Survival outcome differences based on treatments used and knowledge of the primary tumour site for patients with cancer of unknown and known primary in Ontario.

Introduction: Patients with cancer of unknown primary (cup) have pathologically confirmed metastatic tumours with unidentifiable primary tumours. Currently, very little is known about the relationship between the treatment of patients with cup and their survival outcomes. Thus, we compared oncologic treatment and survival outcomes for patients in Ontario with cup against those for a cohort of patients with metastatic cancer of known primary site. Methods: Using the Ontario Cancer Registry and the Same-Day Surgery and Discharge Abstract databases maintained by the Canadian Institute for Health Information, we identified all Ontario patients diagnosed with metastatic cancer between 1 January 2000 and 31 December 2005. Ontario Health Insurance Plan treatment records were linked to identify codes for surgery, chemotherapy, or therapeutic radiation related to oncology. Multivariable Cox regression models were constructed, adjusting for histology, age, sex, and comorbidities. Results: In 45,347 patients (96.3%), the primary tumour site was identifiable, and in 1743 patients (3.7%), cup was diagnosed. Among the main tumour sites, cup ranked as the 6th largest. The mean Charlson score was significantly higher (p < 0.0001) in patients with cup (1.88) than in those with a known primary (1.42). Overall median survival was 1.9 months for patients with cup compared with 11.9 months for all patients with a known-primary cancer. Receipt of treatment was more likely for patients with a known primary site (n= 35,012, 77.2%) than for those with cup (n = 891, 51.1%). Among patients with a known primary site, median survival was significantly higher for treated than for untreated patients (19.0 months vs. 2.2 months, p < 0.0001). Among patients with cup, median survival was also higher for treated than for untreated patients (3.6 months vs. 1.1 months, p < 0.0001). Conclusions: In Ontario, patients with cup experience significantly lower survival than do patients with metastatic cancer of a known primary site. Treatment is associated with significantly increased survival both for patients with cup and for those with metastatic cancer of a known primary site.

Assessment of the functional impact of germline BRCA1/2 variants located in non-coding regions in families with breast and/or ovarian cancer predisposition.

PURPOSE: The molecular mechanism of breast and/or ovarian cancer susceptibility remains unclear in the majority of patients. While germline mutations in the regulatory non-coding regions of BRCA1 and BRCA2 genes have been described, screening has generally been limited to coding regions. The aim of this study was to evaluate the contribution of BRCA1/2 non-coding variants. METHODS: Four BRCA1/2 non-coding regions were screened using high-resolution melting analysis/Sanger sequencing or next-generation sequencing on DNA extracted from index cases with breast and ovarian cancer predisposition (3926 for BRCA1 and 3910 for BRCA2). The impact of a set of variants on BRCA1/2 gene regulation was evaluated by site-directed mutagenesis, transfection, followed by Luciferase gene reporter assay. RESULTS: We identified a total of 117 variants and tested twelve BRCA1 and 8 BRCA2 variants mapping to promoter and intronic regions. We highlighted two neighboring BRCA1 promoter variants (c.-130del; c.-125C > T) and one BRCA2 promoter variants (c.-296C > T) inhibiting significantly the promoter activity. In the functional assays, a regulating region within the intron 12 was found with the same enhancing impact as within the intron 2. Furthermore, the variants c.81-3980A > G and c.4186-2022C > T suppress the positive effect of the introns 2 and 12, respectively, on the BRCA1 promoter activity. We also found some variants inducing the promoter activities. CONCLUSION: In this study, we highlighted some variants among many, modulating negatively the promoter activity of BRCA1 or 2 and thus having a potential impact on the risk of developing cancer. This selection makes it possible to conduct future validation studies on a limited number of variants.

The clinical significance of occult gynecologic primary tumours in metastatic cancer.

OBJECTIVE: We estimated the frequency of occult gynecologic primary tumours (gpts) in patients with metastatic cancer from an uncertain primary and evaluated the effect on disease management and overall survival (os). METHODS: We used Manitoba administrative health databases to identify all patients initially diagnosed with metastatic cancer during 2002-2011. We defined patients as having an "occult" primary tumour if the primary was classified at least 6 months after the initial diagnosis. Otherwise, we considered patients to have "obvious" primaries. We then compared clinicopathologic and treatment characteristics and 2-year os for women with occult and with obvious gpts. We used Cox regression adjustment and propensity score methods to assess the effect on os of having an occult gpt. RESULTS: Among the 5953 patients diagnosed with metastatic cancer, occult primary tumours were more common in women (n = 285 of 2552, 11.2%) than in men (n = 244 of 3401, 7.2%). In women, gpts were the most frequent occult primary tumours (n = 55 of 285, 19.3%). Compared with their counterparts having obvious gpts, women with occult gpts (n = 55) presented with similar histologic and metastatic patterns but received fewer gynecologic diagnostic examinations during diagnostic work-up. Women with occult gpts were less likely to undergo surgery, waited longer for radiotherapy, and received a lesser variety of chemotherapeutic agents. Having an occult compared with an obvious gpt was associated with decreased os (hazard ratio: 1.62; 95% confidence interval: 1.2 to 2.35). Similar results were observed in adjusted analyses. CONCLUSIONS: In women with metastatic cancer from an uncertain primary, gpts constitute the largest clinical entity. Accurate diagnosis of occult gpts early in the course of metastatic cancer might lead to more effective treatment decisions and improved survival outcomes.

Relating centromeric topography in fixed human chromosomes to α-satellite DNA and CENP-B distribution.

Despite extensive analyses on the centromere and its associated proteins, detailed studies of centromeric DNA structure have provided limited information about its topography in condensed chromatin. We have developed a method with correlative fluorescence light microscopy and atomic force microscopy that investigates the physical and structural organization of α-satellite DNA sequences in the context of its associated protein, CENP-B, on human metaphase chromosome topography. Comparison of centromeric DNA and protein distribution patterns in fixed homologous chromosomes indicates that CENP-B and α-satellite DNA are distributed distinctly from one another and relative to observed centromeric ridge topography. Our approach facilitates correlated studies of multiple chromatin components comprising higher-order structures of human metaphase chromosomes.

Predicting severity of haemophilia A and B splicing mutations by information analysis.

Bleeding symptoms and clotting activity vary among mutations that alter mRNA splicing of either the factor VIII or factor IX genes. We analyzed splicing mutations in both genes for changes in individual information (R(i), in bits) involving both donor or acceptor sites. Mutations with low or negative R(i) values (<2.4 bits) or significant changes in R(i) (DeltaR(i) > or = 7 bits) exhibited either reduced protein activity, increased clotting time and bleeding frequency and were predictive of severe disease. Thus, information analysis of splicing mutations may be useful in predicting phenotypes in hemophilia.

Identification and characterization of novel sequence variations in the cytochrome P4502D6 (CYP2D6) gene in African Americans.

Cytochrome P4502D6 (CYP2D6) genotyping reliably predicts poor metabolizer phenotype in Caucasians, but is less accurate in African Americans. To evaluate discordance we have observed in phenotype to genotype correlation studies, select African American subjects were chosen for complete resequencing of the CYP2D6 gene including 4.2 kb of the CYP2D7-2D6 intergenic region. Comparisons were made to a CYP2D6(*)1 reference sequence revealing novel SNPs in the upstream, coding and intervening sequences. These sequence variations, defining four functional alleles (CYP2D6(*)41B, (*)45A and B and (*)46), were characterized for their ability to influence splice site strength, transcription level or catalytic protein activity. Furthermore, their frequency was determined in a population of 251 African Americans. A -692(TGTG) deletion (CYP2D6(*)45B) did not significantly decrease gene expression, nor could any other upstream SNP explain a genotype-discordant case. CYP2D6(*)45 and (*)46 have a combined frequency of 4% and can be identified by a common SNP. Carriers are predicted to exhibit an extensive or intermediate CYP2D6 phenotype.

Sequence-based design of single-copy genomic DNA probes for fluorescence in situ hybridization.

Chromosomal rearrangements are frequently monitored by fluorescence in situ hybridization (FISH) using large, recombinant DNA probes consisting of contiguous genomic intervals that are often distant from disease loci. We developed smaller, targeted, single-copy probes directly from the human genome sequence. These single-copy FISH (scFISH) probes were designed by computational sequence analysis of approximately 100-kb genomic sequences. ScFISH probes are produced by long PCR, then purified, labeled, and hybridized individually or in combination to human chromosomes. Preannealing or blocking with unlabeled, repetitive DNA is unnecessary, as scFISH probes lack repetitive DNA sequences. The hybridization results are analogous to conventional FISH, except that shorter probes can be readily visualized. Combinations of probes from the same region gave single hybridization signals on metaphase chromosomes. ScFISH probes are produced directly from genomic DNA, and thus more quickly than by recombinant DNA techniques. We developed single-copy probes for three chromosomal regions-the CDC2L1 (chromosome 1p36), MAGEL2 (chromosome 15q11.2), and HIRA (chromosome 22q11.2) genes-and show their utility for FISH. The smallest probe tested was 2290 bp in length. To assess the potential utility of scFISH for high-resolution analysis, we determined chromosomal distributions of such probes. Single-copy intervals of this length or greater are separated by an average of 29.2 and 22.3 kb on chromosomes 21 and 22, respectively. This indicates that abnormalities seen on metaphase chromosomes could be characterized with scFISH probes at a resolution greater than previously possible.

[Splice site mutations and atherosclerosis: mechanisms and prediction models]. / Spleissstellenmutationen und Atherosklerose: Mechanismen und Modelle der Prädiktion.

Nucleotide variants in genes of the lipid metabolism influence the risk of premature atherosclerosis. Ten percent of all single nucleotide substitutions in these genes involve splice sites. The effects of these changes on mRNA splicing and phenotypic severity, however, are not inherently obvious from the nucleotide sequence. This review presents various genes of lipid metabolism with splicing mutations known to influence the risk of premature atherosclerosis. Mechanisms of pre-mRNA splicing are illustrated and different models for prediction of the effect of nucleotide substitutions on splice-site function are presented. The role of information theory-based models is emphasized along with its role for prediction of splice-site function and phenotypic severity of atherosclerosis.

Exon skipping in IVD RNA processing in isovaleric acidemia caused by point mutations in the coding region of the IVD gene.

Isovaleric acidemia (IVA) is a recessive disorder caused by a deficiency of isovaleryl-CoA dehydrogenase (IVD). We have reported elsewhere nine point mutations in the IVD gene in fibroblasts of patients with IVA, which lead to abnormalities in IVD protein processing and activity. In this report, we describe eight IVD gene mutations identified in seven IVA patients that result in abnormal splicing of IVD RNA. Four mutations in the coding region lead to aberrantly spliced mRNA species in patient fibroblasts. Three of these are amino acid altering point mutations, whereas one is a single-base insertion that leads to a shift in the reading frame of the mRNA. Two of the coding mutations strengthen pre-existing cryptic splice acceptors adjacent to the natural splice junctions and apparently interfere with exon recognition, resulting in exon skipping. This mechanism for missplicing has not been reported elsewhere. Four other mutations alter either the conserved gt or ag dinucleotide splice sites in the IVD gene. Exon skipping and cryptic splicing were confirmed by transfection of these mutations into a Cos-7 cell line model splicing system. Several of the mutations were predicted by individual information analysis to inactivate or significantly weaken adjacent donor or acceptor sites. The high frequency of splicing mutations identified in these patients is unusual, as is the finding of missplicing associated with missense mutations in exons. These results may lead to a better understanding of the phenotypic complexity of IVA, as well as provide insight into those factors important in defining intron/exon boundaries in vivo.

Maternal uniparental disomy of chromosome 14 confined to an interstitial segment (14q23-14q24.2).

Maternal uniparental disomy for the complete long arm of chromosome 14 has been reported in 14 patients to date and is associated with a specific pattern of malformation. We report a child with clinical features of this syndrome who exhibits maternal uniparental disomy confined to a specific interstitial segment of chromosome 14.

Splice-site mutations in atherosclerosis candidate genes: relating individual information to phenotype.

BACKGROUND: Nucleotide variants in several genes for lipid and methionine metabolism influence the risk of premature atherosclerosis. Ten percent of single nucleotide substitutions in these genes involve mRNA splice sites. The effects of some of these changes on splicing and on phenotypic severity are not inherently obvious. METHODS AND RESULTS: Using an information theory-based model, we measured the individual information content (R(i), in bits) of splice sites adjacent to 289 mutations (including 31 splice-site mutations) in the atherosclerosis candidate genes APOAII, APOB, APOCII, APOE, CBS, CETP, LCAT, LIPA, LDLR, and LPL. The predictions of information analysis were then corroborated by published mRNA analyses. The R(i) values of mutant sites were consistent with either complete (n=17) or partial (n=8) inactivation of these sites. Seven mutations were predicted to activate cryptic splice sites. Predicted inactive mutant sites were associated with either "average" or "severe" dyslipidemia and commensurate reductions in protein levels or activity, whereas mutations expected to exhibit residual splicing had average or "mild" effects on lipid and protein expression. CONCLUSIONS: Information analysis of splice-junction variants in atherosclerosis candidate genes distinguishes inactive from leaky splice sites and identifies activated cryptic sites. Predicted changes in splicing were related to phenotypic severity.

Chromosome breakage in the Prader-Willi and Angelman syndromes involves recombination between large, transcribed repeats at proximal and distal breakpoints.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct neurobehavioral disorders that most often arise from a 4-Mb deletion of chromosome 15q11-q13 during paternal or maternal gametogenesis, respectively. At a de novo frequency of approximately.67-1/10,000 births, these deletions represent a common structural chromosome change in the human genome. To elucidate the mechanism underlying these events, we characterized the regions that contain two proximal breakpoint clusters and a distal cluster. Novel DNA sequences potentially associated with the breakpoints were positionally cloned from YACs within or near these regions. Analyses of rodent-human somatic-cell hybrids, YAC contigs, and FISH of normal or rearranged chromosomes 15 identified duplicated sequences (the END repeats) at or near the breakpoints. The END-repeat units are derived from large genomic duplications of a novel gene (HERC2), many copies of which are transcriptionally active in germline tissues. One of five PWS/AS patients analyzed to date has an identifiable, rearranged HERC2 transcript derived from the deletion event. We postulate that the END repeats flanking 15q11-q13 mediate homologous recombination resulting in deletion. Furthermore, we propose that active transcription of these repeats in male and female germ cells may facilitate the homologous recombination process.

Maternal uniparental disomy of chromosome 21 in a normal child.

Maternal uniparental disomy of chromosome 21 [upd(21)mat] was found previously in a normal female and in 2 cases of early embryonic failure. We present a phenotypically normal child with upd(21)mat due to a de novo der(21;21)(q10;10). This finding suggests that chromosome 21 is not imprinted in the maternal germline.

Imprinting-mutation mechanisms in Prader-Willi syndrome.

Microdeletions of a region termed the "imprinting center" (IC) in chromosome 15q11-q13 have been identified in several families with Prader-Willi syndrome (PWS) or Angelman syndrome who show epigenetic inheritance for this region that is consistent with a mutation in the imprinting process. The IC controls resetting of parental imprints in 15q11-q13 during gametogenesis. We have identified a larger series of cases of familial PWS, including one case with a deletion of only 7.5 kb, that narrows the PWS critical region to <4. 3 kb spanning the SNRPN gene CpG island and exon 1. Identification of a strong DNase I hypersensitive site, specific for the paternal allele, and six evolutionarily conserved (human-mouse) sequences that are potential transcription-factor binding sites is consistent with this region defining the SNRPN gene promoter. These findings suggest that promoter elements at SNRPN play a key role in the initiation of imprint switching during spermatogenesis. We also identified three patients with sporadic PWS who have an imprinting mutation (IM) and no detectable mutation in the IC. An inherited 15q11-q13 mutation or a trans-factor gene mutation are unlikely; thus, the disease in these patients may arise from a developmental or stochastic failure to switch the maternal-to-paternal imprint during parental spermatogenesis. These studies allow a better understanding of a novel mechanism of human disease, since the epigenetic effect of an IM in the parental germ line determines the phenotypic effect in the patient.

Mutations that alter RNA splicing of the human HPRT gene: a review of the spectrum.

The human HPRT gene contains spans approximately 42,000 base pairs in genomic DNA, has a mRNA of approximately 900 bases and a protein coding sequence of 657 bases (initiation codon AUG to termination codon UAA). This coding sequence is distributed into 9 exons ranging from 18 (exon 5) to 184 (exon 3) base pairs. Intron sizes range from 170 (intron 7) to 13,075 (intron 1) base pairs. In a database of human HPRT mutations, 277 of 2224 (12.5%) mutations result in alterations in splicing of the mRNA as analyzed by both reverse transcriptase mediated production of a cDNA followed by PCR amplification and cDNA sequencing and by genomic DNA PCR amplification and sequencing. Mutations have been found in all eight 5' (donor) and 3' (acceptor) splice sequences. Mutations in the 5' splice sequences of introns 1 and 5 result in intron inclusion in the cDNA due to the use of cryptic donor splice sequences within the introns; mutations in the other six 5' sites result in simple exon exclusion. Mutations in the 3' splice sequences of introns 1, 3, 7 and 8 result in partial exon exclusion due to the use of cryptic acceptor splice sequences within the exons; mutations in the other four 3' sites result in simple exon exclusion. A base substitution in exon 3 (209G-->T) creates a new 5' (donor) splice site which results in the exclusion of 110 bases of exon 3 from the cDNA. Two base substitutions in intron 8 (IVS8-16G-->A and IVS8-3T-->G) result in the inclusion of intron 8 sequences in the cDNA due to the creation of new 3' (acceptor) splice sites. Base substitution within exons 1, 3, 4, 6 and 8 also result in splice alterations in cDNA. Those in exons 1 and 6 are at the 3' end of the exon and may directly affect splicing. Those within exons 3 and 4 may be the result of the creation of nonsense codons, while those in exon 8 cannot be explained by this mechanism. Lastly, many mutations that affect splicing of the HPRT mRNA have pleiotropic effects in that multiple cDNA products are found.

Autosomal dominant zonular cataract with sutural opacities is associated with a splice mutation in the betaA3/A1-crystallin gene.

PURPOSE: Congenital cataracts constitute a morphologically and genetically heterogeneous group of diseases that are a major cause of childhood blindness. Autosomal Dominant Zonular Cataracts with Sutural Opacities (CCZS) have been mapped to chromosome 17q11-q12 near the betaA3A1-crystallin gene (CRYBA1). The betaA3A1-crystallin gene was investigated as the causative gene for the cataracts. METHODS: The betaA3/A1-crystallin gene was sequenced in affected and control individuals. Base changes were confirmed and assayed in additional family members and controls using NlaIII restriction digestion of PCR amplified DNA sequences. Base changes were assessed for their effects on splicing by information analysis. RESULTS: The cataracts are associated with a sequence change in the 5' (donor) splice site of intron 3: GC(g->a)tgagt. The sequence change also creates a new NlaIII site. This base change cosegregates with the cataracts in this family, being present in every affected individual. Conversely, this base change was not seen in 140 chromosomes examined in 70 unaffected and unrelated individuals. Information theory mutational analysis shows that the base change lowers the information content of the splice site from 6.0 to -6.8 bits, so that splicing would not be expected to occur at the altered site. CONCLUSIONS: Taken together, these observations suggest that the observed mutation might be causally related to the cataracts in this family.

Information analysis of human splice site mutations.

Splice site nucleotide substitutions can be analyzed by comparing the individual information contents (Ri, bits) of the normal and variant splice junction sequences [Rogan and Schneider, 1995]. In the present study, we related splicing abnormalities to changes in Ri values of 111 previously reported splice site substitutions in 41 different genes. Mutant donor and acceptor sites have significantly less information than their normal counterparts. With one possible exception, primary mutant sites with <2.4 bits were not spliced. Sites with Ri values > or = 2.4 bits but less than the corresponding natural site usually decreased, but did not abolish splicing. Substitutions that produced small changes in Ri probably do not impair splicing and are often polymorphisms. The Ri values of activated cryptic sites were generally comparable to or greater than those of the corresponding natural splice sites. Information analysis revealed preexisting cryptic splice junctions that are used instead of the mutated natural site. Other cryptic sites were created or strengthened by sequence changes that simultaneously altered the natural site. Comparison between normal and mutant splice site Ri values distinguishes substitutions that impair splicing from those which do not, distinguishes null alleles from those that are partially functional, and detects activated cryptic splice sites.

Organization of the ABCR gene: analysis of promoter and splice junction sequences.

Mutations in the human ABCR gene have been associated with the autosomal recessive Stargardt disease (STGD), retinitis pigmentosa (RP19), and cone-rod dystrophy (CRD) and have also been found in a fraction of age-related macular degeneration (AMD) patients. The ABCR gene is a member of the ATP-binding cassette (ABC) transporter superfamily and encodes a rod photoreceptor-specific membrane protein. The cytogenetic location of the ABCR gene was refined to 1p22.3-1p22.2. The intron/exon structure was determined for the ABCR gene from overlapping genomic clones. ABCR spans over 100kb and comprises 50 exons. Intron/exon splice site sequences are presented for all exons and analyzed for information content (Ri). Nine splice site sequence variants found in STGD and AMD patients are evaluated as potential mutations. The localization of splice sites reveals a high degree of conservation between other members of the ABC1 subfamily, e.g. the mouse Abc1 gene. Analysis of the 870-bp 5' upstream of the transcription start sequence reveals multiple putative photoreceptor-specific regulatory elements including a novel retina-specific transcription factor binding site. These results will be useful in further mutational screening of the ABCR gene in various retinopathies and for determining the substrate and/or function of this photoreceptor-specific ABC transporter.