Bertil Lindblom

Lp(a) lipoprotein, IgG, IgA and IgM antibodies to Chlamydia pneumoniae and HLA class II genotype in early coronary artery disease

The associations previously found between lipoprotein(a) (Lp(a)) levels and atherosclerotic disorders, diabetes, rheumatoid arthritis and renal diseases suggest that Lp(a) may be involved in autoimmune reactions. The relation found between Lp(a) levels and the HLA class II genotype in males with early coronary artery disease (CAD) further support that assumption. It was suggested that an autoimmune process, perhaps triggered by a concomitant intracellular infection may occur especially in patients with inherited high Lp(a) levels in combination with certain inherited HLA class II genotypes. In this study a Chlamydia pneumoniae IgG titer > or = 32 was significantly more common (P = 0.036) in CAD patients than in matched controls. This is in agreement with previous reports by other investigators. In addition, an IgG titer > or = 256 in combination with an Lp(a) level > or = 120 mg/l was found to occur significantly more often (P = 0.011) in male patients than in male controls. Certain HLA class II DR genotypes in combination with high Lp(a) levels and C. pneumoniae titers occurred more frequently in both male and female patients than in controls. Some combinations were very common in male patients, and the difference in comparison with male controls was highly significant.

High frequency of occurrence of CYP2D6 gene duplication/multiduplication indicating ultrarapid metabolism among suicide cases.

In Sweden, about 550 individuals die every year of drug intoxication. Many of these drugs are metabolized by CYP enzymes such as CYP2D6 and CYP2C19. A lack of these enzymes, resulting in poor metabolism, can lead to adverse reactions and even to fatality. On the other hand, an ultrarapid metabolism can lead to insufficient drug plasma concentration, resulting in failure of treatment, or it can lead to high concentrations of active/toxic metabolites. The aim of this project was to study the genetic profile of individuals with regard to the presence of CYP2D6 and CYP2C19 genes, in cases of fatal intoxication (242), suicide (intoxications excluded) (262), and natural death (212). PCR, followed by pyrosequencing, was used for all the analyses. We found that, among those who died of suicide (suicide cases), there was a higher number carrying more than two active CYP2D6 genes (corresponding to the phenotype of ultrarapid metabolizer) as compared with those who died of natural causes (natural‐death cases) (P = 0.007).

A nonsense mutation (428G-->A) in the fucosyltransferase FUT2 gene affects the progression of HIV-1 infection.

Background:The human FUT2 gene encodes the α(1,2)fucosyltransferase that determines secretor status. Homozygous for the nonsense mutation are called non-secretors and are unable to express histo-blood group antigens in secretions and on mucosal surfaces. In this study we have investigated the importance of the FUT2 fucosyltransferase activity on the progress of HIV-1 infection. Methods:Swedish blood donors (n = 276), 15 long-term non-progressors (LTNP) and 19 progressors were genotyped with respect to the nonsense mutation 428G→A in the FUT2 gene. In addition 265/276 blood donors and 19 progressors with rapid or expected progression rate were Δ32 CCR5 genotyped. Results:Of 276 blood donors 218 (79%) were found to be secretor positive (se+), either homozygous (se+/+) wild type (30%) or heterozygous (se+/−) (49%) and 58 (21%) were homozygous non-secretors (se−/−). Five LTNP (33%) were found to be secretor-positive (se+/+, se+/−) and 10 (67%) se−/−. Of the 19 individuals with normal HIV-1 progression 15 (79 %) were found to be secretor positive and four (21%) were non-secretors. No frequency differences were found in the Δ32 CCR5 allele among the groups studied. Conclusion:Strong association (P < 0.001) was observed between the nonsense mutation 428G→A in the FUT2 gene and a slow disease progression of HIV-1 infection.

Rapid DNA purification for restriction fragment length polymorphism analysis

This paper describes a method for isolation of DNA from blood samples involving a rapid chemical disintegration of proteins with 8 M urea and with a minimum of exposure to phenol. The DNA is further desalted and purified on Sephadex G-25 prepacked disposable columns. DNA isolated in this way was pure enough to be immediately cleaved by restriction enzymes.

Analysis of linkage and linkage disequilibrium for eight X-STR markers

X-chromosomal short tandem repeats (X-STR) have proven to be informative and useful in complex relationship testing. The main feature of X-STR markers, compared to autosomal forensic markers, is that all loci are located on the same chromosome. Thus, linkage and linkage disequilibrium may occur. The aim of this work was to study population genetic parameters of eight X-STR markers, located in four linkage groups. We present haplotype frequencies, based on 718 Swedish males, for the four linkage groups included in the Argus X-8 kit. Forensic efficiency parameters have been calculated as well as the allelic association between the tested markers for detection of linkage disequilibrium. To study the occurrences of recombination between the loci, both Swedish and Somali families were typed. A mathematical model for the estimation of recombination frequencies is presented and applied on the family samples. Our study showed that the tested markers all have highly informative forensic values and that there is a significant degree of linkage disequilibrium between the STR markers within the four linkage groups. Furthermore, based on the tested families, we also demonstrated that two of the linkage groups are partially linked. A consequence of these findings is that both linkage and linkage disequilibrium should be accounted for when producing likelihood ratios in relationship testing with X-STR markers.

Identification of CYP2D6 alleles by single nucleotide polymorphism analysis using pyrosequencing

ObjectiveTo develop a single nucleotide polymorphism (SNP) analysis for identification of cytochrome P450 (CYP)2D6 alleles by pyrosequencing.MethodsSwedish blood donors (n=282) were typed for a partial CYP2D6 genotype comprising the alleles *1 (wild type), *2 (2850C>T), *3 (2549A>del), *4 (1846G>A) and *6 (1707T>del) using polymerase chain reaction (PCR) and pyrosequencing analysis. CYP2D6*5 (CYP2D6 deleted) was identified using an established long multiplex PCR method. Pyrosequencing is a sequencing-by-synthesis method in which a cascade of enzymatic reactions yields detectable light, which is proportional to the incorporated nucleotides. One feature of typing SNPs by pyrosequencing is that each allelic variant will give a unique sequence. These variants can be readily distinguished by pattern recognition software.ResultsOf 281 individuals analysed, 24 (8.5%) were found to be poor metabolisers with two non-functional alleles. This is in the range of 7–10%, previously reported for Caucasians. A total of 126 individuals (45%) had one functional and one non-functional allele and 131 individuals (47%) had two functional alleles.ConclusionPyrosequencing was found to be a fast and efficient tool for genotyping. The method is robust, reliable, rapid and has high throughput.

Fatal intoxication cases: cytochrome P450 2D6 and 2C19 genotype distributions

Objective Many commonly used pharmaceuticals, such as antidepressants and neuroleptics as well as some illegal drugs, are metabolised by the cytochrome P 450 enzyme debrisoquine 4-hydroxylase (CYP2D6). Of Caucasians, 7–10% lack this enzyme, which can, upon administration of drugs in normal therapeutic doses, lead to adverse reactions and unexpected intoxication, leading in turn even to a fatal outcome in some cases.MethodsIndividuals (n=242) who had died due to intoxication by pharmaceuticals were genotyped for CYP2D6 and CYP2C19 and compared with a reference group of 281 blood donors. A single nucleotide polymorphism (SNP) method was used to identify five CYP2D6 alleles: *1 (wt), *2, *3, *4 and *6. The allele *5, a complete gene deletion, was identified by a multiplex amplification of long DNA fragments. Four CYP2C19 alleles *1 (wt), *2, *3 and *4 were also identified by SNP analysis.ResultsThe prevalence of the CYP2D6 poor metaboliser (PM) genotypes in individuals with fatal intoxication was lower (4.7%) than expected from the frequencies of these genotypes in the blood donors (8.5%). A significantly lower frequency P<0.005 (0.03 with correction according to Bonferroni) was found for the CYP2D6*4 allele among the fatal intoxication cases. The CYP2C19 genotype analyses showed the same results for the fatal intoxication cases and for the blood donors.ConclusionsThe findings in this study confirm our earlier observations of a lower frequency of CYP2D6 PM genotypes in cases of fatal intoxication. To our knowledge, it has not been shown previously that intoxication victims might have a lower frequency of PMs than the general population.

Using X-chromosomal markers in relationship testing: Calculation of likelihood ratios taking both linkage and linkage disequilibrium into account

X-chromosomal markers in forensic genetics have become more widely used during recent years, particularly for relationship testing. Linkage and linkage disequilibrium (LD) must typically be accounted for when using close X-chromosomal markers. Thus, when producing the weight-of-evidence, given by a DNA-analysis with markers that are linked, the normally used product rule is invalid. Here we present an implementation of an efficient model for calculating likelihood ratios (LRs) with markers on the X-chromosome which are linked and in LD. Furthermore, the model was applied on several cases based on data from the eight X-chromosomal loci included in the Mentype(®) Argus X-8 (Biotype). Using a simulation approach we showed that the use of X-chromosome data can offer valuable information for choosing between the alternatives in each of the cases we studied, and that the LR can be high in several cases. We demonstrated that when linkage and LD were disregarded, as opposed to taken into account, the difference in calculated LRs could be considerable. When these differences were large, the estimated haplotype frequencies often had a strong impact and we present a method to estimate haplotype frequencies. Our conclusion is that linkage and LD should be accounted for when using the tested set of markers, and the used model is an efficient way of doing so.

Importance of Lp(a) lipoprotein and HLA genotypes in atherosclerosis and diabetes

Lp(a) lipoprotein [Lp(a)] was found in previous studies to be independently associated with early atherosclerosis and its sequelae. Lp(a) in vitro bound to glucosaminoglycans and was easily aggregated at physiological Ca2+ concentration, and small Lp(a) aggregates were phagocytosed by macrophages. Lp(a) was also found to be related to carbohydrate metabolism, and increased Lp(a) levels have been described in diabetic patients with clinical complications and were recently found in rheumathoid arthritis patients. In this study of nondiabetic male patients with documented CAD before 50 years of age and controls, a significant correlation was found between Lp(a) and IGF‐1 levels. HLA class II DR13 (DR6) was more frequent and DR15 (DR2) was less frequent in patients than in controls. The calculated relative risk for CAD was 4.0 for DR17 (DR3), but the difference was not significant. These differences seem to be related to high Lp(a) levels. It is suggested that phagocytosis of preferably Lp(a) aggregates can induce an immunological tissue response that may contribute in the pathogenesis of Lp(a)‐associated diseases and may be more prominent in combination with some inherited HLA class II haplotypes. Probably due to sex hormone effects, the association may be most pronounced in young males and in older females.

Autosomal SNP typing of forensic samples with the GenPlex TM HID System: Results of a collaborative study

The GenPlex™ HID System (Applied Biosystems - AB) offers typing of 48 of the 52 SNPforID SNPs and amelogenin. Previous studies have shown a high reproducibility of the GenPlex™ HID System using 250-500pg DNA of good quality. An international exercise was performed by 14 laboratories (9 in Europe and 5 in the US) in order to test the robustness and reliability of the GenPlex™ HID System on forensic samples. Three samples with partly degraded DNA and 10 samples with low amounts of DNA were analyzed in duplicates using various amounts of DNA. In order to compare the performance of the GenPlex™ HID System with the most commonly used STR kits, 500pg of partly degraded DNA from three samples was typed by the laboratories using one or more STR kits. The median SNP typing success rate was 92.3% with 500pg of partly degraded DNA. Three of the fourteen laboratories counted for more than two thirds of the locus dropouts. The median percentage of discrepant results was 0.2% with 500pg degraded DNA. An increasing percentage of locus dropouts and discrepant results were observed when lower amounts of DNA were used. Different success rates were observed for the various SNPs. The rs763869 SNP was the least successful. With the exception of the MiniFiler™ kit (AB), GenPlex™ HID performed better than five other tested STR kits. When partly degraded DNA was analyzed, GenPlex™ HID showed a very low mean mach probability, while all STR kits except MiniFiler™ had very limited discriminatory power.