Inhibition of human basophil activation by high dilutions of histamine

A protocol based on anti-IgE induced human basophil activation was set up to allow the intra-assay calculation of basophil count significance. In 6 successive experiments, we demonstrated the normality of the basophil count distribution through a comparison of the mean values by Student's t-test. We confirm the inhibition of anti-IgE induced human basophil activation by histamine dilutions ranging from 10^–10 M to 10^–38 M, the most frequent inhibition observed being for 10^–18, 10^–20, and 10^–32 M, in agreement with our previous results.     

Genetic characterization of 15 autosomal STRs in the interior Sindhi population of Pakistan and their phylogenetic relationship with other populations

Genetic structure of a population can be influenced by evolutionary processes and cultural histories which can alter the frequencies of different variants at particular genetic markers. These characteristics make DNA evidence suitable for forensic applications. Little relevant data are available from the interior Sindhi population; thus, in the current study, we have investigated 15 autosomal STRs in 181 unrelated individuals belonging to the interior parts of Sindh Pakistan, to establish its lineage and parameters of forensic interest. These STRs revealed a high power of discrimination (CPD), power of exclusion (CPE) and matching probability (CMP) are 0.9999999999999999968997, 0.99998612 and 3.1003 × 10^–18 respectively. The genetic distances, neighbour‐joining (NJ) tree, interactivity test and principal component analysis (PCA) based on 15 autosomal STR loci showed that the interior Sindhi population had a closer genetic relationship with Pakistani populations and distant relationships with regional (India and Afghanistan) populations. The present findings exhibited that STRs included in AmpFLSTR Identifiler kit (Applied Biosystems) are genetically polymorphic in the interior Sindhi population of Pakistan. This study provides valuable population genetic data for the genetic information study, forensic human individual identification and paternity testing.     

Predicción del número de linfocitos T CD4 en sangre periférica a partir de teoría de conjuntos y probabilidad en pacientes con VIH/SIDA

Previously a refinement of a methodology was developed based on set theory and probability, with the aim of improving the predictive ability of CD4 T cells (LT-CD4) for HIV/AIDS from the total number of leukocytes and lymphocytes. In this work the clinical applicability of the method was shown by refining prediction in 150 samples. Taking data of leukocytes/mm³, lymphocytes/mm³, and LT-CD4 cells/mm³ of each patient, called triples, they were organized from highest to lowest based on the number of leukocytes, to evaluate ranges of 1,000 leukocytes. The membership of the triples to the set A∪C, B∪D was established, as well as the intersection between the two sets (A∪B)∩(B∪D), in which a prediction of the number of CD4 associated with specific values of leukocytes and lymphocytes is established. The number of elements belonging to each set was counted and the probability of belonging to each of the ranges tested was determined. A total of 7 out of the 9 ranges of leukocytes measured showed a probability of success equal to or greater than 0,76, achieving a probability of 1 in the ranks lower than 4.000/mm³ and 3.000/mm³, respectively. The ability of the methodology for determining the value of the LT-CD4 was demonstrated, achieving a higher predictive capacity than the unrefined methodology. The evidence shows that the applied methodology is effective for clinical use, thus leading to a reduction of costs and resources.     

Improved Molecular Typing Assay for Rhinovirus Species A,B, and C

Human rhinoviruses (RVs), comprising three species (A, B, and C) of the genus Enterovirus, are responsible for the majority of upper respiratory tract infections and are associated with severe lower respiratory tract illnesses such as pneumonia and asthma exacerbations. High genetic diversity and continuous identification of new types necessitate regular updating of the diagnostic assays for the accurate and comprehensive detection of circulating RVs. Methods for molecular typing based on phylogenetic comparisons of a variable fragment in the 5′ untranslated region were improved to increase assay sensitivity and to eliminate nonspecific amplification of human sequences, which are observed occasionally in clinical samples. A modified set of primers based on new sequence information and improved buffers and enzymes for seminested PCR assays provided higher specificity and sensitivity for virus detection. In addition, new diagnostic primers were designed for unequivocal species and type assignments for RV-C isolates, based on phylogenetic analysis of partial VP4/VP2 coding sequences. The improved assay was evaluated by typing RVs in >3,800 clinical samples. RVs were successfully detected and typed in 99% of the samples that were RV positive in multiplex diagnostic assays.     

An improved method for purifying human thymic dendritic cells

Thymic dendritic cells (DC) play a prominent role in the immune response as they constitute a key element involved in the maturation of thymocytes in the thymus. Human thymic DC, like DC from other lymphoid organs, represent a minor cell population (< 2%) of the thymus. Since these cells cannot replicate in vitro, the development of efficient purification methods is an essential prerequisite for extensive functional studies. DC express high levels of HLA-DR, a cell surface marker of the MHC class II antigen which is not exclusive to DC. Since no specific human thymic DC marker has been identified so far, DC purification methods are mainly based on depletion of particular subgroups of cells. We report here an improved method for purifying human thymic dendritic cells. In contrast to prior work, CD2⁺ thymocytes were first depleted by rosetting with neuraminidase treated sheep red blood cells. The nonrosetted cells were separated in a Percoll gradient, and the low-density cells were subsequently depleted of nondendritic cells by using thymocyte and macrophage specific monoclonal antibodies and either magnetic bead depletion or cytofluorometry. Cell populations (18–55 × 10⁶ cells) obtained following magnetic bead purification were at least 80% HLA-DR⁺/CD2⁻ and exhibited ultrastructural morphological features and functional activities such as those described previously for thymic DC. This improved method was compared with different purification approaches that use various combinations of cell density-based separation techniques and cell surface specific markers antibody reactivity. The magnetic beads depletion approach provided higher yields.     

Genetic composition of Teochew population in Chaozhou revealed by 19 STR loci: population polymorphism and interpopulation comparisons

Abstract

Aim: To investigate the genetic polymorphisms of 19 STR loci in the Teochew population of Chaozhou.

Subjects and methods: Nineteen STR loci of 631 unrelated Teochew people in Chaozhou were detected by using the AGCU Expressmarker 20 kit.

Results: The allele frequency ranged from 0.0008–0.5577. The combined power of discrimination and combined probability of excluding paternity is over 0.999,999,999,999,999,999,999,999,999,999 and 0.999,999,992,549,546, respectively. The principal component analysis based on the 11 shared STR loci showed the Teochew population was most genetically related to the Fujian Han population.

Conclusion: The results demonstrate the set of 19 autosomal STRs could provide robust genetics information for individual identification, paternity testing and human genetics research in the Teochew population.     

Direct Identification of Urinary Tract Pathogens from Urine Samples by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been suggested as a reliable method for bacterial identification from cultures. Direct analysis of clinical samples might increase the usefulness of this method, shortening the time for microorganism identification. We compared conventional methods for the diagnosis of urinary tract infections (UTIs) and identification of the urinary tract pathogens (automated screening, plate cultures, and identification based on biochemical characteristics) and a fast method based on conventional screening and MALDI-TOF MS. For this latter method, 4 ml of urine was centrifuged at a low-revolution setting (2,000 × g) to remove leukocytes and then at high revolutions (15,500 × g) to collect bacteria. The pellet was washed and then applied directly to the MALDI-TOF MS plate. Two hundred sixty urine samples, detected as positive by the screening device (UF-1000i), were processed by culture and MALDI-TOF MS. Twenty samples were positive in the screening device but negative in culture, and all of them were also negative by MALDI-TOF MS. Two-hundred thirty-five samples displayed significant growth of a single morphological type in culture. Two-hundred twenty of them showed bacterial growth of >10⁵ CFU/ml. Microorganism identifications in this group were coincident at the species level in 202 cases (91.8%) and at the genus level in 204 cases (92.7%). The most frequent microorganism was Escherichia coli (173 isolates). MALDI-TOF MS identified this microorganism directly from the urine sample in 163 cases (94.2%). Our results show that MALDI-TOF MS allows bacterial identification directly from infected urine in a short time, with high accuracy, and especially when Gram-negative bacteria with high bacterial counts are involved.Urinary tract infections (UTIs) are among the most common human bacterial infections. More than 80% of uncomplicated UTIs are caused by Escherichia coli (9). Other common uropathogens include Staphylococcus saprophyticus in uncomplicated UTIs and Gram-negative rods (enterobacteria other than E. coli or Pseudomonas aeruginosa) and Gram-positive cocci in complicated UTIs (9). UTIs affect an estimated 1 out of 3 women before the age of 24 (7). Up to 40 to 50% of the female population will develop a symptomatic UTI at some time during their life, and about 33% of women with acute uncomplicated UTIs have frequent recurrences (7).Several tests are available for screening patients for UTIs, including urine dipstick testing, urinalysis, Gram staining, and urine culture. Urine culture is the “gold standard” for defining the diagnosis of UTIs, because it allows the quantification and identification of the uropathogenic species. However, this method is time-consuming and expensive. Up to 70% of urine cultures are negative, with high costs for unnecessary testing (4). Thus, automated analyzers for UTI screening to rapidly identify negative samples that can be reported promptly to clinicians are now widely used. Samples positive in this screening are cultured to count colonies and identify the microorganism.Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been suggested as a fast and reliable method for bacterial identification (14), based on the characteristic protein profiles for each microorganism. Databases have been developed that include the main pathogenic microorganisms, thus allowing the use of this method in routine bacterial identification from plate cultures.Direct analysis of clinical samples may further increase the usefulness of this method, since it can significantly shorten the time required for the identification of microorganisms. Here we compared the conventional methods for UTI diagnosis and identification of urinary tract pathogens in microbiology laboratories (automated screening and plate culture) and a fast method based on conventional screening plus MALDI-TOF MS.     

CD11c+ CD103+ cells of Mycobacterium tuberculosis‐infected C57BL/6 but not of BALB/c mice induce a high frequency of interferon‐γ‐ or interleukin‐17‐producing CD4+ cells

The magnitude of the cellular adaptive immune response is critical for the control of Mycobacterium tuberculosis infection in the chronic phase. In addition, the genetic background is equally important for resistance or susceptibility to tuberculosis. In this study, we addressed whether lung populations of dendritic cells, obtained from genetically different hosts, would play a role in the magnitude and function of CD4⁺ populations generated after M. tuberculosis infection. Thirty days post-infection, C57BL/6 mice, which generate a stronger interferon-γ (IFN-γ)-mediated immune response than BALB/c mice, exhibited a higher number and frequency of lung CD11c⁺ CD11b⁻ CD103⁺ cells compared with BALB/c mice, which exhibited a high frequency of lung CD11c⁺ CD11b⁺ CD103⁻ cells. CD11c⁺ CD11b⁻ CD103⁺ cells, purified from lungs of infected C57BL/6 mice, but not from infected BALB/c mice, induced a higher frequency of IFN-γ-producing or interleukin-17 (IL-17)-producing CD4⁺ cells. Moreover, CD4⁺ cells also arrive at the lung of C57BL/6 mice faster than in BALB/c mice. This pattern of immune response seems to be associated with higher gene expression for CCL4, CCL19, CCL20 and CCR5 in the lungs of infected C57BL/6 mice compared with infected BALB/c mice. The results described here show that the magnitude of IFN-γ-producing or IL-17-producing CD4⁺ cells is dependent on CD11c⁺ CD11b⁻ CD103⁺ cells, and this pattern of immune response is directly associated with the host genetic background. Therefore, differences in the genetic background contribute to the identification of immunological biomarkers that can be used to design human assays to predict progression of M. tuberculosis infection.     

Molecular phylogeny of leader proteinase gene of type A of <Emphasis Type="Italic">Foot-and-mouth disease virus</Emphasis> from India

Summary. We previously demonstrated the presence of three genotypes (IV, VI and VII) of type A (subtype A22) of Foot-and-mouth disease virus (FMDV) in India based on 1D gene sequence analysis. In the present study, the leader proteinase (L^pro) gene sequences of 35 type A FMDV field isolates sampled over a period of 24 years (1977–2000) have been analyzed. Maximum-likelihood (ML) phylogenetic analysis revealed four distinct genetic lineages (A–D), indicating high divergence in L gene of type A FMDV. Lineages A and D correspond to the earlier genotypes IV, and VII, respectively. The genotype VI isolates were divided into two separate lineages (B and C) in the tree with high confidence values (96–99%). One isolate (IND 21/90) showed incongruous grouping between the (1D and L) gene-based analyses, which could be due to intergenotypic recombination. The ML molecular clock hypothesis was easily rejected on the data set studied indicating the absence of clock-like evolution in the L gene. ML codon-substitution models identified positive selection in the amino acid site 194 of C-terminal extension of the Lb^pro with high posterior probability (>99%). The catalytic triad of the L^pro at C-51, H-148 and D-163 were conserved across all the Indian isolates studied. Finally, amino acid differences between the lineages have been discussed briefly.     

Immunohistochemical Localization of Somatostatin Receptors sst2A in Human Tumors

Human tumors frequently express somatostatin receptors. However, none of the receptor subtype proteins have been individually visualized in normal or neoplastic human tissues. Here, the distribution of the sst2A receptor was investigated using immunohistochemistry with the specific anti-peptide antibody R2–88 in 47 human tumors. All tumors selected for their abundance of sst2 mRNA and/or strong binding of the sst2-preferring ligand ¹²⁵I-labeled Tyr³-octreotide were specifically immunostained with R2–88. Conversely, all tumors without somatostatin binding or expressing predominantly other somatostatin receptor subtype mRNAs (sst1 or sst3) were not specifically immunostained by R2–88. Specificity was shown in immunoblots, demonstrating receptor migration as a 70-kd broad band. In immunohistochemical and immunoblotting experiments, the abolition of staining after antibody blockade with antigen peptide was demonstrated. Immunostaining was identified in cryostat and in formalin-fixed, paraffin-embedded sections. Heat-induced epitope retrieval was necessary to visualize sst2A receptors in formalin-fixed sections. Moreover, because of occasional high nonspecific staining, the demonstration of complete abolition of immunostaining by treatment with antigen peptide was a prerequisite for the correct identification of sst2A-positive tumors. The sst2A receptors were clearly located at the membrane of the tumor cells. These results provide the first localization of a somatostatin receptor subtype in human tissues at the cellular level. The sst2A receptor identification and visualization in tumors with simple immunohistochemical methods in formalin-fixed, paraffin-embedded material will open new diagnostic opportunities for pathologists.     

Branched-DNA Assay for Detection of the mecA Gene in Oxacillin-Resistant and Oxacillin-Sensitive Staphylococci

The identification of methicillin-resistant staphylococcus isolates in the clinical laboratory has typically been performed by using methods that detect phenotypic expression of resistance determinants. However, these methods may be difficult to interpret and some isolates do not express resistance until selective pressure is administered. Assays that detect genetic determinants are not subject to these limitations and have been effective in distinguishing isolates that are capable of expressing the resistance phenotype. In this study, a novel branched-DNA (bDNA) hybridization assay was used to test for the mecA gene in 416 clinical staphylococcal isolates. The results were compared with those obtained by a PCR-based assay and oxacillin disk diffusion. For 155 Staphylococcus aureus and 261 coagulase-negative Staphylococcus isolates, the bDNA assay and PCR results were 100% concordant. Among the S. aureus isolates, 20 were MecA⁺ and 135 were MecA⁻. For the coagulase-negative staphylococci, 150 were MecA⁺ and 111 were MecA⁻. The results from the genotypic detection methods were compared with those obtained by oxacillin disk diffusion. No discrepancies were detected among the S. aureus isolates; however, 10 coagulase-negative isolates were MecA⁺ but oxacillin sensitive and 1 isolate was MecA⁻ but oxacillin resistant. Oxacillin resistance was induced in 6 of the 10 MecA⁺ isolates previously classified as oxacillin sensitive. These results suggest that the bDNA method described here is a sensitive and efficient method for detection of methicillin resistance in staphylococci and that genetic detection methods may be useful for detection of potential methicillin resistance in the clinical laboratory.Methicillin resistance in clinical isolates of Staphylococcus is thought to occur as a combined result of the expression of the mecA gene, which codes for the cell wall surrogate enzyme penicillin binding protein (PBP) 2a or 2′ and several factors such as the fem gene series or auxiliary (aux) genes (reviewed in reference 5). In clinical laboratories, antibiotic resistance is usually detected by using methods that require a viable culture of the organism and phenotypic expression of resistance genes. However, studies indicate that there is heterogeneous expression of PBP 2a that is dependent on environmental conditions (1, 10, 16). In addition, some isolates have been shown to exhibit low- or moderate-level methicillin resistance due to overproduction of β-lactamase, modifications in the PBP binding affinities, or the presence of expression factors not related to the mecA gene (2, 9, 12, 20). Variations in laboratory reporting of high-level methicillin resistance, which requires treatment with vancomycin, may be responsible for unnecessary vancomycin usage. The current guidelines from the Centers for Disease Control and Prevention suggest restriction of vancomycin use in order to slow the occurrence of vancomycin resistance in staphylococci (4).Molecular diagnostic assays, which detect genetic targets irrespective of expression level, have proven useful for the identification of isolates containing mecA. In recent years, several genotypic detection methods have been described (3, 7, 11, 14, 17). Most are PCR based, and some are multiplexed with broad-range 16S ribosomal DNA primers to assess the lysis efficiency of each assay. While these assays are highly sensitive and specific, we have found that they are time-consuming and PCR failures may occasionally occur due to lysis inefficiency or inhibitory substances.In the past, assays utilizing branched-DNA (bDNA) technology have been developed to detect antibiotic resistance markers as well as pathogenic agents in clinical samples (6, 19). This assay uses multiple probes that cause an amplification of chemiluminescent signal rather than the amplification of a genetic target that is observed in PCR-based assays. To avoid the complications observed with other tests, we developed a mecA-specific assay that uses bDNA technology to detect the gene in lysates derived from bacterial colonies isolated on solid media and directly from blood cultures. The assay is performed in a 96-well microtiter plate format and takes approximately 6 h to complete, thereby allowing same-day results for cultures containing staphylococci.(Portions of this work were presented at the Conference on Molecular Diagnostics and Therapeutics, Kananaskis, Alberta, Canada, 15–19 August 1997, and the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Ontario, Canada, 28 September–1 October 1997.)     

Integrating genetic and gene expression evidence into genome-wide association analysis of gene sets

Single variant or single gene analyses generally account for only a small proportion of the phenotypic variation in complex traits. Alternatively, gene set or pathway association analyses are playing an increasingly important role in uncovering genetic architectures of complex traits through the identification of systematic genetic interactions. Two dominant paradigms for gene set analyses are association analyses based on SNP genotypes and those based on gene expression profiles. However, gene-disease association can manifest in many ways, such as alterations of gene expression, genotype, and copy number; thus, an integrative approach combining multiple forms of evidence can more accurately and comprehensively capture pathway associations. We have developed a single statistical framework, Gene Set Association Analysis (GSAA), that simultaneously measures genome-wide patterns of genetic variation and gene expression variation to identify sets of genes enriched for differential expression and/or trait-associated genetic markers. Simulation studies illustrate that joint analyses of genomic data increase the power to detect real associations when compared with gene set methods that use only one genomic data type. The analysis of two human diseases, glioblastoma and Crohn's disease, detected abnormalities in previously identified disease-associated pathways, such as pathways related to PI3K signaling, DNA damage response, and the activation of NFKB. In addition, GSAA predicted novel pathway associations, for example, differential genetic and expression characteristics in genes from the ABC transporter family in glioblastoma and from the HLA system in Crohn's disease. These demonstrate that GSAA can help uncover biological pathways underlying human diseases and complex traits.     

Human Y chromosome haplogroup R-V88: a paternal genetic record of early mid Holocene trans-Saharan connections and the spread of Chadic languages

Although human Y chromosomes belonging to haplogroup R1b are quite rare in Africa, being found mainly in Asia and Europe, a group of chromosomes within the paragroup R-P25^* are found concentrated in the central-western part of the African continent, where they can be detected at frequencies as high as 95%. Phylogenetic evidence and coalescence time estimates suggest that R-P25^* chromosomes (or their phylogenetic ancestor) may have been carried to Africa by an Asia-to-Africa back migration in prehistoric times. Here, we describe six new mutations that define the relationships among the African R-P25^* Y chromosomes and between these African chromosomes and earlier reported R-P25 Eurasian sub-lineages. The incorporation of these new mutations into a phylogeny of the R1b haplogroup led to the identification of a new clade (R1b1a or R-V88) encompassing all the African R-P25^* and about half of the few European/west Asian R-P25^* chromosomes. A worldwide phylogeographic analysis of the R1b haplogroup provided strong support to the Asia-to-Africa back-migration hypothesis. The analysis of the distribution of the R-V88 haplogroup in >1800 males from 69 African populations revealed a striking genetic contiguity between the Chadic-speaking peoples from the central Sahel and several other Afroasiatic-speaking groups from North Africa. The R-V88 coalescence time was estimated at 9200–5600 kya, in the early mid Holocene. We suggest that R-V88 is a paternal genetic record of the proposed mid-Holocene migration of proto-Chadic Afroasiatic speakers through the Central Sahara into the Lake Chad Basin, and geomorphological evidence is consistent with this view.     

Complementation of genetic disease: A velocity sedimentation procedure for the enrichment of heterokaryons

Methodology is described to enrich for heterokaryons after mammalian cell fusion. A heterogeneous cell mixture can be separated on a Sta-Put apparatus into fractions of uniform size cells by sedimentation through a 1% bovine serum albumin-5% Ficoll gradient. Unfused RAG and LM/TK^– cells, differing by 10% in diameter, have been sorted by size; following fusion, larger and faster sedimenting cells were shown to be hybrids. This methodology can be utilized in genetic complementation studies of human genetic diseases where selection procedures for proliferating hybrids do not exist. When fibroblasts from individuals with Tay-Sachs disease [deficient in hexosaminidase A (HEX A^–)] and Sandhoff-Jatzkewitz disease (HEX A^– and HEX B^–) are fused, HEX A is generated, demonstrating complementation of two different mutations. After Sta-Put fractionation, the HEX A complementation product was associated with the faster sedimenting multinuclear cells and not with the mononuclear parental cells. This methodology will facilitate detection of genetic differences in fibroblasts from related inherited disorders.     

A rapid scoring tool to assess mutation probability in patients with inherited cardiac disorders

Aims

To explore clinical features and relationship with positive mutation ascertainment in inherited heart diseases in order to develop a clinical tool to assist identification of individuals in whom to offer genetic testing. A clinical tool that increases pre test probability of mutation detection would have the benefits of improving patient counselling, prioritising cases for MPS and allowing equity in decision making.

Current advances in humanized mouse models

Humanized mouse models that have received human cells or tissue transplants are extremely useful in basic and applied human disease research. Highly immunodeficient mice, which do not reject xenografts and support cell and tissue differentiation and growth, are indispensable for generating additional appropriate models. Since the early 2000s, a series of immunodeficient mice appropriate for generating humanized mice has been successively developed by introducing the IL-2Rγ^null gene (e.g., NOD/SCID/γc^null and Rag2^nullγc^null mice). These strains show not only a high rate of human cell engraftment, but also generate well-differentiated multilineage human hematopoietic cells after human hematopoietic stem cell (HSC) transplantation. These humanized mice facilitate the analysis of human hematology and immunology in vivo. However, human hematopoietic cells developed from HSCs are not always phenotypically and functionally identical to those in humans. More recently, a new series of immunodeficient mice compensates for these disadvantages. These mice were generated by genetically introducing human cytokine genes into NOD/SCID/γc^null and Rag2^nullγc^null mice. In this review, we describe the current knowledge of human hematopoietic cells developed in these mice. Various human disease mouse models using these humanized mice are summarized.     

The B6.SW bilineal congenic sucrose octaacetate (SOA)-Taster mice

SWR/J inbred mice (Tasters) reliably avoid, whereas C57BL/6J inbred mice (Nontasters) are indifferent to, sucrose octaacetate (SOA) at certain concentrations. From these strains we have developed a set of bilineal congenic Taster mice. Approximately 4000 mice, from 2 isogenic and 12 segregating generations, were tested in a program designed to evaluate genetic models for SOA tasting during development of congenic strains. The criterion phenotype was avoidance or nonavoidance in preference tests of the bitter tastant SOA at concentrations of 10^–4 and 10^–5 M. Across the 12 segregating generations, the results were consistent with Mendelian expectations for a single autosomal locus with complete dominance of the Taster phenotype. The breeding program produced 12 replicate B6.SW lines containing the taster allele on the B6-Nontaster genomic background. The congenic Taster mice may facilitate a functional analysis of the sense of taste.This research was supported in part by Grant NS 15560 from the NINCDS.     

Allele frequencies for 40 autosomal SNP loci typed for US population samples using electrospray ionization mass spectrometry

Aim

To type a set of 194 US African American, Caucasian, and Hispanic samples (self-declared ancestry) for 40 autosomal single nucleotide polymorphism (SNP) markers intended for human identification purposes.

Methods

Genotyping was performed on an automated commercial electrospray ionization time-of-flight mass spectrometer, the PLEX-ID. The 40 SNP markers were amplified in eight unique 5plex PCRs, desalted, and resolved based on amplicon mass. For each of the three US sample groups statistical analyses were performed on the resulting genotypes.

Results

The assay was found to be robust and capable of genotyping the 40 SNP markers consuming approximately 4 nanograms of template per sample. The combined random match probabilities for the 40 SNP assay ranged from 10⁻¹⁶ to 10⁻²¹.

Conclusion

The multiplex PLEX-ID SNP-40 assay is the first fully automated genotyping method capable of typing a panel of 40 forensically relevant autosomal SNP markers on a mass spectrometry platform. The data produced provided the first allele frequencies estimates for these 40 SNPs in a National Institute of Standards and Technology US population sample set. No population bias was detected although one locus deviated from its expected level of heterozygosity.The forensic community has addressed the application of autosomal single nucleotide polymorphism (SNP) markers for human identification (1-4). SNPs may be of utility when working with highly degraded DNA because they can be assayed with very small polymerase chain reaction (PCR) amplicons. Over the past 10 years, various SNP assays and candidate marker panels have been described (5-10). One set of interest is a panel of 40 autosomal SNP markers intended as a universal individual identification panel. These markers were selected for high heterozygosity and low F_st values in studies of 40 populations to complement CODIS STR loci (8). Initially these markers were screened and typed for world populations by singleplex TaqMan-based assays. More recently, there have been attempts to develop multiplex assays for typing the 40 SNP panel (11). One of these is the PLEX-ID SNP-40 comprised of 8 unique 5plex PCRs developed by Abbott Molecular. The PLEX-ID instrument platform is a commercial electrospray ionization mass spectrometer capable of automated analysis of short PCR amplicons (less than 140 bp) generated by proprietary assays (see SNP-40, mtDNA 2.0). The instrument desalts each PCR reaction through the use of magnetic bead chemistry and injects the desalted PCR reaction into the mass spectrometer. The peaks are separated and resolved based on time-of-flight analysis. With the emerging development of ultra-high throughput sequencing applied to forensics it will be more commonplace to utilize these “core” SNP maker sets. Here we report the assay performance and allele frequencies for a subset of our National Institute of Standards and Technology (NIST) US population samples.     

Consistent count region–copy number variation (CCR-CNV): an expandable and robust tool for clinical diagnosis of copy number variation at the exon level using next-generation sequencing data

PurposeDespite the importance of exonic copy number variations (CNVs) in human genetic diseases, reliable next-generation sequencing–based methods for detecting them are unavailable. We developed an expandable and robust exonic CNV detection tool called consistent count region (CCR)–CNV.MethodsIn total, about 1000 samples of the truth set were used for validating CCR-CNV. We compared CCR-CNV performance with 2 well-known CNV tools. Finally, to overcome the limitations of CCR-CNV, we devised a combined approach.ResultsThe mean sensitivity and specificity of CCR-CNV alone were above 95%, which was superior to that of other CNV tools, such as DECoN and Atlas-CNV. However, low covered region and positive predictive value and high false discovery rate act as obstacles to its use in clinical settings. The combined approach showed much improved performance than CCR-CNV alone.ConclusionIn this study, we present a novel diagnostic tool that allows the identification of exonic CNVs with high confidence using various reagents and clinical next-generation sequencing platforms. We validated this method using the largest multiple ligation-dependent probe amplification–confirmed data set, including sufficient copy normal control data. The approach, combined with existing CNV tools, allows the implementation of CCR-CNV in clinical settings.