion, and the prevention of progression of glomerular diseases.

Distinctive molecular profiles of high-grade and low-grade gliomas based on oligonucleotide microarray analysis.

Astrocytomas are heterogeneous intracranial glial neoplasms ranging from the highly aggressive malignant glioblastoma multiforme (GBM) to the indolent, low-grade pilocytic astrocytoma. We have investigated whether DNA microarrays can identify gene expression differences between high-grade and low-grade glial tumors. We compared the transcriptional profile of 45 astrocytic tumors including 21 GBMs and 19 pilocytic astrocytomas using oligonucleotide-based microarrays. Of the approximately 6800 genes that were analyzed, a set of 360 genes provided a molecular signature that distinguished between GBMs and pilocytic astrocytomas. Many transcripts that were increased in GBM were not previously associated with gliomas and were found to encode proteins with properties that suggest their involvement in cell proliferation or cell migration. Microarray-based data for a subset of genes was validated using real-time quantitative reverse transcription-PCR. Immunohistochemical analysis also localized the protein products of specific genes of interest to the neoplastic cells of high-grade astrocytomas. Our study has identified a large number of novel genes with distinct expression patterns in high-grade and low-grade gliomas.

Down syndrome congenital heart disease: a narrowed region and a candidate gene.

PURPOSE: Down syndrome (DS) is a major cause of congenital heart disease (CHD) and the most frequent known cause of atrioventricular septal defects (AVSDs). Molecular studies of rare individuals with CHD and partial duplications of chromosome 21 established a candidate region that included D21S55 through the telomere. We now report human molecular and cardiac data that narrow the DS-CHD region, excluding two candidate regions, and propose DSCAM (Down syndrome cell adhesion molecule) as a candidate gene. METHODS: A panel of 19 individuals with partial trisomy 21 was evaluated using quantitative Southern blot dosage analysis and fluorescence in situ hybridization (FISH) with subsets of 32 BACs spanning the region defined by D21S16 (21q11.2) through the telomere. These BACs span the molecular markers D21S55, ERG, ETS2, MX1/2, collagen XVIII and collagen VI A1/A2. Fourteen individuals are duplicated for the candidate region, of whom eight (57%) have the characteristic spectrum of DS-CHD. RESULTS: Combining the results from these eight individuals suggests the candidate region for DS-CHD is demarcated by D21S3 (defined by ventricular septal defect), through PFKL (defined by tetralogy of Fallot). CONCLUSIONS: These data suggest that the presence of three copies of gene(s) from the region is sufficient for the production of subsets of DS-CHD. This region does not include genes located near D21S55, previously proposed as a "DS critical region," or the genes encoding collagens VI and XVIII. Of the potential gene candidates in the narrowed DS-CHD region, DSCAM is notable in that it encodes a cell adhesion molecule, spans more than 840 kb of the candidate region, and is expressed in the heart during cardiac development. Given these properties, we propose DSCAM as a candidate for DS-CHD.

The gene for the axonal cell adhesion molecule TAX-1 is amplified and aberrantly expressed in malignant gliomas.

The human TAX-1 gene encodes a Mr 135,000 glycoprotein that is transiently expressed on the surface of a subset of neurons during development and is involved in neurite outgrowth. The TAX-1 gene has been mapped to a region on chromosome 1 that has been implicated in microcephaly and the Van der Woude syndrome. Using restriction landmark genome scanning to search for amplified genes in gliomas, we found TAX-1 to be amplified in 2 high-grade gliomas among a group of 26 gliomas investigated. Real-time reverse transcription-quantitative PCR analysis detected high levels of TAX-1 mRNA in glial tumors, even in the absence of TAX-1 gene amplification. Immunohistochemical analysis revealed abundant levels of TAX-1 in neoplastic glial cells of glioblastoma multiforme tumors. Because glial tumors are highly invasive and in view of the role of TAX-1 in neurite outgrowth, we investigated the potential role of TAX-1 in glioma cell migration. Using an in vitro assay, we found that the migration of glioma tumor cells is profoundly reduced in the presence of either an anti-TAX-1 antibody or a TAX-1 antisense oligonucleotide. Our findings suggest that TAX-1 plays a role in glial tumorigenesis and may provide a potential target for therapeutic intervention.

Recombination-based assay (RBA) for screening bacteriophage lambda libraries.

The recombination-based assay represents a convenient way to screen a complex library constructed in bacteriophage lambda for homology to a given sequence cloned into a specially designed plasmid. The technique serves to screen a bacteriophage library rapidly and efficiently with a sequence cloned into a plasmid; counterselection then yields the gene product of interest with its plasmid carrier deleted. Because 10(6) to 10(7) plaque-forming units (pfu) may be screened using several petri dishes, and the homology for crossing-over need only be greater than 25 bp, the RBA represents an efficient way to screen complex lambda libraries rapidly for homology to a given sequence. In this procedure, a lambda library is screened using a specially designed plasmid carrying the desired target sequence. Recombinants arising from cross-over events between the plasmid and a bacteriophage carrying a corresponding region of homology are selected by their ability to grow on strain DM21. Growth of lambda on DM21 requires the presence of an allele encoded on the plasmid to suppress an amber mutation in the host strain that prevents lambda propagation. Recovery of the original phage carrying the target sequence requires a reversal of the homologous recombination event. This reversal occurs spontaneously, and is detected by PCR amplification using primers that flank the cloning site in the lambda vector.

Complementary signaling pathways regulate the unfolded protein response and are required for C. elegans development.

The unfolded protein response (UPR) is a transcriptional and translational intracellular signaling pathway activated by the accumulation of unfolded proteins in the lumen of the endoplasmic reticulum (ER). We have used C. elegans as a genetic model system to dissect UPR signaling in a multicellular organism. C. elegans requires ire-1-mediated splicing of xbp-1 mRNA for UPR gene transcription and survival upon ER stress. In addition, ire-1/xbp-1 acts with pek-1, a protein kinase that mediates translation attenuation, in complementary pathways that are essential for worm development and survival. We propose that UPR transcriptional activation by ire-1 as well as translational attenuation by pek-1 maintain ER homeostasis. The results demonstrate that the UPR and ER homeostasis are essential for metazoan development.

A fluorescent quantitative PCR approach to map gene deletions in the Drosophila genome.

We report the application of TaqMan quantitative PCR (QPCR) to map Drosophila chromosome deficiencies by discrimination of twofold copy number differences. For a model system, we used this technology to confirm the X chromosomal mapping of Dspt6 given the autosomal mapping of Dspt4. We then used this technique on both preexisting deletion mutant flies and flies that we generated with deletions to demonstrate the presence or absence of Dspt6, Dspt4, and swa in various deletion mutant flies. In contrast with in situ hybridization studies, QPCR both vitiates the need to do these more intricate studies, and it is more accurate as the site of deletion can be known down to the 10(2)-bp level. We then successfully applied the technique to the analysis of transcription, demonstrating that the amount of Dspt6 or Dspt4 transcriptional product depended directly on the dosage of the Dspt6 or Dspt4 gene, respectively. The rapidity and precision of this method demonstrates its applicability in Drosophila genetics, the rapid and accurate mapping of Drosophila deletion mutants.

Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukaemia.

Familial platelet disorder with predisposition to acute myelogenous leukaemia (FPD/AML, MIM 601399) is an autosomal dominant disorder characterized by qualitative and quantitative platelet defects, and propensity to develop acute myelogenous leukaemia (AML). Informative recombination events in 6 FPD/AML pedigrees with evidence of linkage to markers on chromosome 21q identified an 880-kb interval containing the disease gene. Mutational analysis of regional candidate genes showed nonsense mutations or intragenic deletion of one allele of the haematopoietic transcription factor CBFA2 (formerly AML1) that co-segregated with the disease in four FPD/AML pedigrees. We identified heterozygous CBFA2 missense mutations that co-segregated with the disease in the remaining two FPD/AML pedigrees at phylogenetically conserved amino acids R166 and R201, respectively. Analysis of bone marrow or peripheral blood cells from affected FPD/AML individuals showed a decrement in megakaryocyte colony formation, demonstrating that CBFA2 dosage affects megakaryopoiesis. Our findings support a model for FPD/AML in which haploinsufficiency of CBFA2 causes an autosomal dominant congenital platelet defect and predisposes to the acquisition of additional mutations that cause leukaemia.

Detection of erbB-2 amplifications in tumors and sera from esophageal carcinoma patients.

We used TaqMan PCR to detect quantitative anomalies of tumor markers in both tumor and serum DNA from esophageal cancer patients. We demonstrated the potential of this methodology by detecting erbB-2 amplifications in a plurality of esophageal tumor samples. These amplifications were corroborated by Southern blots. We then showed the potential of this methodology to detect quantitative anomalies of erbB-2 in serum DNA from individuals with a corresponding amplification in the tumor. The capability of TaqMan PCR to detect abnormalities in serum of esophageal cancer patients creates an opportunity to diagnose esophageal cancer and to monitor the outcome of treatment with a blood test.

Comparison of murine Supt4h and a nearly identical expressed, processed gene: evidence of sequence conservation through gene conversion extending into the untranslated regions.

We show herein the transcription of a processed gene that originated from a spliced transcript. Recently, we isolated the human and murine homologues of the yeast chromatin protein, SPT4. The Supt4h gene is spliced normally from five exons encoded by chromosome 11. Here we show that a related sequence on chromosome 10 encodes Supt4h2, a processed intronless gene (with a polyA tail and a tandemly-duplicated 13 bp insertion site in the genome) with a different 5' control region. Both the spliced gene, Supt4h , and the processed gene, Supt4h2 , are expressed in each of four tissues we examined. Supt4h2 encodes a 117 amino acid protein nearly identical to the Supt4h gene product with only one amino acid difference, indicating extreme conservation of this expressed processed gene with the spliced gene over evolutionary time. This illustrates another potential complexity of the mammalian genome, i.e. the use of a processed gene under the control of a different promoter region than the spliced gene.

A comparison between microsatellite and quantitative PCR analyses to detect frequent p16 copy number changes in primary bladder tumors.

We tested 70 primary bladder tumors for altered copy number of p16 (D9S1752) by microsatellite analysis and by a quantitative PCR (QPCR) assay. These two approaches were fully concordant for 53 tumors, including all 39 tumors in which microsatellite analysis detected loss. In addition, the QPCR method detected useful anomalies in 17 additional cases, including those in which D9S1752 was uninformative. QPCR was abnormal in 56 of 70 (80%) cases, whereas microsatellite analysis was abnormal in 39 of 70 (56%) cases. Although QPCR uses more DNA than microsatellite analysis, it represents a rapid, informative technique that can readily detect both chromosome 9p21 deletions and amplifications in primary bladder tumors without the need for electrophoretic separation.

Identification of a novel Ran binding protein 2 related gene (RANBP2L1) and detection of a gene cluster on human chromosome 2q11-q12.

The giant 358-kDa protein Ran binding protein 2 (RanBP2/Nup358) is localized at the cytoplasmic side of the nuclear pore complex and likely constitutes the Ran-GTP binding site at the cytoplasmic face of the complex. RanBP2/Nup358 furthermore acts as a chaperone for red/green opsin molecules. Here, we report on the physical mapping of human RanBP2 between markers D2S340 and D2S1893. A duplication of the 5'-end sequence of RanBP2 occurs within 3 Mb distal to RanBP2. Detailed sequence analysis resulted in primers specific for this distal duplication. Polymerase chain reaction-based screening of cDNA libraries indicates that this transcript, called RanBP2alpha (HGMW-approved symbol RANBP2L1), is expressed in several tissues. Screening of a fetal brain cDNA library yielded a 4057-bp partial cDNA clone for RanBP2alpha. Its 5'-end is almost identical to RanBP2, whereas its 3'-part is distinct from RanBP2. Northern blot analysis using a probe of the 3'-untranslated sequence of RanBP2alpha detected in several tissues an 8-kb transcript representing the full length of the transcript. In pancreas and placenta, an additional transcript of 14 kb was detected. PAC clones containing the bona fide RanBP2 sequences were localized to 2q11-q12 by FISH analysis, and a region of high similarity was detected on 2p11-p12. In summary, we have identified a RanBP2 gene cluster on 2q11-q12 together with a novel gene termed RanBP2alpha, with high sequence similarity to RanBP2.

Molecular cloning of the interleukin-1 gene cluster: construction of an integrated YAC/PAC contig and a partial transcriptional map in the region of chromosome 2q13.

Genes of the interleukin-1 (IL-1) gene cluster localized on chromosome 2q13 are implicated in many physiological and pathophysiological processes. We present here a high-resolution physical map of this region between markers D2S2008 and D2S4/PAX8. An integrated YAC/PAC contig and a partial transcriptional map were constructed by STS-constent mapping using the CEPH YAC library and three PAC libraries. A total of 3 YACs, 34 PACs, and 56 STSs were integrated: 33 newly generated probes to PAC end sequences, 9 polymorphic and 4 nonpolymorphic markers, 5 known genes, 4 expressed sequence tags, and 1 pseudogene. Within the map, a complete PAC contig of > 1 Mb encompasses the IL-1 gene cluster and PAX8, a paired-box-containing gene. This allowed us to define the transcriptional orientation of GLVR1, IL1B, and IL1RN and to show that PAX8 is localized outside the IL-1 gene cluster. FISH analysis localized PAC clones containing the IL-1 gene cluster to 2q12-q13. The data provide the basis for further characterization of the IL-1 gene cluster and for the construction of a sequence-ready PAC contig of this region.

Multiple independent molecular etiology for limb-girdle muscular dystrophy type 2A patients from various geographical origins.

Limb-girdle muscular dystrophies (LGMDs) are a group of neuromuscular diseases presenting great clinical heterogeneity. Mutations in CANP3, the gene encoding muscle-specific calpain, were used to identify this gene as the genetic site responsible for autosomal recessive LGMD type 2A (LGMD2A; MIM 253600). Analyses of the segregation of markers flanking the LGMD2A locus and a search for CANP3 mutations were performed for 21 LGMD2 pedigrees from various origins. In addition to the 16 mutations described previously, we report 19 novel mutations. These data indicate that muscular dystrophy caused by mutations in CANP3 are found in patients from all countries examined so far and further support the wide heterogeneity of molecular defects in this rare disease.

The murine Dyrk protein maps to chromosome 16, localizes to the nucleus, and can form multimers.

We mapped the murine copy of the Dyrk gene and examined its subcellular localization and self-interaction. We found that: (1) Dyrk maps to the distal portion of MMU chromosome 16, consistent with previous mapping of the human DYRK gene to the Down syndrome critical region on HSA 21q22.2. (2) The Dyrk protein localizes to the cell nucleus, affording the potential of controlling the expression of other gene(s). (3) The Dyrk protein can self-associate in a two-hybrid system, in accord with the presence of a leucine zipper motif noted in the original sequence. In particular, its expression pattern in frontal brain nuclei during murine embryogenesis, its subcellular localization and its ability to interact with other proteins all suggest that this protein remains a good candidate to mediate some of the pleiotropic effects of Down syndrome.