Chromosome aberrations in pressure-induced triploid Atlantic salmon.

BACKGROUND: Triploid organisms have three sets of chromosomes. In Atlantic salmon, hydrostatic pressure treatment of newly fertilized eggs has been extensively used to produce triploids which are functionally sterile due to their unpaired chromosomes. These fish often perform poorly on commercial farms, sometimes without explanation. Inheritance patterns in individuals subjected to pressure treatment have not been investigated in Atlantic salmon thus far. However, work on other species suggests that this treatment can result in aberrant inheritance. We therefore studied this in Atlantic salmon by genotyping 16 polymorphic microsatellites in eyed eggs and juveniles which had been subjected to pressure-induction of triploidy. Communally reared juveniles including fish subjected to pressure-induction of triploidy and their diploid siblings were included as a control. RESULTS: No diploid offspring were detected in any of the eggs or juveniles which were subjected to hydrostatic pressure; therefore, the induction of triploidy was highly successful. Aberrant inheritance was nevertheless observed in 0.9% of the eggs and 0.9% of the juveniles that had been subjected to pressure treatment. In the communally reared fish, 0.3% of the fish subjected to pressure treatment displayed aberrant inheritance, while their diploid controls displayed 0% aberrant inheritance. Inheritance errors included two eyed eggs lacking maternal DNA across all microsatellites, and, examples in both eggs and juveniles of either the maternal or paternal allele lacking in one of the microsatellites. All individuals displaying chromosome aberrations were otherwise triploid. CONCLUSIONS: This is the first study to document aberrant inheritance in Atlantic salmon that have been subjected to pressure-induction of triploidy. Our experiments unequivocally demonstrate that even when induction of triploidy is highly successful, this treatment can cause chromosome aberrations in this species. Based upon our novel data, and earlier studies in other organisms, we hypothesize that in batches of Atlantic salmon where low to modest triploid induction rates have been reported, aberrant inheritance is likely to be higher than the rates observed here. Therefore, we tentatively suggest that this could contribute to the unexplained poor performance of triploid salmon that is occasionally reported in commercial aquaculture. These hypotheses require further investigation.

Influence of nalidixic acid on tandem heterotrophic-autotrophic kinetics in a "NIPHO" activated sludge reactor.

This work analyzes the effect of nalidixic acid (NAL) on the kinetics of the heterotrophic and autotrophic biomass growth within a "NIPHO" activated sludge reactor treating municipal wastewater. Thus, the effect of this chemical in the degradation rates of carbon and nitrogen sources and net biomass growth rate is evaluated. Activated sludge samples were taken at three different operation conditions, changing the values of hydraulic retention time (2.8-3.8 h), biomass concentration (1400-1700 mgVSS L-1), temperature (12.6-14.8 °C), and sludge retention time (11.0-12.6 day). A respirometric method was applied to model the kinetic performance of heterotrophic and autotrophic biomass in absence and presence of NAL, and a multivariable statistical analysis was carried out to characterize the influence of the operation variables on the kinetic response of the system, which was finally optimized. The results showed that there was no inhibitory effect of NAL on heterotrophic biomass, with an increase of net heterotrophic biomass growth rate from 1.70 to 6.73 mgVSS L-1 h-1 at the most favorable period. By contrast, the autotrophic biomass was negatively affected by NAL, reducing the value of net autotrophic biomass growth rate from 25.37 to 10.29 mgVSS L-1 h-1 at the best operation conditions. In general, biomass concentration and temperature had the highest influence on the degradation rate of carbon and nitrogen sources, whereas hydraulic retention time and sludge retention time were the most influential on net heterotrophic and autotrophic biomass growth rates.

Performance and precision of double digestion RAD (ddRAD) genotyping in large multiplexed datasets of marine fish species.

The development of Genotyping-By-Sequencing (GBS) technologies enables cost-effective analysis of large numbers of Single Nucleotide Polymorphisms (SNPs), especially in "non-model" species. Nevertheless, as such technologies enter a mature phase, biases and errors inherent to GBS are becoming evident. Here, we evaluated the performance of double digest Restriction enzyme Associated DNA (ddRAD) sequencing in SNP genotyping studies including high number of samples. Datasets of sequence data were generated from three marine teleost species (>5500 samples, >2.5 × 1012 bases in total), using a standardized protocol. A common bioinformatics pipeline based on STACKS was established, with and without the use of a reference genome. We performed analyses throughout the production and analysis of ddRAD data in order to explore (i) the loss of information due to heterogeneous raw read number across samples; (ii) the discrepancy between expected and observed tag length and coverage; (iii) the performances of reference based vs. de novo approaches; (iv) the sources of potential genotyping errors of the library preparation/bioinformatics protocol, by comparing technical replicates. Our results showed use of a reference genome and a posteriori genotype correction improved genotyping precision. Individual read coverage was a key variable for reproducibility; variance in sequencing depth between loci in the same individual was also identified as an important factor and found to correlate to tag length. A comparison of downstream analysis carried out with ddRAD vs single SNP allele specific assay genotypes provided information about the levels of genotyping imprecision that can have a significant impact on allele frequency estimations and population assignment. The results and insights presented here will help to select and improve approaches to the analysis of large datasets based on RAD-like methodologies.

Evolution and conservation of Characidium sex chromosomes.

Fish species exhibit substantial variation in the degree of genetic differentiation between sex chromosome pairs, and therefore offer the opportunity to study the full range of sex chromosome evolution. We used restriction-site associated DNA sequencing (RAD-seq) to study the sex chromosomes of Characidium gomesi, a species with conspicuous heteromorphic ZW/ZZ sex chromosomes. We screened 9863 single-nucleotide polymorphisms (SNPs), corresponding to ~1 marker/100 kb distributed across the genome for sex-linked variation. With this data set, we identified 26 female-specific RAD loci, putatively located on the W chromosome, as well as 148 sex-associated SNPs showing significant differentiation (average FST=0.144) between males and females, and therefore in regions of more recent divergence between the Z and W chromosomes. In addition, we detected 25 RAD loci showing extreme heterozygote deficiency in females but which were in Hardy-Weinberg equilibrium in males, consistent with degeneration of the W chromosome and therefore female hemizygosity. We validated seven female-specific and two sex-associated markers in a larger sample of C. gomesi, of which three localised to the W chromosome, thereby providing useful markers for sexing wild samples. Validated markers were evaluated in other populations and species of the genus Characidium, this exploration suggesting a rapid turnover of W-specific repetitive elements. Together, our analyses point to a complex origin for the sex chromosome of C. gomesi and highlight the utility of RAD-seq for studying the composition and evolution of sex chromosomes systems in wild populations.

The antidepressant drug carbamazepine induces differential transcriptome expression in the brain of Atlantic salmon, Salmo salar.

Concerns are being expressed recently over possible environmental effects of human pharmaceuticals. Although the likelihood of acute toxicity is low, the continuous discharge of pharmaceuticals into the aquatic environment means that sublethal effects on non-target organisms need to be seriously considered. One-year-old Atlantic salmon parr were exposed to 7.85±0.13µgL(-1) of the antidepressant drug Carbamazepine (CBZ) for five days to investigate changes of mRNA expression in the brain by means of a custom 17k Atlantic salmon cDNA microarray. The selected concentration is similar to upper levels that can be found in hospital and sewage treatment plant effluents. After treatment, 373 features were differently expressed with 26 showing up- or down-regulation of ≥2-fold (p≤0.05). Among the mRNAs showing the highest change were the pituitary hormones encoding features somatolactin, prolactin and somatotropin, or growth hormone. Functional enrichment and network analyses of up- and down-regulated genes showed that CBZ induced a highly different gene expression profile in comparison to untreated organisms. CBZ induced expression of essential genes of the focal adhesion and extracellular matrix - receptor interaction pathways most likely through integrin alpha-6 (itga6) activation. Negative regulation of apoptotic process, extracellular matrix organization and heme biosynthesis were the most enriched biological process related GO-terms, with the simultaneous enrichment of collagen and extracellular region related cellular component GO-terms, and extracellular matrix structural constituent, hormone activity and chromatin binding molecular function related GO-terms. These results show that relatively low doses of CBZ may affect brain physiology in exposed salmon parr, targeting similar processes as in human, indicating a high degree of conservation of targets of CBZ action. However, and since the mRNAs showing most changes in expression are critical for adaptation to different stressors and life history transitions in Atlantic salmon, more research should be undertaken to assess CBZ effects to avoid impairment of normal development and maintenance of natural populations.

Bone marrow levels of 25 hydroxy vitamin D are not depressed in cases of hip fracture compared with controls.

There is little information on tissue as distinct from plasma levels of vitamin D metabolites in cases of hip fracture compared with controls. Femoral neck fractures in the elderly are associated with increased cortical remodelling and endosteal resorption, leading to regional increases in porosity and reduced cortical thickness. Vitamin D metabolites play a central role in the maintenance of normal serum calcium levels and may, through interactions with parathyroid hormone, exert an important influence on bone structure. To investigate whether hip fracture might be associated with tissue vitamin D deficiency, we have measured by radioimmunoassay the levels of 25 hydroxy vitamin D (25 (OH)D) in bone marrow samples extracted from the proximal femurs of 16 female subjects who had suffered fracture (mean age = 82.1 years, standard error (se) 1.9) and nine sex matched post mortem controls (mean age = 83.8 years, se 2.5). Twenty five (OH)D concentrations were significantly greater in the fracture cases (median = 3.7, IQR = 2.5-3.9 ng/g) than in the control group (median = 1.5, IQR = 0.9-2.3 ng/g; P = 0.0007, non-parametric Wilcoxon/Kruskal-Wallis test). It was suggested in the 1970s that bone loss and hip fracture risk in the UK were driven by vitamin D deficiency. Our results suggest that the alterations in femoral neck bone microstructure and remodelling in hip fracture cannot be assigned to the single cause of relative deficiency of vitamin D. Vitamin D deficiency or insufficiency may nevertheless increase remodelling and loss of bone tissue and contribute causally to a minority of hip fractures.

Differential vasodilation of human placental and myometrial arteries related to myofilament Ca(2+)-desensitization and the expression of Hsp20 but not MYPT1.

Endothelial-dependent regulation of vascular tone occurs in part via protein kinase G1α-mediated changes in smooth muscle myofilament sensitivity to Ca(2+). Tissue-specific differences in PKG-dependent relaxation have been attributed to altered expression of myofilament-associated proteins that are substrates for PKG binding. These include the alternative splicing of the myosin targeting subunit (MYPT1) of myosin light chain phosphatase to yield leucine zipper positive (LZ(+)) and negative (LZ(-)) isovariants, with the former being required for PKG-mediated relaxation, and/or altered expressions of telokin, vasodilator-stimulated phosphoprotein (VASP) or heat shock protein Hsp20. During human pregnancy the uterine and placental circulations remain distinct entities and, as such, their mechanisms of vascular tone regulation may differ. Indeed, the sensitivity of myometrial arteries to endothelial-dependent agonists has been suggested to be greater than that of placental arteries. We tested the hypothesis that this was related to tissue-specific changes in PKG-mediated myofilament Ca(2+)-desensitization and/or the expressions of PKG-interacting myofilament-associated proteins. Permeabilized human placental and myometrial arteries were constricted with maximal activating Ca(2+) (pCa 4.5), or sub-maximal Ca(2+) (pCa 6.7) and the thrombane mimetic U46619, and exposed to 8-Br-cGMP. In each case, relaxation was significantly greater in myometrial arteries (e.g. relaxation in pCa 4.5 to 8-Br-cGMP was 49 ± 9.7%, n = 7) than placental arteries (relaxation of 23 ± 6.6%, n = 6, P < 0.05). MYPT1 protein levels, or MYPT1 LZ(+)/LZ(-) mRNA ratios, were similar for both artery types. Of other proteins examined, only Hsp20 expression was significantly elevated in myometrial arteries than placental arteries. These results demonstrate that the reduced human placental artery relaxation to PKG stimulation lies partly at the level of myofilament (de)activation and may be related to a lower expression of Hsp20 than in myometrial arteries.

The Teamwork Study: enhancing the role of non-GP staff in chronic disease management in general practice.

There is evidence for a team-based approach in the management of chronic disease in primary health care. However, the standard of care is variable, probably reflecting the limited organisational capacity of health services to provide the necessary structured and organised care for this group of patients. This study aimed to evaluate the impact of a structured intervention involving non-GP staff in GP practices on the quality of care for patients with diabetes or cardiovascular disease. A cluster randomised trial was undertaken across 60 GP practices. The intervention was implemented in 30 practices with staff and patients interviewed at baseline and at 12-15 months follow up. The change in team roles was evaluated using a questionnaire completed by practice staff. The quality of care was evaluated using the Patient Assessment of Chronic Illness Care questionnaire. We found that although the team roles of staff improved in the intervention practices and there were significant differences between practices, there was no significant difference between those in the intervention and control groups in patient-assessed quality of care after adjusting for baseline-level score and covariates at the 12-month follow up. Practice team roles were not significantly associated with change in Patient Assessment of Chronic Illness Care scores. Patients with multiple conditions were more likely to assess their quality of care to be better. Thus, although previous research has shown a cross-sectional association between team work and quality of care, we were unable to replicate these findings in the present study. These results may be indicative of insufficient time for organisational change to result in improved patient-assessed quality of care, or because non-GP staff roles were not sufficiently focussed on the aspects of care assessed. The findings provide important information for researchers when designing similar studies.

Towards an ontology for data quality in integrated chronic disease management: a realist review of the literature.

PURPOSE: Effective use of routine data to support integrated chronic disease management (CDM) and population health is dependent on underlying data quality (DQ) and, for cross system use of data, semantic interoperability. An ontological approach to DQ is a potential solution but research in this area is limited and fragmented. OBJECTIVE: Identify mechanisms, including ontologies, to manage DQ in integrated CDM and whether improved DQ will better measure health outcomes. METHODS: A realist review of English language studies (January 2001-March 2011) which addressed data quality, used ontology-based approaches and is relevant to CDM. RESULTS: We screened 245 papers, excluded 26 duplicates, 135 on abstract review and 31 on full-text review; leaving 61 papers for critical appraisal. Of the 33 papers that examined ontologies in chronic disease management, 13 defined data quality and 15 used ontologies for DQ. Most saw DQ as a multidimensional construct, the most used dimensions being completeness, accuracy, correctness, consistency and timeliness. The majority of studies reported tool design and development (80%), implementation (23%), and descriptive evaluations (15%). Ontological approaches were used to address semantic interoperability, decision support, flexibility of information management and integration/linkage, and complexity of information models. CONCLUSION: DQ lacks a consensus conceptual framework and definition. DQ and ontological research is relatively immature with little rigorous evaluation studies published. Ontology-based applications could support automated processes to address DQ and semantic interoperability in repositories of routinely collected data to deliver integrated CDM. We advocate moving to ontology-based design of information systems to enable more reliable use of routine data to measure health mechanisms and impacts.

The University of NSW electronic practice based research network: disease registers, data quality and utility.

INTRODUCTION: Accurate well-maintained registers are a prerequisite to co-ordinated care of patients with chronic diseases. Their effectiveness in enabling improved management is dependent on the quality of the information captured. This paper provides an overview into the methodology and data quality of the electronic Practice Based Research Network. METHODS: Clinical records with no identifying information are routinely extracted from four general practices. The data are linked in the data warehouse. Data quality is assessed for completeness, correctness and consistency. Reports on data quality are given back to practices and semi-structured interviews provide information to interpret the results and discuss how data quality could be improved. FINDINGS: Data quality is mostly complete for sex and date of birth but indigenous status, smoking and weight were incomplete. There are generally high levels of correctness and internal consistency. Completeness of records in assisting the management of diabetes patients using the annual cycle of care was poor. GPs often use the progress notes to enter information during the consultation and coding diagnoses was considered onerous. DISCUSSION: The routine capture of electronic clinical health records from primary health care and health services can be used to monitor performance and improve the quality of clinical records. There is a need for accurate and comprehensive clinical records to ensure the safety and quality of clinical practice. Understanding the true reasons for poor data quality is complex. Having a community-based research network may assist in answering some of these questions. CONCLUSION: Electronic health records are increasingly being used for secondary research and evaluation, beyond the primary purpose of supporting clinical care. The data must be of sufficient quality to support these purposes.

Readiness for organisational change among general practice staff.

BACKGROUND: Increasing demands on general practice to manage chronic disease may warrant organisational change at the practice level. Staff's readiness for organisational change can act as a facilitator or barrier to implementing interventions aimed at organisational change. OBJECTIVES: To explore general practice staff readiness for organisational change and its association with staff and practices characteristics. METHODS: This is a cross-sectional study of practices in three Australian states involved in a randomised control trial on the effectiveness of an intervention to enhance the role of non-general practitioner staff in chronic disease management. Readiness for organisational change, job satisfaction and practice characteristics were assessed using questionnaires. RESULTS: 502 staff from 58 practices completed questionnaires. Practice characteristics were not associated with staff readiness for change. A multilevel regression analysis showed statistically significant associations between staff readiness for organisational change (range 1 to 5) and having a non-clinical staff role (vs general practitioner; B=-0.315; 95% CI -0.47 to -0.16; p<0.001), full-time employment (vs part-time; B=0.175, 95% CI 0.06 to 0.29; p<0.01) and lower job satisfaction (B=-0.277, 95% CI -0.40 to -0.15; p<0.001). CONCLUSIONS: The results suggest that different approaches are needed to facilitate change which addresses the mix of practice staff. Moderately low job satisfaction may be an opportunity for organisational change.

The susceptibility of Atlantic salmon fry to freshwater infectious pancreatic necrosis is largely explained by a major QTL.

Infectious pancreatic necrosis (IPN) is a viral disease with a significant negative impact on the global aquaculture of Atlantic salmon. IPN outbreaks can occur during specific windows of both the freshwater and seawater stages of the salmon life cycle. Previous research has shown that a proportion of the variation seen in resistance to IPN is because of host genetics, and we have shown that major quantitative trait loci (QTL) affect IPN resistance at the seawater stage of production. In the current study, we completed a large freshwater IPN challenge experiment to allow us to undertake a thorough investigation of the genetic basis of resistance to IPN in salmon fry, with a focus on previously identified QTL regions. The heritability of freshwater IPN resistance was estimated to be 0.26 on the observed scale and 0.55 on the underlying scale. Our results suggest that a single QTL on linkage group 21 explains almost all the genetic variation in IPN mortality under our experimental conditions. A striking contrast in mortality is seen between fry classified as homozygous susceptible versus homozygous resistant, with QTL-resistant fish showing virtually complete resistance to IPN mortality. The findings highlight the importance of the major QTL in the genetic regulation of IPN resistance across distinct physiological lifecycle stages, environmental conditions and viral isolates. These results have clear scientific and practical implications for the control of IPN.

Detection of QTL affecting harvest traits in a commercial Atlantic salmon population.

Genetic variation in performance and quality traits measured at harvest has previously been demonstrated in Atlantic salmon aquaculture populations. To map major loci underlying this variation, we utilized data from 10 families from a commercial breeding programme. Significant QTL were detected affecting harvest weight and length traits on linkage group 1, and affecting waste weight on linkage group 5. In total, 11 of the 29 linkage groups examined showed at least suggestive evidence for a QTL. These data suggest that major loci affecting economically important harvest characteristics are segregating in commercial salmon populations.

Evaluation of the INNO-LiPA mycobacteria v2 assay for identification of aquatic mycobacteria.

Fifty-seven isolates of mycobacteria comprising 10 reference strains, 47 field isolates and one non-Mycobacterium isolate were screened using commercial INNO-LiPA v2 assay kits. All mycobacteria isolates tested hybridized with the Mycobacterium genus probe on the LiPA strip. All M. marinum, M. fortuitum and M. chelonae reference and field strains and three out of the four M. gordonae isolates hybridized to their corresponding species- or complex-specific probes. Two cultures (a type strain and a field isolate) yielded mixed growth of two mycobacterial species, i.e. M. chelonae and M. fortuitum. A Mycobacterium isolate from one of these cultures was subsequently purified and correctly identified with the kit. However, sequence analysis of the 16S-23S rRNA internal transcribed spacer (ITS) region of various mycobacteria isolates revealed a misidentification of M. shottsii and M. pseudoshottsii with the kit because these isolates reacted with the M. marinum/M. ulcerans probe. Moreover, nine of the 13 field isolates presumed to be M. fortuitum from the results of the kit had closer ITS sequence homology with M. conceptionense, a species which, to our knowledge, has never been reported in fish. These findings highlight the need to redesign the M. fortuitum-M. peregrinum probe included in the INNO-LiPA assay and to introduce additional complex-specific probes into the kit. Nevertheless, the kit proved to be a rapid and reliable method for identifying mycobacteria in the aquatic environment and would be particularly useful in laboratories without sequencing facilities.

Detection and confirmation of a major QTL affecting resistance to infectious pancreatic necrosis (IPN) in Atlantic salmon (Salmo salar).

Infectious pancreatic necrosis (IPN) is a viral disease currently presenting a major problem to the aquaculture of Atlantic salmon (Salmon salar), during both the freshwater and seawater stages of production. Genetic variation in resistance to IPN has previously been demonstrated and the purpose of this study was to determine whether this variation includes loci of major effect. The initial QTL detection methodology utilized the limited recombination seen in male salmon to detect QTL in ten large full-sib families, using a genome-wide scan of two to three markers per linkage group. QTL were then positioned by adding additional markers to the significant linkage groups in a female-based analysis. The most significant QTL was mapped to LG 21, and further confirmation of the LG 21 QTL is provided in an analysis of the QTL flanking markers in an additional nine full-sib families from the same population. The size of QTL effect is such that the QTL flanking markers can be immediately applied in marker-assisted selection programmes to improve the resistance of salmon populations to IPN, thus reducing mortality due to the disease.

A description of the origins, design and performance of the TRAITS-SGP Atlantic salmon Salmo salar L. cDNA microarray.

The origins, design, fabrication and performance of an Atlantic salmon microarray are described. The microarray comprises 16 950 Atlantic salmon-derived cDNA features, printed in duplicate and mostly sourced from pre-existing expressed sequence tag (EST) collections [SALGENE and salmon genome project (SGP)] but also supplemented with cDNAs from suppression subtractive hybridization libraries and candidate genes involved in immune response, protein catabolism, lipid metabolism and the parr-smolt transformation. A preliminary analysis of a dietary lipid experiment identified a number of genes known to be involved in lipid metabolism. Significant fold change differences (as low as 1.2x) were apparent from the microarray analysis and were confirmed by quantitative real-time polymerase chain reaction analysis. The study also highlighted the potential for obtaining artefactual expression patterns as a result of cross-hybridization of similar transcripts. Examination of the robustness and sensitivity of the experimental design employed demonstrated the greater importance of biological replication over technical (dye flip) replication for identification of a limited number of key genes in the studied system. The TRAITS (TRanscriptome Analysis of Important Traits of Salmon)-salmon genome project microarray has been proven, in a number of studies, to be a powerful tool for the study of key traits of Atlantic salmon biology. It is now available for use by researchers in the wider scientific community.

Interferon type I and type II responses in an Atlantic salmon (Salmo salar) SHK-1 cell line by the salmon TRAITS/SGP microarray.

Interferons (IFNs) are cytokines that have proinflammatory, antiviral, and immunomodulatory effects and play a central role during a host response to pathogens. The IFN family contains both type I and type II molecules. While there are a number of type I IFNs, there is only one type II IFN. Recently both type I and type II IFN genes have been cloned in salmonid fish and recombinant proteins produced showing IFN activity. We have stimulated an Atlantic salmon cell line (SHK-1) with both type I and type II recombinant salmonid IFNs and analyzed the transcriptional response by microarray analysis. Cells were exposed to recombinant IFNs for 6 or 24 h or left unexposed as controls. RNA was hybridized to an Atlantic salmon cDNA microarray (salmon 17K feature TRAITS/SGP array) in order to assess differential gene expression in response to IFN exposure. For IFN I and II, 47 and 72 genes were stimulated, respectively; most genes were stimulated by a single IFN type, but some were affected by both IFNs, indicating coregulation of the IFN response in fish. Real-time PCR analysis was employed to confirm the microarray results for selected differentially expressed genes in both a cell line and primary leukocyte cultures.

Application of real time PCR determination to assess interanimal variabilities in CYP1A induction in the European flounder (Platichthys flesus).

In both laboratory experiments and field investigations with fish a large interanimal variability in CYP1A expression has been observed which may be attributed to variations in environmental inducer exposure and/or inducer response. We are carrying out laboratory investigations to assess the contribution of a potential genetic component in inducer response of flounder (Platichthy sflesus) CYP1A to PCB exposure and in this paper we report development of a sensitive quantitative RT-PCR procedure (real time PCR) where accumulation of the intercalated dye SYBR Green I is followed during cycling. Preliminary experiments using this procedure with artificially reared Arochlor 1254-treated flounders showed large interanimal differences in response for a single family group indicating that variability does have a genetic basis.