Calculating LRs for presence of body fluids from mRNA assay data in mixtures.

Messenger RNA (mRNA) profiling can identify body fluids present in a stain, yielding information on what activities could have taken place at a crime scene. To account for uncertainty in such identifications, recent work has focused on devising statistical models to allow for probabilistic statements on the presence of body fluids. A major hurdle for practical adoption is that evidentiary stains are likely to contain more than one body fluid and current models are ill-suited to analyse such mixtures. Here, we construct a likelihood ratio (LR) system that can handle mixtures, considering the hypotheses H1: the sample contains at least one of the body fluids of interest (and possibly other body fluids); H2: the sample contains none of the body fluids of interest (but possibly other body fluids). Thus, the LR-system outputs an LR-value for any combination of mRNA profile and set of body fluids of interest that are given as input. The calculation is based on an augmented dataset obtained by in silico mixing of real single body fluid mRNA profiles. These digital mixtures are used to construct a probabilistic classification method (a 'multi-label classifier'). The probabilities produced are subsequently used to calculate an LR, via calibration. We test a range of different classification methods from the field of machine learning, ways to preprocess the data and multi-label strategies for their performance on in silico mixed test data. Furthermore, we study their robustness to different assumptions on background levels of the body fluids. We find logistic regression works as well as more flexible classifiers, but shows higher robustness and better explainability. We test the system's performance on lab-generated mixture samples, and discuss practical usage in case work.

Systemic analysis and zygosity determination of the RHD gene in a D-negative Chinese Han population reveals a novel D-negative RHD gene.

BACKGROUND AND OBJECTIVES: The aim of this study was to systemically analyse the genetic background of D negativity in a Chinese Han population. MATERIALS AND METHODS: DNA of 74 D-negative samples was analysed by using an RHD multiplex polymerase chain reaction (MPX PCR) for the presence of RHD and by PCR-restriction fragment length polymorphism (PCR-RFLP) for RHD zygosity determination. Sixty-five samples were additionally analysed by using real-time quantitative PCR on RHD exon 7. RHD exon-specific sequencing was performed on discrepant samples. RESULTS: Forty-six samples (62%) showed the absence of RHD-specific exons by RHD MPX PCR and homozygous RHD negativity by PCR-RFLP. Twenty-two samples (30%) showed a 1227G>A mutation, characteristic for the Del phenotype. Five (7%) samples showed all characteristics of the RHD(1-2)-CE(3-9)-D(10) hybrid gene. One sample (1.4%) showed a novel 933C>A nonsense mutation in RHD exon 6, which resulted in a premature stop codon. CONCLUSIONS: The RHD gene deletion, RHD-CE-D hybrid genes and one novel 93C>A mutation were found to be the three mechanisms that cause D negativity in our samples. The 1227G>A Del mutation was found to be the major cause of discrepant results between genotyping and phenotyping strategies, favouring genotyping of D-negative samples.

The applicability of different PCR-based methods for fetal RHD and K1 genotyping: a prospective study.

The applicability of different PCR-based assays for fetal RHD and K1 genotyping using DNA isolated from uncultured amniotic fluid cells has been tested prospectively: cord blood serotyping served as a control. For RHD genotyping, DNA was amplified with PCRs specific for RHD exon 7, the 3'-non-coding region and intron 4, using standard conditions. The results of these three separate assays were compared to those of a newly-developed multiplex PCR, simultaneously amplifying six regions of RHD. The PCRs analysing the 3'-non-coding region or intron 4 often yielded false-negative results or no results at all. Results of the exon 7 PCR and of the multiplex PCR always corresponded with postnatal serotyping, the multiplex PCR having the advantage of analysing six RHD-specific exons simultaneously. For K1 genotyping, two different PCR-based assays, both analysing the presence of T578C in the KEL gene, were applied. With the first method, a consensus 740-bp product of the KEL gene was amplified and subsequently specifically digested. As we were not able to obtain any PCR product from amniotic fluid DNA, we developed a new K1-specific PCR, amplifying a fragment of 91 bp only in cases of K1-positivity. With this PCR, all K1 genotyping results (n=30) correctly predicted the phenotypes. We conclude that fetal RHD and K1 genotyping can be performed reliably with DNA from uncultured amniotic fluid cells.

Rhd and C/cE/e genotyping in Slovenian population.

The Rhesus (Rh) blood group system is, after ABO, clinically most important. Alloantibodies directed against Rh antigens are the major cause of a haemolytic disease of newborn (HDN) and of transfusion reactions. In search for novel methods for Rh genotyping we started to compare Rh genotypes identified from different tissues and Rh phenotypes. Genotypes determined from blood samples with PCR based RhD, C/c and E/e genotyping methods were compared with serologically identified phenotypes (N=32). With two exceptions the results of phenotyping and genotyping were in concordance. Two Rh serotypes from a Slovenian family that were unexpected according to the Mendelian laws were characterised genotypically. The two family members were suspected to have a chromosomal deletion on RH gene locus.

DAR, a new RhD variant involving exons 4, 5, and 7, often in linkage with ceAR, a new Rhce variant frequently found in African blacks.

The highly polymorphic Rh system is encoded by 2 homologous genes RHD and RHCE. Gene rearrangements, deletions, or point mutations may cause partial D and CE antigens. In this study, a new RHD variant, DAR, and a new RHCE variant, ceAR, are described in 4 Dutch African Blacks. Serologically, DAR showed weaker reactions with a monoclonal antibody and polyclonal antiserum against D. The DAR phenotype was characterized by complete loss of at least 9 of 37 Rh D epitopes. Erythrocytes expressing ceAR were all typed as VS(-), V(+). DNA analysis showed a partial D allele with only 3 mutations: C602G (exon 4), T667G (exon 5), and T1025C (exon 7). The ceAR allele carried G48C (exon 1), a hybrid exon 5 (A712G, C733G, A787G, and T800A), and A916G (exon 6). To study the frequency of these variants, 326 South-African Blacks was screened genomically. Of the 326 donors, 16 (4.9%) carried the DAR allele, 20 (6.1%) the ceAR allele, and 14 (4.3%) both mutated alleles. Five of these donors (1.5%) had the DAR phenotype, indicating that they carried the DAR allele homozygously or next to a D-negative allele. Immunogenicity of the D antigen for individuals with the DAR phenotype was proven, because 1 of the 4 Dutch individuals produced allo-antibodies against D after multiple transfusions with D-positive blood. In a multiethnic society, the prevalence of this D phenotype will increase and is therefore relevant in transfusion practice and in prevention of hemolytic disease of the newborn.

[Prenatal typing of Rh- and Kell- blood group system antigens]. / Prenatale typering van de bloedgroepantigenen van het Rh- en het Kell-systeem.

Rhesus (Rh) and Kell blood group immunisations are the most frequent causes of haemolytic disease of the newborn. Recently, the molecular bases of the Rh and Kell antigens have been elucidated. Subsequently, specific polymerase chain reactions (PCRs) could be developed to determine the RhD, RhC/Rhc and RhE/Rhe genotypes as well as the KI genotype (from the Kell blood group) with genomic DNA. The tests were applied to genomically determine the foetal Rh and Kell blood groups with DNA obtained from amniotic fluid cells. The genotypes obtained were compared with the Rh phenotypes established by cord blood red cell serology. The PCRs to determine the RhD, Rhc, RhE and Rhe and KI genotypes were found to be reliable. The test for RhC however, resulted in false-positive C genotypes. Indeed, more than half of the subsequently tested C-negative Negroid donors were false-positive with the DNA test. Thus, except for RhC, it is possible to reliably determine the Rh and KI genotypes of a foetus with DNA isolated from amniotic fluid cells. Amniocentesis, however, carries a risk for the pregnancy and therefore the tests will only be justified in pregnant women in whom an antibody has been detected and the father of the foetus is heterozygous for the specific antigen. Recently foetal RhD genotypes were determined in foetal DNA circulating in the plasma of RhD-negative pregnant women. This could eventually lead to the introduction of assays with which the foetal blood group can be determined without any risk to the foetus.

Genotyping of RHD by multiplex polymerase chain reaction analysis of six RHD-specific exons.

BACKGROUND: Qualitative RHD variants are the result of the replacement of RHD exons by their RHCE counterparts or of point mutations in RHD causing amino acid substitutions. For RHD typing, the use of at least two RHD typing polymerase chain reaction (PCR) assays directed at different regions of RHD is advised to prevent discrepancies between phenotyping and genotyping results, but even then discrepancies occur. A multiplex RHD PCR based on amplification of six RHD-specific exons in one reaction mixture is described. STUDY DESIGN AND METHODS: Six RHD-specific primer sets were designed to amplify RHD exons 3, 4, 5, 6, 7, and 9. DNA from 119 donors (87 D+, 14 D- and 18 with known D variants; whites and nonwhites) with known Rh phenotypes was analyzed. RESULTS: All six RHD-specific exons from 85 D+ individuals were amplified, whereas none of the RHD exons from 13 D- individuals were amplified. Multiplex PCR analysis showed that the genotypes of two donors typed as D+ were DIVa and DVa. Red cell typing confirmed these findings. From all D variants tested (DIIIc, DIVa, DIVb, DVa, DVI, DDFR, DDBT) and from RoHar, RHD-specific exons were amplified as expected from the proposed genotypes. CONCLUSION: The multiplex PCR assay is reliable in determining genotypes in people who have the D+ and partial D phenotypes as well as in discovering people with new D variants. Because the multiplex PCR is directed at six regions of RHD, the chance of discrepancies is markedly reduced. The entire analysis can be performed in one reaction mixture, which results in higher speed, higher accuracy, and the need for smaller samples. This technique might be of great value in prenatal RHD genotyping.

The VS and V blood group polymorphisms in Africans: a serologic and molecular analysis.

BACKGROUND: VS and V are common red cell antigens in persons of African origin. The molecular background of these Rh system antigens is poorly understood. STUDY DESIGN AND METHODS: Red cells from 100 black South Africans and 43 black persons from Amsterdam, the Netherlands, were typed serologically for various Rh system antigens. Allele-specific polymerase chain reaction and sequencing of polymerase chain reaction products were used to analyze C733G (Leu245Val) and G1006T (Gly336Cys) polymorphisms in exons 5 and 7 of RHCE and the presence of a D-CE hybrid exon 3. RESULTS: The respective frequencies of all VS+ and of VS+ V-(r's) phenotypes were 43 percent and 9 percent in the South Africans and 49 percent and 12 percent in the Dutch donors. All VS+ donors had G733 (Val245), but six with G733 were VS- (4 V+w, 2 V-). The four VS- V+w donors with G733 appeared to have a CE-D hybrid exon 5. T1006 (Cys336) was present in 12 percent and 16 percent of donors from the two populations. With only a few exceptions, T1006, a D-CE hybrid exon 3, and a C410T (Ala137Val) substitution were associated with a VS+ V-phenotype ((C)ces or r's haplotype). Two VS+ V-individuals, with the probable genotype, (C)ces/(C)ces), were homozygous for G733 and for T1006. CONCLUSIONS: It is likely that anti-VS and anti-V recognize the conformational changes created by Val245, but that anti-V is sensitive to additional conformational changes created by Cys336.

Effects of corticotrophin-releasing hormone, vasopressin and insulin-like growth factor-I on proliferation of and adrenocorticotrophic hormone secretion by canine corticotrophic adenoma cells in vitro.

Extrinsic factors such as hypothalamic hormones or intrapituitary growth factors may stimulate clonal expansion of a genomically altered cell and therefore play a role in pituitary tumorigenesis. Here we report on the effects of the hypophysiotrophic hormones corticotrophin-releasing hormone (CRH) and vasopressin (AVP) and the intrapituitary growth factor insulin-like growth factor-I (IGF-I) on the proliferation of, as measured by the bromodeoxyuridine labelling index, and ACTH secretion by normal canine pituitary cells and corticotrophic adenoma cells of dogs with pituitary-dependent hyperadrenocorticism. The sensitivity to inhibition by cortisol was analysed under various conditions. Under basal conditions, no significant differences were found in the bromodeoxyuridine labelling indices between control cells and tumour cells. CRH, AVP, IGF-I and cortisol had no effect on the proliferation of canine pituitary cells or canine corticotrophic adenoma cells. In contrast with normal pituitary cells, the proliferation of corticotrophic adenoma cells was stimulated by fetal calf serum (FCS). This FCS-induced proliferation was not inhibited by cortisol. The CRH-induced ACTH secretion by corticotrophic adenoma cells was significantly (P < 0.05) lower than that by normal pituitary cells after 4 h incubation with CRH. Incubation with cortisol for 24 h resulted in reduced ACTH secretion under basal and AVP- or IGF-I-stimulated conditions. The relative inhibition was, however, significantly (P < 0.05) lower in ACTH-producing tumour cells than in normal pituitary cells. Cortisol did not inhibit the CRH-induced ACTH secretion in normal pituitary cells after 24 h. In conclusion, canine corticotrophic adenomas are less sensitive to stimulation by CRH and less sensitive to inhibition by glucocorticoids. These tumours have an aberrant sensitivity to a growth-promoting factor present in FCS. This factor may have an important role in the growth promotion of canine corticotrophic tumours.

Molecular screening for somatic mutations in corticotropic adenomas of dogs with pituitary-dependent hyperadrenocorticism.

Pituitary tumorigenesis is now generally regarded as a multistep process of genomic damage leading to uncoupling of interdependent systems that control cell proliferation and differentiation. The alterations include mutations in genes encoding for proteins involved in signal transduction pathways, such as G-proteins and the p21 protein encoded for by the ras genes. Apart from their excessive secretion of ACTH, corticotropic adenomas are characterized by decreased sensitivity to inhibition by glucocorticoids. Therefore, mutations in the glucocorticoid receptor leading to decreased sensitivity to glucocorticoids may contribute to corticotropic tumor formation. In this study, 16 corticotropic adenomas of dogs with pituitary-dependent hyperadrenocorticism were screened for mutations in the Gs alpha, H-, K-, N-ras genes and the coding region of the DNA-binding domain of the glucocorticoid receptor. The cDNA fragment of the Gs alpha gene encompassed codons 159-240. The K-, and N-ras fragments spanned codons 1-71. The H-ras gene was only screened for mutations in codons 12/13 by direct sequencing of the PCR product. The cDNA fragment of the DNA-binding domain of the glucocorticoid receptor encompassed codons 410-500. The Gs alpha, K-ras, N-ras genes and the DNA-binding domain of the glucocorticoid receptor were screened by single-strand conformation polymorphism analysis. No mutations were found in the Gs alpha gene, the ras genes and the DNA-binding domain of the glucocorticoid receptor. It is concluded that mutations in the Gs alpha gene (codons 159-240), the K- and N-ras genes (codons 1-71), the H-ras gene (codons 12/13) and mutations in the DNA-binding domain of the glucocorticoid receptor do not play a role in the tumorigenesis of canine corticotropic adenomas.

Proliferation of the murine corticotropic tumour cell line AtT20 is affected by hypophysiotrophic hormones, growth factors and glucocorticoids.

In pituitary-dependent hyperadrenocorticism (Cushing's disease), the disturbed regulation of ACTH secretion is associated with neoplastic transformation of corticotropic cells. As these two phenomena are almost indissolubly connected, it is of prime importance to elucidate the factor(s) that induce corticotropic cell proliferation. Here we report on the effects of hypophysiotrophic hormones and intrapituitary growth factors on the proliferation and hormone secretion of the murine corticotropic tumour cell line AtT20/D16v, as measured by DNA content, and ACTH concentration in culture media. In addition, sensitivity to the inhibitory effect of cortisol was assessed under various conditions. Corticotropin releasing hormone (CRH) and vasopressin (AVP) induced proliferation of AtT20-cells. In contrast to that caused by AVP, the CRH-induced proliferation was associated with increased ACTH secretion, which could be inhibited by cortisol. Insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) also stimulated the proliferation of AtT20-cells. The proliferation of AtT20-cells was significantly inhibited by cortisol in all tests. The IGF-I-induced proliferation was the least sensitive to inhibition by cortisol. The growth factors did not stimulate ACTH secretion but IGF-I differed in that it prevented the inhibition of basal ACTH secretion by cortisol. Additional experiments (Western ligand blot analysis) concerning the relative insensitivity of IGF-I induced proliferation to inhibition by cortisol revealed that IGF-I increased the concentration of a 29 kDa IGF binding protein (IGFBP) in the culture medium. The concentration of the 29 kDa IGFBP was slightly decreased by cortisol.(ABSTRACT TRUNCATED AT 250 WORDS)

Responsiveness to corticotropin-releasing hormone and vasopressin in canine Cushing's syndrome.

Corticotropin-releasing hormone (CRH) and vasopressin are the most important hypothalamic factors regulating adrenocorticotropic hormone (ACTH) secretion. In this study we have investigated the responsiveness of the pituitary-adrenocortical axis to intravenous administration of CRH or lysine vasopressin (LVP) in 16 control dogs, 22 dogs with pituitary-dependent hyperadrenocorticism and five dogs with hyperadrenocorticism due to an adrenocortical tumor, using doses of CRH and LVP that caused equivalent ACTH responses in the control dogs. After CRH administration, the increment in plasma ACTH was significantly (p < 0.05) lower in dogs with pituitary-dependent hyperadrenocorticism (221 +/- 53 ng/l) than that in control dogs (279 +/- 41 ng/l). In the dogs with pituitary-dependent hyperadrenocorticism, the relative increases in ACTH after CRH were significantly (p < 0.05) lower than those after LVP. Despite the absence of an increase in ACTH following LVP administration in dogs with hyperadrenocorticism due to an adrenocortical tumor, there was a significant increase in plasma cortisol, the increment (790 +/- 238 nmol/l) being not statistically different from that in the control dogs (412 +/- 37 nmol/l). We conclude that in spite of the changes inherent to pituitary-dependent hyperadrenocorticism, i.e. neoplastic transformation of corticotropic cells and hypercortisolism, there is persistence of responsiveness to hypophysiotropic hormones. The ACTH secretion by corticotropic cells in pituitary-dependent hyperadrenocorticism was relatively less sensitive to stimulation with CRH than with LVP. Adrenocortical tumors develop an aberrant sensitivity to LVP.

Corticotropin-releasing hormone and adrenocorticotropic hormone concentrations in cerebrospinal fluid of dogs with pituitary-dependent hyperadrenocorticism.

There is still some controversy concerning the question of whether Cushing's disease in man is caused by a primary dysfunction of the pituitary or a hypothalamic disorder. In the latter option, excessive hypothalamic stimulation of pituitary corticotropes would cause or contribute to the genesis of POMC-secreting adenomas. In the present study cerebrospinal fluid (CSF) CRH levels and levels of ACTH and cortisol in CSF and plasma were measured in clinically healthy dogs, in dogs with pituitary-dependent hyperadrenocorticism (PDH), and in dogs with hyperadrenocorticism due to an adrenocortical tumor (ATH). In CSF from dogs with PDH, CRH concentrations (226.6 +/- 14.4 ng/liter) were significantly (P < 0.05) lower than those in control dogs (309.5 +/- 20.3 ng/liter). In the dogs with ATH, CSF CRH concentrations (211.0 +/- 40.3 ng/liter) were in the range of those in PDH dogs. In dogs with ATH, CSF ACTH levels (13.0 +/- 3.0 ng/liter) were significantly (P < 0.05) lower than those in control dogs (63.4 +/- 3.5 ng/liter), whereas in dogs with PDH, the levels (116.8 +/- 47.5 ng/liter) were not different from those in the control group. In control dogs, the concentrations of CSF CRH and plasma ACTH were significantly correlated (r = 0.635; P < 0.01). This functional dependency appeared to be disturbed in dogs with PDH, as in these dogs CSF CRH concentrations did not correlate with plasma ACTH concentrations. It is concluded that continuous hyperstimulation of pituitary corticotropes with hypothalamic CRH is probably not the cause of excessive ACTH secretion in dogs with pituitary-dependent hyperadrenocorticism.