Inter-laboratory evaluation of the EUROFORGEN Global ancestry-informative SNP panel by massively parallel sequencing using the Ion PGM™.

The EUROFORGEN Global ancestry-informative SNP (AIM-SNPs) panel is a forensic multiplex of 128 markers designed to differentiate an individual's ancestry from amongst the five continental population groups of Africa, Europe, East Asia, Native America, and Oceania. A custom multiplex of AmpliSeq™ PCR primers was designed for the Global AIM-SNPs to perform massively parallel sequencing using the Ion PGM™ system. This study assessed individual SNP genotyping precision using the Ion PGM™, the forensic sensitivity of the multiplex using dilution series, degraded DNA plus simple mixtures, and the ancestry differentiation power of the final panel design, which required substitution of three original ancestry-informative SNPs with alternatives. Fourteen populations that had not been previously analyzed were genotyped using the custom multiplex and these studies allowed assessment of genotyping performance by comparison of data across five laboratories. Results indicate a low level of genotyping error can still occur from sequence misalignment caused by homopolymeric tracts close to the target SNP, despite careful scrutiny of candidate SNPs at the design stage. Such sequence misalignment required the exclusion of component SNP rs2080161 from the Global AIM-SNPs panel. However, the overall genotyping precision and sensitivity of this custom multiplex indicates the Ion PGM™ assay for the Global AIM-SNPs is highly suitable for forensic ancestry analysis with massively parallel sequencing.

Population genetic study in Hungarian canine populations using forensically informative STR loci.

Allele frequencies for 10 short tandem repeats (STRs) were determined using the StockMarks® Dog Genotyping Kit (Applied Biosystems) from a pool of 668 unrelated dogs, consisting of 79 different breeds or breed variants from the Hungarian canine population. For the comparative statistical analysis, four pure bred, one mixed group - all individuals except from the four breeds - and considering to unequal representation of breeds the group of all pooled individuals ("All breeds") were distinguished. The forensically informative values - Hardy-Weinberg equilibrium (HWE), observed heterozygosity (H(obs)), polymorphism information content (PIC), power of discrimination (PD), power of paternity exclusion (PE), linkage disequilibrium (LD) and fixation index (F) were determined. The Hungarian pure bred dog populations could be distinguished by comparing the allele frequency values using G-statistics and calculating the F(ST) indices with pair-wise comparisons of inter-population molecular variance (AMOVA). The results showed that these 10 loci can be adequate for individual identification in forensic cases even in relatively inbred dog populations.

Migration rates and genetic structure of two Hungarian ethnic groups in Transylvania, Romania.

Transylvania's ethnic mosaic is composed of Romanians, German Saxons and Hungarians. The ethnic groups of the Hungarian minority that settled in Romania show differences in dialects, customs and religious affiliations. In this study entire mtDNA control region sequences from 360 individuals of Hungarian ethnicity from two populations (the Csángó and the Székely), settled in the historical region of Transylvania in Romania, were generated and analyzed following high quality sequencing standards. Phylogenetic analyses were used for haplogroup determination, quasi-median network analyses were applied for the visualization of character conflicts, and median joining reconstructions were used for depicting haplotype structures. Affiliation of haplotypes to major west Eurasian haplogroups was confirmed using coding region SNPs. Gene flow between the two populations was low and biased towards a higher migration rate from the Csángó to the Székely than vice versa. Phylogeographic analyses revealed effects of genetic isolation within the Csángó population, which is, in its genetic structure, clearly different from the Székely population. The pronounced genetic divergence between the two populations is in sharp contrast to the expectation of high genetic similarity due to the close geographic proximity of their native homelands. The population data will be incorporated in the EMPOP database (http://www.empop.org).

Increased serum albumin, gamma globulin, immunoglobulin IgG, and IgG2 and IgG4 in autism.

BACKGROUND: Research on the biological pathophysiology of autism has found some evidence that immune alterations may play a role in the pathophysiology of that illness. As a consequence we expected to find that autism is accompanied by abnormalities in the pattern obtained in serum protein electrophoresis and in the serum immunoglobulin (Ig) and IgG subclass profile. METHOD: We examined whether subjects with autism showed changes in total serum protein (TSP) and the serum concentrations of albumin, alpha1 globulin, alpha2 globulin, beta globulin and gamma globulins, IgA, IgM and IgG and the IgG subclasses IgG 1, IgG2, IgG3 and IgG4, compared with normal controls. RESULTS: We found significantly increased concentrations of TSP in autistic subjects, which were attributable to increased serum concentrations of albumin and gamma globulin. Serum IgG, IgG2 and IgG4 were also significantly raised. In autism there were significant and positive correlations between social problems and TSP and serum gamma globulin and between withdrawal symptoms and TSP and serum albumin and IgG. CONCLUSIONS: The results suggest that autism is characterized by increased TSP, a unique pattern obtained in serum protein electrophoresis, i.e. increased serum albumin and IgG, and by a specific IgG subclass profile, i.e. increased serum IgG2 and IgG4. The increased serum concentrations of IgGs in autism may point towards an underlying autoimmune disorder and/or an enhanced susceptibility to infections resulting in chronic viral infections, whereas the IgG subclass skewing may reflect different cytokine-dependent influences on autoimmune B cells and their products.

Canine STR analyses in forensic practice. Observation of a possible mutation in a dog hair.

In a case of the death of a 7-year-old boy, the police investigations revealed a possible dog attack contrary to the witness testimonies. DNA investigations were carried out from hairs, saliva and bloodstains with 10 canine-specific STR loci by the use of fluorescently labelled multiplex PCR and the ABI PRISM 310 genetic analyzer. The analysis of one hair sample revealed one allele deviation from the profile of the putative Rottweiler perpetrator possibly caused by a mutation. The PCR fragments in question at the PEZ20 locus were sequenced and compared with the alleles detected in the Hungarian canine population and identified on a repeat number basis. The allele frequencies were determined based on typing of 242 genetically independent canine individuals from 72 breeds. The results suggested that two of the canine individuals could be the perpetrators.

Immunotherapy with interferon-alpha in patients affected by chronic hepatitis C induces an intercorrelated stimulation of the cytokine network and an increase in depressive and anxiety symptoms.

Immunotherapy with interferon-alpha (IFNalpha) may induce depressive symptoms, anxiety and major depression when administered for at least 1-3 months at a dose of 3-10 MUI daily, twice or three times a week. Previously, it has been shown that immunotherapy with interleukin-2 (IL-2) significantly induces the cytokine network, as measured by increases in serum IL-6, IL-10 and the IL-2 receptor (IL-2R), and that the immunotherapy-induced changes in the cytokine network are significantly correlated with the increases in depression ratings. The main aim of this study was to examine the effects of immunotherapy with IFNalpha on the cytokine network in relation to changes in depression and anxiety ratings. Fourteen patients, affected by chronic active C-hepatitis, were treated with IFNalpha (3-6 MUI s.c. three/six times a week for 6 months) and had measurements of serum IFN-gamma (IFNgamma), IL-2, IL-6, IL-6R, IL-8 and IL-10 before starting therapy and 2, 4, 16 and 24 weeks after immunotherapy with IFNalpha. Severity of depression and anxiety were measured with the Montgomery-Asberg Depression Rating Scale (MADRS) and the Hamilton Anxiety Rating Scale (HAM-A), respectively. Repeated measure (RM) design ANOVAs showed significantly higher MADRS and HAM-A scores 2-4 weeks and 4-6 months after starting IFNalpha-based immunotherapy than at baseline. RM design ANOVAs showed significantly higher serum IL-6 and IL-8 levels 2-4 weeks after starting IFNalpha-based immunotherapy and higher serum IL-10 levels 2-4 weeks and 4-6 months after starting therapy than at baseline. There were significant relationships between the IFNalpha-induced changes in serum IL-6 or IL-8 and the depression and anxiety scores. The findings show that IFNalpha-based immunotherapy induces the cytokine network and that IFNalpha-induced increases in IL-6 predicts the development of depressive symptoms. Depressive symptoms following IFNalpha treatment may be secondary to cytokine induction, including that of IL-6.

Resolution of parentage in dogs by examination of microsatellites after death of putative sire: case report.

A case of disputed paternity in dogs is reported. DNA examinations were carried out from hair samples of the individuals several months after the death of the putative sire. Ten short tandem repeat (STR) loci were analysed by fluorescence-labelled multiplex PCR using ABI PRISM 310 Genetic Analyser. Based on the results the candidate sire was included in the pedigree records as the biological sire. In spite of the genetic homogeneity of pedigree dogs due to inbreeding, canine microsatellites can provide an adequate basis for assigning paternity in pure breeds.

Canine microsatellite polymorphisms as the resolution of an illegal animal death case in a Hungarian zoological gardens.

Several animal carcasses were found in the paddocks of a Hungarian County Zoo during 1 week. The 14 animals killed were thought to be the victims of a dogfight training. The primary suspect was the security guard of the Zoo with his guard dogs. DNA tests were carried out on hairs and bloodstains and 10 canine-specific STR loci were analysed by fluorescently labelled multiplex PCR using the ABI PRISM 310 Genetic Analyzer. The results confirmed that the killer was a single animal and all of the guard dogs were excluded.

Variations in primer sequences are the origin of allele drop-out at loci D13S317 and CD4.

STRs have become almost the exclusive tool of genetic scientists in forensic typing work. Consequently, large numbers of samples are genotyped and the detection of rare abnormalities is to be expected. We found rare losses of alleles, also known as drop-out, at the two STR loci D13S317 and CD4. Drop-out at D13S317 was accidentally found in typing of suspects in a murder case and three other examples of drop-out were found at locus CD4 during paternity testing. The lost alleles reappeared when alternative PCR primer pairs were used. Sequences of lost alleles were characterised at the molecular level after cloning. Variations were found in the primer sequences and these are believed to prevent amplification or to reduce amplification yield and to be the origin of the allele drop-out.

Lowered serum dipeptidyl peptidase IV activity is associated with depressive symptoms and cytokine production in cancer patients receiving interleukin-2-based immunotherapy.

There is some evidence that treatment with interleukin-2 (IL-2) and interferon-alpha (IFNalpha) frequently induces depressive symptoms and activation of the inflammatory response system (IRS). There is evidence that major depression is accompanied by lowered serum activity of dipeptidyl peptidase IV (DPP IV; EC 3.4.14.5), a membrane-bound serine protease which catalyses the cleavage of some cytokines and neuro-active peptides and which modulates T cell activation and the production of cytokines, such as IL-2. This study was carried out to examine the effects of immunochemotherapy with IL-2 and IFNalpha, alone and together, in cancer patients on serum DPP IV activity in relation to changes in depressive symptoms and the IRS. The Montgomery and Asberg Rating Scale (MADRS), serum DPP IV activity, and the serum IL-6, and IL-2 receptor (IL-2R) concentrations were measured in 26 patients with metastatic cancers before and three and five days after treatment with IL-2 and IFNalpha, alone or together. Treatment with IL-2 with or without IFNalpha significantly suppressed serum DPP IV activity. The MADRS scores were significantly elevated by treatment with IL-2 with or without IFNalpha, but not IFNalpha alone. The immunochemotherapy-induced decreases in serum DPP IV were significantly and inversely correlated with the increases in the MADRS. Treatment with IL-2 alone or combined with IFNalpha also elevated serum IL-6 and IL-2R. There were significant and inverse correlations between the immuchemotherapy-induced decreases in serum DPP IV and the elevations in serum IL-6 or IL-2R. In conclusion, treatment with IL-2/IFNalpha decreases serum DPP IV activity within 3-5 days and the immunochemotherapy-induced decreases in serum DPP IV activity are significantly and inversely related to treatment-induced increases in severity of depression and signs of activation of the IRS.

Immunological differences between patients with major depression and somatization syndrome.

There is some evidence that major depression is accompanied by activation of the inflammatory response system (IRS). There is also evidence that proinflammatory cytokines and induction of IRS activation are associated with sickness behavior in experimental animals. However, no research has examined the IRS in somatization disorder. The aim of this study was to examine possible immunological differences between major depression, somatization and healthy controls. We measured the following IRS variables in patients with major depression (n=36), somatization syndrome (SSI-8; n=37), major depression and somatization (n=40) and healthy controls (n=37): interleukin-6 (IL-6); interleukin-1-receptor-antagonist (IL-1RA); plasma soluble interleukin-6 receptor (IL-6R); soluble suppressor/cytotoxic antigen (CD8); leukemia inhibitory factor (LIF-R); and Clara cell protein (CC16), an endogenous anticytokine. Serum CD8 concentrations were significantly increased in patients with major depression compared with concentrations in patients with somatization syndrome, whereas concentrations in normal controls were intermediate between those of the two groups of patients. Serum CC16 was significantly lower in major depression than in healthy controls. The highest CC16 scores were found in patients with somatization syndrome. Somatizing patients have significantly lower serum IL-6 values than normal controls and depressed patients. The present results indicate (1) an activation of the IRS in depression with signs of T-cell activation (increased CD8), monocytic activation (IL-1RA) and a lowered anti-inflammatory capacity of the serum (lower CC16) and (2) an immune alteration in somatizing syndrome, such as monocytic activation (increased IL-1RA) and indicators of lowered T-lymphocytic activity (lowered CD8 and IL-6). These results suggest different immune alterations in somatization syndrome and depression.

The effects of noradrenaline and alpha-2 adrenoceptor agents on the production of monocytic products.

There are some reports that catecholamines may modulate the production of monocytic cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF alpha). The present study was carried out in order to examine the effects of noradrenaline (10(-5), 10(-6) and 10(-7) M), clonidine (10(-5), 10(-6) and 10(-7) M), an alpha2-adrenoceptor agonist, and yohimbine (10(-5), 10(-6) and 10(-7) M), an alpha 2-adrenoceptor antagonist, on the production of IL-6, the IL-1 receptor antagonist (IL-1RA) and TNF alpha by stimulated whole blood of normal humans. We measured the in vitro production of IL-6, TNF alpha and IL-1RA by stimulated (phytohemagglutinin+lipopolysaccharide), diluted whole blood of 16 normal volunteers. The results show that noradrenaline, 10(-5) M, significantly suppressed the production of IL-6; noradrenaline, 10(-5) and 10(-6) M, significantly suppressed the production of IL-1RA and TNF alpha; clonidine, 10(-5) M, significantly suppressed the production of TNF alpha; and yohimbine, 10(-5) and 10(-6) M, significantly suppressed the production of IL-1RA. It is concluded that (1) noradrenaline has significant negative immunoregulatory effects in humans through suppression of the production of (monocytic) proinflammatory cytokines, e.g. IL-6 and TNF alpha, and (2) the suppression of the production of TNF alpha may be related to alpha(2)-adrenoceptor-related mechanisms.

Analysis of eight STR loci in two Hungarian populations.

A multiplex reaction for the eight STR loci D3S1358, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820 was used to generate allele frequency databases for two Hungarian population samples, Caucasians from the Budapest area and Romanies from Baranya county. During the analysis two intermediate-sized alleles and a sequence variant allele were observed at the D7S820 locus. All three types of allelic variants were found to have modifications in the same block of a (T)9 stretch located within the 3' flanking region of each allele, which may indicate a possible higher mutation rate of this (T)9 block. For the loci D3S 1358 and D7S820 the Romany population database showed departures from Hardy-Weinberg equilibrium. The forensic efficiency values for the Romany population were slightly different from those found in the Hungarian Caucasian population. Comparing the allele frequency values by G-statistic, calculating the F(ST) indices and with the pair-wise comparisons of interpopulation variance, the two Hungarian populations could be distinguished using data from the eight STR loci.

Analysis of eight STR loci in two Hungarian populations.

A collection of eight STR loci (D3S1358, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820) was used to generate allele frequency databases for two Hungarian population samples, Caucasians from the Budapest area and Romanies from Baranya county. During the analysis two intermediate sized alleles and a sequence variant allele were observed at the D7S820 locus. All three types of allelic variants were found to have modification (deletion, insertion, transversion) in the same block of a (T)(9) stretch located within the 3' flanking region of each allele, which may indicate a possible higher mutation rate of this (T)(9) block. For the loci D3S1358 and D7S820 the Romany population database showed departures from Hardy-Weinberg equilibrium. The forensic efficiency values for the Romany population were slightly different from those found in the Hungarian Caucasian population. Comparing the allele frequency values by G-statistic, calculating the F(st) indices and with the pairwise comparisons of inter-population variance, the two Hungarian populations could be distinguished using data of the eight STR loci.

Measurement of electrical resistance after nerve injuries of the hand.

The authors measured electrical resistance of skin to define the sensory loss. A significant increase of the skin resistance was observed in the zone of sensory loss, as compared with the skin surfaces of normal innervation. The sensory map, sweating map (ninhydrine test) and the skin resistance map were also compared by the authors. The main advantages of the electrical skin resistance test are that it is a quantitative one, and takes less time than the other methods.