Øystein Ariansen Haaland

Genotyping errors in a calibrated DNA register: implications for identification of individuals

BackgroundThe use of DNA methods for the identification and management of natural resources is gaining importance. In the future, it is likely that DNA registers will play an increasing role in this development. Microsatellite markers have been the primary tool in ecological, medical and forensic genetics for the past two decades. However, these markers are characterized by genotyping errors, and display challenges with calibration between laboratories and genotyping platforms. The Norwegian minke whale DNA register (NMDR) contains individual genetic profiles at ten microsatellite loci for 6737 individuals captured in the period 1997-2008. These analyses have been conducted in four separate laboratories for nearly a decade, and offer a unique opportunity to examine genotyping errors and their consequences in an individual based DNA register. We re-genotyped 240 samples, and, for the first time, applied a mixed regression model to look at potentially confounding effects on genotyping errors.ResultsThe average genotyping error rate for the whole dataset was 0.013 per locus and 0.008 per allele. Errors were, however, not evenly distributed. A decreasing trend across time was apparent, along with a strong within-sample correlation, suggesting that error rates heavily depend on sample quality. In addition, some loci were more error prone than others. False allele size constituted 18 of 31 observed errors, and the remaining errors were ten false homozygotes (i.e., the true genotype was a heterozygote) and three false heterozygotes (i.e., the true genotype was a homozygote).ConclusionsTo our knowledge, this study represents the first investigation of genotyping error rates in a wildlife DNA register, and the first application of mixed models to examine multiple effects of different factors influencing the genotyping quality. It was demonstrated that DNA registers accumulating data over time have the ability to maintain calibration and genotyping consistency, despite analyses being conducted on different genotyping platforms and in different laboratories. Although errors were detected, it is demonstrated that if the re-genotyping of individual samples is possible, these will have a minimal effect on the databases primary purpose, i.e., to perform individual identification.

Quantitative proteomics comparison of arachnoid cyst fluid and cerebrospinal fluid collected perioperatively from arachnoid cyst patients

BackgroundThere is little knowledge concerning the content and the mechanisms of filling of arachnoid cysts. The aim of this study was to compare the protein content of arachnoid cysts and cerebrospinal fluid by quantitative proteomics to increase the understanding of arachnoid cysts.MethodsArachnoid cyst fluid and cerebrospinal fluid from five patients were analyzed by quantitative proteomics in two separate experiments.In a label-free experiment arachnoid cyst fluid and cerebrospinal fluid samples from individual patients were trypsin digested and analyzed by Orbitrap mass spectrometry in a label-free manner followed by data analysis using the Progenesis software.In the second proteomics experiment, a patient sample pooling strategy was followed by MARS-14 immunodepletion of high abundant proteins, trypsin digestion, iTRAQ labelling, and peptide separation by mix-phase chromatography followed by Orbitrap mass spectrometry analysis. The results from these analyzes were compared to previously published mRNA microarray data obtained from arachnoid membranes.ResultsWe quantified 348 proteins by the label-free individual patient approach and 1425 proteins in the iTRAQ experiment using a pool from five patients of arachnoid cyst fluid and cerebrospinal fluid. This is by far the largest number of arachnoid cyst fluid proteins ever identified, and the first large-scale quantitative comparison between the protein content of arachnoid cyst fluid and cerebrospinal fluid from the same patients at the same time. Consistently in both experiment, we found 22 proteins with significantly increased abundance in arachnoid cysts compared to cerebrospinal fluid and 24 proteins with significantly decreased abundance. We did not observe any molecular weight gradient over the arachnoid cyst membrane. Of the 46 proteins we identified as differentially abundant in our study, 45 were also detected from the mRNA expression level study. None of them were previously reported as differentially expressed. We did not quantify any of the proteins corresponding to gene products from the ten genes previously reported as differentially abundant between arachnoid cysts and control arachnoid membranes.ConclusionsFrom our experiments, the protein content of arachnoid cyst fluid and cerebrospinal fluid appears to be similar. There were, however, proteins that were significantly differentially abundant between arachnoid cyst fluid and cerebrospinal fluid. This could reflect the possibility that these proteins are affected by the filling mechanism of arachnoid cysts or are shed from the membranes into arachnoid cyst fluid. Our results do not support the proposed filling mechanisms of oncotic pressure or valves.

Congenital laryngomalacia is related to exercise-induced laryngeal obstruction in adolescence

Objectives Congenital laryngomalacia (CLM) is the major cause of stridor in infants. Most cases are expected to resolve before 2 years of age, but long-term respiratory prospects are poorly described. We aimed to investigate if CLM was associated with altered laryngeal structure or function in later life. Methods Twenty of 23 (87%) infants hospitalised at Haukeland University Hospital during 1990–2000 for CLM without comorbidities and matched controls were assessed at mean age 13 years. Past and current respiratory morbidity was recorded in a questionnaire, and spirometry performed according to standard quality criteria. Laryngoscopy was performed at rest and continuously throughout a maximal treadmill exercise test (continuous laryngoscopy exercise test (CLE-test)), and scored and classified in a blinded fashion according to preset criteria. Results In the CLM group, laryngeal anatomy supporting CLM in infancy was described at rest in nine (45%) adolescents. Eleven (55%) reported breathing difficulties in relation to exercise, of whom 7 had similarities to CLM at rest and 10 had supraglottic obstruction during CLE-test. Overall, 6/20 had symptoms during exercise and similarities to CLM at rest and obstruction during CLE-test. In the control group, one adolescent reported breathing difficulty during exercise and two had laryngeal obstruction during CLE-test. The two groups differed significantly from each other regarding laryngoscopy scores, obtained at rest and during exercise (p=0.001 or less). Conclusions CLM had left footprints that increased the risk of later exercise-induced symptoms and laryngeal obstruction. The findings underline the heterogeneity of childhood respiratory disease and the importance of considering early life factors.

Protein profiling reveals inter-individual protein homogeneity of arachnoid cyst fluid and high qualitative similarity to cerebrospinal fluid

BackgroundThe mechanisms behind formation and filling of intracranial arachnoid cysts (AC) are poorly understood. The aim of this study was to evaluate AC fluid by proteomics to gain further knowledge about ACs. Two goals were set: 1) Comparison of AC fluid from individual patients to determine whether or not temporal AC is a homogenous condition; and 2) Evaluate the protein content of a pool of AC fluid from several patients and qualitatively compare this with published protein lists of cerebrospinal fluid (CSF) and plasma.MethodsAC fluid from 15 patients with temporal AC was included in this study. In the AC protein comparison experiment, AC fluid from 14 patients was digested, analyzed by LC-MS/MS using a semi-quantitative label-free approach and the data were compared by principal component analysis (PCA) to gain knowledge of protein homogeneity of AC. In the AC proteome evaluation experiment, AC fluid from 11 patients was pooled, digested, and fractionated by SCX chromatography prior to analysis by LC-MS/MS. Proteins identified were compared to published databases of proteins identified from CSF and plasma. AC fluid proteins not found in these two databases were experimentally searched for in lumbar CSF taken from neurologically-normal patients, by a targeted protein identification approach called MIDAS (Multiple Reaction Monitoring (MRM) initiated detection and sequence analysis).ResultsWe did not identify systematic trends or grouping of data in the AC protein comparison experiment, implying low variability between individual proteomic profiles of AC.In the AC proteome evaluation experiment, we identified 199 proteins. When compared to previously published lists of proteins identified from CSF and plasma, 15 of the AC proteins had not been reported in either of these datasets. By a targeted protein identification approach, we identified 11 of these 15 proteins in pooled CSF from neurologically-normal patients, demonstrating that the majority of abundant proteins in AC fluid also can be found in CSF. Compared to plasma, as many as 104 proteins in AC were not found in the list of 3017 plasma proteins.ConclusionsBased on the protein content of AC fluid, our data indicate that temporal AC is a homogenous condition, pointing towards a similar AC filling mechanism for the 14 patients examined. Most of the proteins identified in AC fluid have been identified in CSF, indicating high similarity in the qualitative protein content of AC to CSF, whereas this was not the case between AC and plasma. This indicates that AC is filled with a liquid similar to CSF. As far as we know, this is the first proteomics study that explores the AC fluid proteome.

A calibration study of SAPS II with Norwegian intensive care registry data

Mortality prediction is important in intensive care. The Simplified Acute Physiology Score (SAPS) II is a tool for predicting such mortality. However, the original SAPS II is poorly calibrated to current intensive care unit (ICU) populations because it draws on data, which is more than 20 years old. We aimed to improve the calibration of SAPS II using data from the Norwegian Intensive Care Registry (NIR). This is the first recalibration of SAPS II for Nordic data.

A new approach to chromosome-wide analysis of X-linked markers identifies new associations in Asian and European case-parent triads of orofacial clefts

Background GWAS discoveries on the X-chromosome are underrepresented in the literature primarily because the analytical tools that have been applied were originally designed for autosomal markers. Our objective here is to employ a new robust and flexible tool for chromosome-wide analysis of X-linked markers in complex traits. Orofacial clefts are good candidates for such analysis because of the consistently observed excess of females with cleft palate only (CPO) and excess of males with cleft lip with or without cleft palate (CL/P). Methods Genotypes for 14,486 X-chromosome SNPs in 1,291 Asian and 1,118 European isolated cleft triads were available from a previously published GWAS. The R-package HAPLIN enables genome-wide–level analyses as well as statistical power simulations for a range of biologic scenarios. We analyzed isolated CL/P and isolated CPO for each ethnicity in HAPLIN, using a sliding-window approach to haplotype analysis and two different statistical models, with and without X-inactivation in females. Results There was a larger number of associations in the Asian versus the European sample, and similar to previous reports that have analyzed the same GWAS dataset using different methods, we identified associations with EFNB1/PJA1 and DMD. In addition, new associations were detected with several other genes, among which KLHL4, TBX22, CPXCR1 and BCOR were noteworthy because of their roles in clefting syndromes. A few of the associations were only detected by one particular X-inactivation model, whereas a few others were only detected in one sex. Discussion/Conclusion We found new support for the involvement of X-linked variants in isolated clefts. The associations were specific for ethnicity, sex and model parameterization, highlighting the need for flexible tools that are capable of detecting and estimating such effects. Further efforts are needed to verify and elucidate the potential roles of EFNB1/PJA1, KLHL4, TBX22, CPXCR1 and BCOR in isolated clefts.

Atherosclerosis in Trial of Org 10172 in Acute Stroke Treatment Subtypes among Young and Middle-Aged Stroke Patients: The Norwegian Stroke in the Young Study

BACKGROUND Ischemic stroke patients subtyped as of undetermined cause (SUC) usually outnumber those with determined cause subtypes. Etiological stroke classifications may lead to neglect of parallel, noncausative findings. Atherosclerosis progresses over decades and is associated with high morbidity and mortality in young stroke patients in long-term follow-up studies. We compared the prevalence of carotid atherosclerosis in all TOAST subtypes among young patients with acute ischemic stroke. METHODS We investigated 150 patients aged 15-60 years with documented acute ischemic stroke, and 84 controls free of cardiovascular disease. Stroke etiology was classified according to TOAST criteria. Carotid intima-media thickness (cIMT) measurements were obtained from 12 standardized multiangle measurements in the common carotid artery, carotid bifurcation, and internal carotid artery. RESULTS The causes of stroke were 5.3% large-artery atherosclerosis (LAA), 26.7% cardioembolism, 21.3% small-artery occlusion (SAO), 10% stroke of other determined cause, and 36.7% stroke of undetermined cause (SUC). cIMT was increased in patients with LAA (1.56 mm, P = .002), SAO (1.11 mm, P = .006), and SUC (1.10 mm, P = .004) compared to controls (cIMT 0.86 mm). Segmental cIMT distribution differed across stroke subtypes, age groups, and sexes. CONCLUSIONS Atherosclerotic disease is prevalent in the majority of young and middle-aged ischemic stroke patients, requiring determined investigation and aggressive treatment of modifiable risk factors.

Targeted Program for Provision of Mother’s Own Milk to Very Low Birth Weight Infants

OBJECTIVE: Evaluate the effect of an evolving targeted program to encourage mothers to provide own milk (MM) to their very low birth weight (VLBW) infants in a traditional open-bay NICU. METHODS: Retrospective review of medical records on all VLBW infants (birth weight <1500 g) born in a geographical region of Norway in 1986/1987, 1996, and 2007/2008 (n = 203). Types of nutrition and data on maternal and infant health were prospectively and similarly recorded during all time periods. Between each period, targeted programs were initiated to encourage provision of MM. RESULTS: The rates of providing MM (exclusively MM in parenthesis) for the 3 periods were 55% (33%), 85% (60%), and 89% (62%) when achieving full enteral feeds; 48% (11%), 76% (39%), and 92% (60%) at discharge; 15%, 42%, and 62% at 2 to 4 months’ corrected age; and 10%, 40%, and 53% at 6 to 8 months’ corrected age (P < .001 at all end points). Neither maternal or pregnancy disorders nor neonatal morbidity had significant effects on provision of MM, but smoking was associated with a lower rate after discharge. CONCLUSIONS: Both early and long-term provision of MM for their VLBW infants were strongly associated with targeted programs to encourage provision. We suggest that almost all mothers are able to provide their own milk if given targeted encouragement and guidance, even in crowded open-bay NICUs.

Parent-of-origin-environment interactions in case-parent triads with or without independent controls

With case–parent triad data, one can frequently deduce parent of origin of the childs alleles. This allows a parent‐of‐origin (PoO) effect to be estimated as the ratio of relative risks associated with the alleles inherited from the mother and the father, respectively. A possible cause of PoO effects is DNA methylation, leading to genomic imprinting. Because environmental exposures may influence methylation patterns, gene–environment interaction studies should be extended to allow for interactions between PoO effects and environmental exposures (i.e., PoOxE). One should thus search for loci where the environmental exposure modifies the PoO effect.

Genome-wide analysis of parent-of-origin interaction effects with environmental exposure (PoOxE): An application to European and Asian cleft palate trios

Cleft palate only is a common birth defect with high heritability. Only a small fraction of this heritability is explained by the genetic variants identified so far, underscoring the need to investigate other disease mechanisms, such as gene-environment (GxE) interactions and parent-of-origin (PoO) effects. Furthermore, PoO effects may vary across exposure levels (PoOxE effects). Such variation is the focus of this study. We upgraded the R-package Haplin to enable direct tests of PoOxE effects at the genome-wide level. From a previous GWAS, we had genotypes for 550 case-parent trios, of mainly European and Asian ancestry, and data on three maternal exposures (smoking, alcohol, and vitamins). Data were analyzed for Europeans and Asians separately, and also for all ethnicities combined. To account for multiple testing, a false discovery rate method was used, where q-values were generated from the p-values. In the Europeans-only analyses, interactions with maternal smoking yielded the lowest q-values. Two SNPs in the ‘Interactor of little elongation complex ELL subunit 1’ (ICE1) gene had a q-value of 0.14, and five of the 20 most significant SNPs were in the ‘N-acetylated alpha-linked acidic dipeptidase-like 2’ (NAALADL2) gene. No evidence of PoOxE effects was found in the other analyses. The connections to ICE1 and NAALADL2 are novel and warrant further investigation. More generally, the new methodology presented here is easily applicable to other traits and exposures in which a family-based study design has been implemented.