The reliability of the disabled children's quality-of-life questionnaire in Swedish children with diabetes.

AIM: To determine the reliability of the disabled children's quality-of-life measure (DISABKIDS) chronic generic questionnaire and diabetes module in children. The questionnaire is being evaluated for repeated routine health-related quality-of-life (HrQoL) assessment and in association with the Swedish national paediatric diabetes registry (Swediabkids), which is a tool for regular clinical use. METHODS: Children and parents completed the questionnaire during a routine visit to the diabetes clinic. In total, 120 families completed the test and retest. Split-half reliability correlation and intraclass correlation (ICC) coefficients were calculated. Bland & Altman plots were calculated on the generic HRQoL domain. RESULTS: Both child and parent versions showed good internal consistency. Test-retest ICC coefficients for the generic HrQoL module were 0.913 for the children and 0.820 for the parent version. All generic domains independently showed good reliability. The diabetes module had a score of 0.855 for children and 0.823 for parents. Split-half correlation for generic and diabetes modules was 0.930 and 0.848 for children, 0.953 and 0.903 for parents. Bland and Altman plots showed substantial agreement between the two administrations for both children and parents. CONCLUSION: The DISABKIDS questionnaire is a reliable instrument for the repeated measurements of HrQoL in children with diabetes.

Extended evaluation of the safety and efficacy of GAD treatment of children and adolescents with recent-onset type 1 diabetes: a randomised controlled trial.

AIMS/HYPOTHESIS: The aim of this study was to investigate the safety and efficacy of alum formulated glutamic acid decarboxylase GAD(65) (GAD-alum) treatment of children and adolescents with type 1 diabetes after 4 years of follow-up. METHODS: Seventy children and adolescents aged 10-18 years with recent onset type 1 diabetes participated in a phase II, double-blind, randomised placebo-controlled clinical trial. Patients identified as possible participants attended one of eight clinics in Sweden to receive information about the study and for an eligibility check, including a medical history. Participants were randomised to one of the two treatment groups and received either a subcutaneous injection of 20 µg of GAD-alum or placebo at baseline and 1 month later. The study was blinded to participants and investigators until month 30. The study was unblinded at 15 months to the sponsor and statistician in order to evaluate the data. At follow-up after 30 months there was a significant preservation of residual insulin secretion, as measured by C-peptide, in the group receiving GAD-alum compared with placebo. This was particularly evident in patients with <6 months disease duration at baseline. There were no treatment-related serious adverse events. We have now followed these patients for 4 years. Overall, 59 patients, 29 who had been treated with GAD-alum and 30 who had received placebo, gave their informed consent. RESULTS: One patient in each treatment group experienced an episode of keto-acidosis between months 30 and 48. There were no treatment-related adverse events. The primary efficacy endpoint was the change in fasting C-peptide concentration from baseline to 15 months after the prime injection for all participants per protocol set. In the GAD-alum group fasting C-peptide was 0.332 ± 0.032 nmol/l at day 1 and 0.215 ± 0.031 nmol/l at month 15. The corresponding figures for the placebo group were 0.354 ± 0.039 and 0.184 ± 0.033 nmol/l, respectively. The decline in fasting C-peptide levels between day 1 and month 1, was smaller in the GAD-alum group than the placebo group. The difference between the treatment groups was not statistically significant. In those patients who were treated within 6 months of diabetes diagnosis, fasting C-peptide had decreased significantly less in the GAD-alum group than in the placebo-treated group after 4 years. CONCLUSION/INTERPRETATION: Four years after treatment with GAD-alum, children and adolescents with recent-onset type 1 diabetes continue to show no adverse events and possibly to show clinically relevant preservation of C-peptide. TRIAL REGISTRATION: ClinicalTrials.gov NCT00435981 FUNDING: The study was funded by The Swedish Research Council K2008-55X-20652-01-3, Barndiabetesfonden (The Swedish Child Diabetes Foundation), the Research Council of Southeast Sweden, and an unrestricted grant from Diamyd Medical AB.

Analysis of short stature homeobox-containing gene ( SHOX) and auxological phenotype in dyschondrosteosis and isolated Madelung deformity.

Dyschondrosteosis (DCO; also called Léri-Weill syndrome) is a skeletal dysplasia characterised by disproportionate short stature because of mesomelic shortening of the limbs. Madelung deformity is a feature of DCO that is distinctive, variable in expressivity and frequently observed. Mutations of the SHOX (short stature homeobox-containing) gene have been previously described as causative in DCO. Isolated Madelung deformity (IMD) without the clinical characteristics of DCO has also been described in sporadic and a few familial cases but the genetic defect underlying IMD is unknown. In this study, we have examined 28 probands with DCO and seven probands with IMD for mutations in the SHOX gene by using polymorphic CA-repeat analysis, fluorescence in situ hybridisation (FISH), Southern blotting, direct sequencing and fibre-FISH analyses. This was combined with auxological examination of the probands and their family members. Evaluation of the auxological data showed a wide intra- and interfamilial phenotype variability in DCO. Out of 28 DCO probands, 22 (79%) were shown to have mutations in the SHOX gene. Sixteen unrelated DCO families had SHOX gene deletions. Four novel DCO-associated mutations were found in different families. In two additional DCO families, the previously described nonsense mutation (Arg195Stop) was detected. We conclude that mutations in the SHOX gene are the major factor in the pathogenesis of DCO. In a female proband with severe IMD and her unaffected sister, we detected an intrachromosomal duplication of the SHOX gene.

Linkage disequilibrium mapping of the bolting gene in sea beet using AFLP markers.

The possibility of using linkage disequilibrium mapping in natural plant populations was assessed. In studying linkage disequilibrium among 137 mapped AFLP markers in four populations of sea beet (Beta vulgaris ssp. maritima (L.) Arcang.) it was shown that tightly linked loci could be detected by screening for associations. It was hypothesized that the short distances spanned by linkage disequilibrium enable markers that are very tightly linked to a target gene to be identified. The hypothesis was tested by whole-genome screening of AFLP markers for association with the gene for the annual growth habit, the B gene, in a sample of 106 sea beets. Despite the dominant nature of AFLP, two markers showing significant linkage disequilibrium with the B gene were detected. The results indicate the potential use of linkage disequilibrium for gene mapping in natural plant populations.

Effects of distance and pollen competition on gene flow in the wind-pollinated grass Festuca pratensis Huds.

Pollen dispersal and gene flow in the grass meadow fescue (Festuca pratensis Huds.) were studied using two populations which were homozygous for different allozymes at the Gpi-2 locus. The populations were established in a concentric donor-acceptor field experiment. Gene flow was found mainly to be affected by the distance between the donor and acceptor plants. Analysing 21 132 acceptor plant progenies, gene flow was shown to decrease rapidly with distance to the donor field up to 75 m, and beyond this distance much more slowly. The ability of donor pollen to fertilize acceptor plants depended very much on the density of the acceptor plants. Pairs of acceptor plants produced more compatible pollen locally, and captured significantly less donor pollen than single-plants. Despite the higher seed production of acceptor plants planted in pairs, the absolute number of heterozygous seeds carrying the donor allele was always lower than for single-plants. Wind direction had only a slight effect upon the type of pollen captured. Because of pollen production within the two plant populations being continuous and overlapping, the time when anthesis occurred had little effect on gene flow between the populations. Vigorous and tall acceptor plants with many panicles, high seed yield and high 1000-seed weight were able to capture more donor pollen than shorter plants. The results may be used to assess the risk of gene flow and to develop strategies for monitoring the spread of transgenes from genetically modified grasses.

Positive correlation between recombination rates and levels of genetic variation in natural populations of sea beet (Beta vulgaris subsp. maritima).

The relation between the level of genetic variation and the rate of recombination per physical unit was investigated in sea beet (Beta vulgaris subsp. maritima). The rate of recombination per physical unit was estimated indirectly through marker density in an RFLP linkage map of sugar beet. From this map, we also selected RFLP markers covering two of the nine chromosomes in Beta. The markers were used to estimate the level of genetic variation in three populations of sea beet, two from Italy and one from England. Two estimates of genetic variation were employed, one based on the number of alleles in the sample and the other on heterozygosity. A statistically significant positive correlation was found between recombination rate and genetic variation. Several theoretical explanations for this are discussed, background selection being one. A correlation similar to this has been observed previously in Drosophila, one that was higher than what we obtained for Beta. This is consistent with various biological differences between the two species.

Comparing the distribution of RAPD and RFLP markers in a high density linkage map of sugar beet.

The distribution of RAPD markers was compared with that of RFLP markers in a high density linkage map of sugar beet. The same mapping population of 161 F2 individuals was used to generate all the marker data. The total map comprises 160 RAPD and 248 RFLP markers covering 508 cM. Both the RAPD and the RFLP markers show a high degree of clustering over the nine linkage groups. The pattern is compatible with a strong distal localization of recombination in the sugar beet. It leads generally to one major cluster of markers in the centre of each linkage group. In regions of high marker density, dominant RAPD markers present in either linkage phase and codominant RFLP markers are subclustered relative to each other. This phenomenon is shown to be attributable to: (i) effects of the mapping procedure when dominant and codominant data are combined, (ii) effects of the mapping procedure when dominant data in both linkage phases are combined, and (iii) genuine differences in the way RAPD and RFLP markers are recruited.

A high density RFLP linkage map of sugar beet.

A high density sugar beet RFLP map with an average distance of 1.5 cM between markers has been constructed. The map covers 621 cM and includes 413 markers distributed over the nine linkage groups of sugar beet. The map is based on two F2 populations representing two different pairs of parents. The two sets of data were integrated into a single map using 90 markers that were common to both data sets. The quality of the map was assessed in several ways. The common markers were used to investigate how often the loci had been mapped in the same order in the two F2 populations. For closely situated markers (<1.5 cM) the order specified in the map is uncertain, but for markers separated by more than 2 cM the locus order is highly reliable. The error rate of the overall process was estimated at 0.3% by independently repeating the analysis of 41 markers. The map is comparatively short, with a map length corresponding to approximately 1.4 crossovers per bivalent. Another feature of the map is a high degree of clustering of markers along the linkage groups. With the possible exception of linkage group 2, each linkage group shows one major cluster, which in most cases is situated in the centre of the linkage group. Our interpretation is that sugar beet, in comparison with most other species, has an extreme localization of recombination. Key words : sugar beet, linkage, RFLP, clustering.

Competition as a source of errors in RAPD analysis.

We have used artificial 1∶1 DNA mixtures of all pairwise combinations of four doubled haploid Brassica napus lines to test the ability of RAPDs to function as reliable dominant genetic markers. In situations where a specific RAPD band is present in one homozygous line but absent in the other, the band is expected in the artificial heterozygote, i.e. in the 1∶1 DNA mixture. In 84 of all 613 heterozygous situations analysed, the expected band failed to amplify in the RAPD reaction. Thus, RAPD markers will lead to an erroneous genetic interpretation in 14% of all cases. In contrast, the formation of non-parental heteroduplex bands was found at a frequency of only 0.2%. Analysis of 1∶ 1 mixtures using (1) a different set of optimized reaction conditions and (2) a material with low genomic complexity (Bacillus cereus) gave identical results. Serial dilutions of one genome into another, in steps of 10%, showed that all of the polymorphic bands decreased in intensity as a linear function of their respective proportion in the mixture. In dilutions with water no differences in band intensity were detected. Thus, competition occurs in the amplification of all RAPD fragments and is a major source of genotyping errors in RAPD analysis.

A model of chiasma reduction of closely formed crossovers.

A model of chiasma reduction is developed, evaluated by computer simulation and discussed in relation to the evolution of interference. The model assumes that adjacently formed crossovers can interact, if there is incomplete sister chromatid cohesion between them, and give rise to a reduced number of chiasmata. In the absence of crossover position interference this leads to a considerable risk of non-disjunction for an average sized chromosome. It is suggested that an important function of crossover interference is to reduce this effect. The question is raised whether chiasma reduction takes place today. Some available cytological data can be interpreted as showing that chiasma reduction occurs in normal meiosis. The possibility of chiasma reduction therefore needs further attention.

The robustness of recombination frequency estimates in intercrosses with dominant markers.

The robustness of the maximum likelihood estimates of recombination frequencies has been investigated in double intercrosses with complete dominance at both loci. The robustness was investigated with respect to bias in the recombination frequency estimates due to: (1) limited sample sizes, (2) heterogeneity in recombination frequencies between sexes or among meioses and (3) factors that distort the segregation-misclassification or differential viability. In the coupling phase, the recombination frequency estimates are quite robust with respect to most of the investigated factors. Potentially, the most serious cause of a bias is misclassifications, which tend to increase the recombination frequency estimates. In the repulsion phase, misclassifications are particularly serious, leading to extreme discrepancies between true and observed values. In addition, limited sample size and sex differences in recombination can also bias recombination frequency estimates in repulsion. These effects may pose serious problem in genetic mapping with random amplified polymorphic DNA (RAPD) markers.

Crossover distribution in barley analysed through RFLP linkage data.

The pattern of recombination in barley with regard to (1) the distribution of crossover points among whole gametes, (2) the distribution of crossover points among individual chromosomes and (3) the distribution of crossover points within chromosomes has been analysed using data sets underlying two recently published restriction fragment length polymorphism (RFLP) linkage maps representing male and female meiosis, respectively. The data indicated that the process of recombination had been random with no interference. The two data sets gave similar results, indicating that male and female meiosis in barley do no differ significantly. The possibility of using RFLP data in studies of crossover distribution is stressed.

Evaluation of RFLP and RAPD markers in a comparison of Brassica napus breeding lines.

RFLP and RAPD markers were evaluated and compared for their ability to determine genetic relationships in a set of three B. napus breeding lines. Using a total of 50 RFLP and 92 RAPD markers, the relatedness between the lines was determined. In total, the RFLP and the RAPD analysis revealed more than 500 and 400 bands, respectively. The relative frequencies of loci with allele differences were estimated from the band data. The RFLP and RAPD marker sets detected very similar relationships among the three lines, consistent with known pedigree data. Bootstrap analyses showed that the use of approximately 30 probes or primers would have been sufficient to achieve these relationships. This indicates that RAPD markers have the same resolving power as RFLP markers when used on exactly the same set of B. napus genotypes. Since RAPD markers are easier and quicker to use, these markers may be preferred in applications where the relationships between closely-related breeding lines are of interest. The use of RAPD markers in fingerprinting applications may, however, not be warranted, and this is discussed in relation to the reliability of RAPD markers.

Chiasma and recombination data in plants: are they compatible?

It has long been considered that the number of chiasmata formed during meiosis corresponds to the number of crossovers indicated by the genetic map. However, recent investigations in plants show an unexpected discrepancy in the results obtained when calculating the total number of crossover events per meiosis by these two methods. Is this discrepancy due to methodological difficulties? Or is there something fundamentally wrong with our understanding of crossovers and chiasmata?

Effects of prolonged food restriction on some aspects of lipid metabolism in Norwegian and Svalbard reindeer.

The high-arctic Svalbard reindeer (SR) deposit great amounts of body fat in autumn for subsequent use during winter when food is often in short supply. Captive SR and, for comparative reasons, the sub-arctic Norwegian reindeer (NR) were offered 15% of their ad libitum food intake during a 21-day period in September/October and its effect on fat metabolism was investigated. Plasma free fatty acids (FFA), glycerol, glucose, insulin and urea as well as lipogenic and lipolytic activity of isolated adipocytes were determined. Levels of FFA and glycerol increased immediately when food intake was restricted, reaching the highest levels in SR. Plasma glucose was fairly constant in NR, but decreased in SR. Plasma insulin decreased in both species. Plasma urea increased steadily from day 5 in NR and from day 11 in SR, after a transient rise on day 1 in both. Lipogenic activity had vanished completely in both NR and SR adipocytes when tested after 13 days of food restriction, while lipolytic activity was initially increased, after which it decreased in adipocytes from both species. After 21 days of food restriction SR adipocytes exhibited another marked increase in lipolytic activity, while the fat deposits of NR at that time were too small to allow examination. Thus, reindeer do not differ from other species in their lipogenic responses, although they show some hitherto undescribed lipolytic responses to prolonged food restriction.

Seasonal changes in lipogenesis and lipolysis in isolated adipocytes from Svalbard and Norwegian reindeer.

Arctic reindeer exhibit marked seasonal changes in fat deposition and mobilization. At intervals throughout the year, therefore, we have measured feed intake of both Svalbard (SR) and Norwegian reindeer (NR) together with the seasonal changes in size, lipogenic and lipolytic capacity of isolated adipocytes from both sub-species. Feed intake of both NR and SR was maximal in August, but declined thereafter, reaching minimum values in January (NR) and March (SR), 55 and 69% below the August value, respectively. NR and SR adipocyte volume changed in parallel and were reduced to the same extent (69%) from their maximum in August to their minimum in May. Adipocyte lipogenic capacity, measured as acetate incorporation into cellular lipid at saturated acetate concentrations, was lowest in January (NR adipocytes) and March (SR adipocytes), 92 and 90%, respectively, below the maximum values, which were obtained in August. Lipolytic capacity, measured as maximum adrenaline-stimulated glycerol release, was high in SR adipocytes from March through to October and in NR adipocytes from July through to January. Minimum lipolytic capacity, on the other hand, was found in January (SR adipocytes) and March (NR adipocytes). The present findings may be explained by alterations in lipogenic enzyme activity and in the lipolytic activation system.