Sigve Nakken

A comprehensive assessment of somatic mutation detection in cancer using whole-genome sequencing

As whole-genome sequencing for cancer genome analysis becomes a clinical tool, a full understanding of the variables affecting sequencing analysis output is required. Here using tumour-normal sample pairs from two different types of cancer, chronic lymphocytic leukaemia and medulloblastoma, we conduct a benchmarking exercise within the context of the International Cancer Genome Consortium. We compare sequencing methods, analysis pipelines and validation methods. We show that using PCR-free methods and increasing sequencing depth to ∼100 × shows benefits, as long as the tumour:control coverage ratio remains balanced. We observe widely varying mutation call rates and low concordance among analysis pipelines, reflecting the artefact-prone nature of the raw data and lack of standards for dealing with the artefacts. However, we show that, using the benchmark mutation set we have created, many issues are in fact easy to remedy and have an immediate positive impact on mutation detection accuracy.

Cancer incidence and survival in Lynch syndrome patients receiving colonoscopic and gynaecological surveillance: first report from the prospective Lynch syndrome database.

Objective Estimates of cancer risk and the effects of surveillance in Lynch syndrome have been subject to bias, partly through reliance on retrospective studies. We sought to establish more robust estimates in patients undergoing prospective cancer surveillance. Design We undertook a multicentre study of patients carrying Lynch syndrome-associated mutations affecting MLH1, MSH2, MSH6 or PMS2. Standardised information on surveillance, cancers and outcomes were collated in an Oracle relational database and analysed by age, sex and mutated gene. Results 1942 mutation carriers without previous cancer had follow-up including colonoscopic surveillance for 13 782 observation years. 314 patients developed cancer, mostly colorectal (n=151), endometrial (n=72) and ovarian (n=19). Cancers were detected from 25 years onwards in MLH1 and MSH2 mutation carriers, and from about 40 years in MSH6 and PMS2 carriers. Among first cancer detected in each patient the colorectal cancer cumulative incidences at 70 years by gene were 46%, 35%, 20% and 10% for MLH1, MSH2, MSH6 and PMS2 mutation carriers, respectively. The equivalent cumulative incidences for endometrial cancer were 34%, 51%, 49% and 24%; and for ovarian cancer 11%, 15%, 0% and 0%. Ten-year crude survival was 87% after any cancer, 91% if the first cancer was colorectal, 98% if endometrial and 89% if ovarian. Conclusions The four Lynch syndrome-associated genes had different penetrance and expression. Colorectal cancer occurred frequently despite colonoscopic surveillance but resulted in few deaths. Using our data, a website has been established at http://LScarisk.org enabling calculation of cumulative cancer risks as an aid to genetic counselling in Lynch syndrome.

Variants of the genes encoding AQP4 and Kir4.1 are associated with subgroups of patients with temporal lobe epilepsy

OBJECTIVE The etiopathogenesis of temporal lobe epilepsy (TLE) and its subgroups - mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) and TLE with antecedent febrile seizures (TLE-FS) - is poorly understood. It has been proposed that the water channel aquaporin-4 (AQP4) and the potassium channel Kir4.1 (KCNJ10 gene) act in concert to regulate extracellular K(+) homeostasis and that functional alterations of these channels influence neuronal excitability. The current study was designed to identify variants of the AQP4 and KCNJ10 genes associated with TLE and subgroups of this condition. MATERIAL AND METHODS We included 218 Norwegian patients with TLE and 181 ethnically matched healthy controls. An association study was established in which all TLE patients were compared with healthy controls. Additionally, subgroups of 56 MTLE-HS patients were compared with 162 TLE patients without HS, and 102 TLE-FS patients were compared with 105 TLE without FS. RESULTS We found eight single SNPs, seven in KCNJ10 and one between KCNJ10 and KCNJ9, associated with TLE-FS (nominal p-values from 0.009 to 0.041). Seven of the SNPs segregate into one large haplotype block expanding from KCNJ10 to KCNJ9, including the region interposed those genes. One haplotype was overrepresented in the TLE-FS cases (nominal p-value 0.014). These results were confirmed by explorative multivariate analysis indicating that a combination of SNPs from KCNJ10, the region between KCNJ10 and KCNJ9, and the AQP4 gene is associated with TLE-FS. For the TLE cohort as a whole, explorative multivariate analysis indicated a combination of SNPs from the KCNJ10 and AQP4 genes in association with TLE. CONCLUSION Variations in the AQP4 and the KCNJ10/KCNJ9 region are likely to be associated with TLE, particularly TLE-FS, supporting the suggestion that perturbations of water and K(+) transport are involved in the etiopathogenesis of TLE.

Effects of intronic mutations in the LDLR gene on pre-mRNA splicing: Comparison of wet-lab and bioinformatics analyses

Screening for mutations in the low density lipoprotein receptor (LDLR) gene has identified more than 1000 mutations as the cause of familial hypercholesterolemia (FH). In addition, numerous intronic mutations with uncertain effects on pre-mRNA splicing have also been identified. In this study, we have selected 18 intronic mutations in the LDLR gene for comprehensive studies of their effects on pre-mRNA splicing. Epstein-Barr virus (EBV) transformed lymphocytes from subjects heterozygous for these mutations were established and mRNA was studied by Northern blot analyses and reverse transcription polymerase chain reactions. Furthermore, functional studies of the LDLRs were performed by flow cytometry. The results of the wet-lab analyses were compared to the predictions obtained from bioinformatics analyses using the programs MaxEntScan, NetGene2 and NNSplice 0.9, which are commonly used software packages for prediction of abnormal splice sites. Thirteen of the 18 intronic mutations were found to affect pre-mRNA splicing in a biologically relevant way as determined by wet-lab analyses. Skipping of one or two exons was observed for eight of the mutations, intron inclusion was observed for four of the mutations and activation of a cryptic splice site was observed for two of the mutations. Transcripts from eight of the mutant alleles were subjected to degradation. The computational analyses of the normal and mutant splice sites, predicted abnormal splicing with a sensitivity of 100% and a specificity of 60%. Thus, bioinformatics analyses are valuable tools as a first screening of the effects of intronic mutations in the LDLR gene on pre-mRNA splicing.

The disruptive positions in human G-quadruplex motifs are less polymorphic and more conserved than their neutral counterparts

Specific guanine-rich sequence motifs in the human genome have considerable potential to form four-stranded structures known as G-quadruplexes or G4 DNA. The enrichment of these motifs in key chromosomal regions has suggested a functional role for the G-quadruplex structure in genomic regulation. In this work, we have examined the spectrum of nucleotide substitutions in G4 motifs, and related this spectrum to G4 prevalence. Data collected from the large repository of human SNPs indicates that the core feature of G-quadruplex motifs, 5′-GGG-3′, exhibits specific mutational patterns that preserve the potential for G4 formation. In particular, we find a genome-wide pattern in which sites that disrupt the guanine triplets are more conserved and less polymorphic than their neutral counterparts. This also holds when considering non-CpG sites only. However, the low level of polymorphisms in guanine tracts is not only confined to G4 motifs. A complete mapping of DNA three-mers at guanine polymorphisms indicated that short guanine tracts are the most under-represented sequence context at polymorphic sites. Furthermore, we provide evidence for a strand bias upstream of human genes. Here, a significantly lower rate of G4-disruptive SNPs on the non-template strand supports a higher relative influence of G4 formation on this strand during transcription.

Computational prediction of the effects of non-synonymous single nucleotide polymorphisms in human DNA repair genes

Non-synonymous single nucleotide polymorphisms (nsSNPs) represent common genetic variation that alters encoded amino acids in proteins. All nsSNPs may potentially affect the structure or function of expressed proteins and could therefore have an impact on complex diseases. In an effort to evaluate the phenotypic effect of all known nsSNPs in human DNA repair genes, we have characterized each polymorphism in terms of different functional properties. The properties are computed based on amino acid characteristics (e.g. residue volume change); position-specific phylogenetic information from multiple sequence alignments and from prediction programs such as SIFT (Sorting Intolerant From Tolerant) and PolyPhen (Polymorphism Phenotyping). We provide a comprehensive, updated list of all validated nsSNPs from dbSNP (public database of human single nucleotide polymorphisms at National Center for Biotechnology Information, USA) located in human DNA repair genes. The list includes repair enzymes, genes associated with response to DNA damage as well as genes implicated with genetic instability or sensitivity to DNA damaging agents. Out of a total of 152 genes involved in DNA repair, 95 had validated nsSNPs in them. The fraction of nsSNPs that had high probability of being functionally significant was predicted to be 29.6% and 30.9%, by SIFT and PolyPhen respectively. The resulting list of annotated nsSNPs is available online (http://dna.uio.no/repairSNP), and is an ongoing project that will continue assessing the function of coding SNPs in human DNA repair genes.

ABCB4 sequence variations in young adults with cholesterol gallstone disease

Background and Aims: Mutations in the gene encoding the ABCB4 [adenosine triphosphate (ATP)‐binding cassette, sub‐family B (MDR/TAP), member 4] transporter lower phosphatidylcholine output into bile and contribute to cholesterol gallstone formation by decreasing the solubility of cholesterol in bile. Mutations in ABCB4 have been identified in patients with low phospholipid‐associated cholelithiasis. The aim of the present study was to determine the types and frequencies of ABCB4 mutations in cholecystectomized patients aged <40 years.

Incidence of and survival after subsequent cancers in carriers of pathogenic MMR variants with previous cancer: a report from the prospective Lynch syndrome database

Objective Today most patients with Lynch syndrome (LS) survive their first cancer. There is limited information on the incidences and outcome of subsequent cancers. The present study addresses three questions: (i) what is the cumulative incidence of a subsequent cancer; (ii) in which organs do subsequent cancers occur; and (iii) what is the survival following these cancers? Design Information was collated on prospectively organised surveillance and prospectively observed outcomes in patients with LS who had cancer prior to inclusion and analysed by age, gender and genetic variants. Results 1273 patients with LS from 10 countries were followed up for 7753 observation years. 318 patients (25.7%) developed 341 first subsequent cancers, including colorectal (n=147, 43%), upper GI, pancreas or bile duct (n=37, 11%) and urinary tract (n=32, 10%). The cumulative incidences for any subsequent cancer from age 40 to age 70 years were 73% for pathogenic MLH1 (path_MLH1), 76% for path_MSH2 carriers and 52% for path_MSH6 carriers, and for colorectal cancer (CRC) the cumulative incidences were 46%, 48% and 23%, respectively. Crude survival after any subsequent cancer was 82% (95% CI 76% to 87%) and 10-year crude survival after CRC was 91% (95% CI 83% to 95%). Conclusions Relative incidence of subsequent cancer compared with incidence of first cancer was slightly but insignificantly higher than cancer incidence in patients with LS without previous cancer (range 0.94–1.49). The favourable survival after subsequent cancers validated continued follow-up to prevent death from cancer. The interactive website http://lscarisk.org was expanded to calculate the risks by gender, genetic variant and age for subsequent cancer for any patient with LS with previous cancer.

Cancer risk and survival in path_MMR carriers by gene and gender up to 75 years of age: a report from the Prospective Lynch Syndrome Database

Background Most patients with path_MMR gene variants (Lynch syndrome (LS)) now survive both their first and subsequent cancers, resulting in a growing number of older patients with LS for whom limited information exists with respect to cancer risk and survival. Objective and design This observational, international, multicentre study aimed to determine prospectively observed incidences of cancers and survival in path_MMR carriers up to 75 years of age. Results 3119 patients were followed for a total of 24 475 years. Cumulative incidences at 75 years (risks) for colorectal cancer were 46%, 43% and 15% in path_MLH1, path_MSH2 and path_MSH6 carriers; for endometrial cancer 43%, 57% and 46%; for ovarian cancer 10%, 17% and 13%; for upper gastrointestinal (gastric, duodenal, bile duct or pancreatic) cancers 21%, 10% and 7%; for urinary tract cancers 8%, 25% and 11%; for prostate cancer 17%, 32% and 18%; and for brain tumours 1%, 5% and 1%, respectively. Ovarian cancer occurred mainly premenopausally. By contrast, upper gastrointestinal, urinary tract and prostate cancers occurred predominantly at older ages. Overall 5-year survival for prostate cancer was 100%, urinary bladder 93%, ureter 85%, duodenum 67%, stomach 61%, bile duct 29%, brain 22% and pancreas 0%. Path_PMS2 carriers had lower risk for cancer. Conclusion Carriers of different path_MMR variants exhibit distinct patterns of cancer risk and survival as they age. Risk estimates for counselling and planning of surveillance and treatment should be tailored to each patient’s age, gender and path_MMR variant. We have updated our open-access website www.lscarisk.org to facilitate this.

Is the brain water channel aquaporin‐4 a pathogenetic factor in idiopathic intracranial hypertension? Results from a combined clinical and genetic study in a Norwegian cohort

Purpose:  Idiopathic intracranial hypertension (IIH) is a condition of increased intracranial pressure of unknown aetiology. Patients with IIH usually suffer from headache and visual disturbances. High intracranial pressure despite normal ventricle size and negative MRI indicate perturbed water flux across cellular membranes, which is provided by the brain water channel aquaporin‐4 (AQP4). IIH could be associated with malfunctioning intracerebral water homeostasis and cerebrospinal fluid (CSF) reabsorption based on functional or regulatory alterations of AQP4.