Parallel Genetic Analysis Research Articles

Which tissue to take? A retrospective study of the identification success of altered human remains

In forensic medicine, deceased are usually identified by comparing ante- and post-mortem dental or radiological features. However, in severe putrefaction, burning or absent reference data, the remaining tool for identifying human remains is DNA genotyping. But even a DNA-based identification can be challenging when confronted with a high post-mortem interval or heat impacts because it can lead to undesirable degradation of the DNA that varies among tissue types.This retrospective study investigated the identification success in 402 altered human corpses over seven years by comparing the examined tissue types from decomposed, skeletonised and burnt corpses as well as bodies found in water. For each tissue, the STR genotyping results and the number of additional or parallel genetic analyses were evaluated. By comparing the amplification success in samples from altered and unaltered remains, condition-based and tissue-specific differences were observed. With a mean number of 1.6 additional amplifications in cases with well-preserved corpses and 4.5 in altered corpses, the results showed significantly more DNA analyses for altered remains. In 83% of the cases, extra amplifications were performed to identify the corpse. The tissue-specific differences revealed an uncertainty in choosing suitable material from altered corpses for a successful DNA profile. Especially for bone and muscle samples, the genotyping success was the most unpredictable. Furthermore, comparing the retrospective outcome with other research findings, a remarkable variety of recommendations for the “best tissue choice” exists in the forensic community. Thus, our survey highlights the advantages of a broader and systematic approach on hard and soft tissues for successful DNA-based identification of altered human remains at first attempt.

PD-1 Overexpression in Richter's Transformation (RT) and Aggressive Chronic Lymphocytic Leukemia (CLL) after Progression on Ibrutinib Increases Bcl-2 Expression Via Akt/mTOR Pathway

Introduction: Given the established role of PD-1 in mediating immune suppression in chronic lymphocytic leukemia (CLL), we tested and reported the efficacy of PD-1 blocking antibody pembrolizumab in relapsed and transformed CLL patients. A selective response of pembrolizumab (~40%) in patients with RT, particularly after prior ibrutinib, was observed (Ding, Blood, 129:3419). Correlative analysis showed PD-1 expression in tumor B-cells of patients with RT and aggressive CLL after progression on ibrutinib. PD-1, an inhibitory receptor expressed on CLL T-cells, inhibits the immune synapse and cytotoxic T cell functions via the interactions with its ligands. However, the expression pattern and role of PD-1 in tumor B-cells is not well defined. In this study we investigated the functional implication of the PD-1 signaling axis in B-cell pathobiology in CLL and RT patients.Methods: 26 CLL-involved lymph node (LN) and 20 RT-involved LN were tested for PD-1 expression by immunohistochemistry (mouse clone NAT105, Abcam). For in vitro study, we checked PD-1 expression in 11 lymphoma cell lines and 1 CLL cell line by both flow cytometry and Western blot (WB) analysis. Effect of PD-1 knockdown using CRISPR/Cas9 system (Addgene) and over-expression of PD-1 using pLEX-lentiviral (Thermoscientific) or pRetro-retroviral (Clonetech) system were evaluated on pro-survival and apoptotic signaling pathways by Western blot analysis. Gene expression signatures in CLL and RT patients were also evaluated by Illumina-based RNA sequencing using FFPE-nodal tissue obtained by clinical biopsy (Tempus Labs; Chicago, IL).Results: The expression of PD-1 was significantly increased in RT-LN compared to CLL-LN. (mean ± SEM in RT vs. CLL, 30.6 ± 4.7 vs. 11.5 ± 2.8, p < 0.001). PD-1 expression was highest in patients with RT where the immediate prior CLL therapy was ibrutinib (Figure 1A). Among all cell lines tested for PD-1 expression, the expression of PD-1 by WB and flow was highest in Mino (mantle cell lymphoma line), followed by moderate expression in Jvm2 (B-PLL line) and Mec1 (CLL line), and very low-level expression in both Jeko-1 (B-NHL line) and lymphoma line ‘Karpas299‘. CRISPR/Cas9 mediated depletion of PD-1 in Mino cells inhibited constitutively active Akt, p70S6K and mTOR pathway, accompanied by significant downregulation of the anti-apoptotic proteins, Bcl-2, Mcl-1 and XIAP, but P-ERK1/2 was not affected. Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in Jeko-1 and doxycycline regulated inducible retroviral (pRetro-PD-1) mediated overexpression of PD-1 in Karpas299 activated Akt, mTOR and p70S6K pathway. Overexpression of PD-1 in Jeko-1 significantly increased Bcl-2 and Mcl-1 and in Karpas299 increased Bcl-2, Mcl-1 and XIAP expression (Figure 1B). A parallel genetic analysis using RNA sequencing was performed on 5 nodal tissues involved by either RT or progressive CLL after these patients developed clinical progression after prior ibrutinib therapy. In all 5 patients, overexpression of PD-1 was associated with increased expression of Bcl-2 and mTOR regardless of the genetic mutations detected (including TP53, ATM, BTK, NOTCH1, XPO1, SF3B1, TET2 etc). However, in 2 patients who received prior chemoimmunotherapy, similar overexpression of gene signature was not observed by RNA sequencing analysis, alternative pathways including Met or NFkB overexpression was detected. Given these clinical and laboratory findings, we have treated 2 RT patients with a combination of BTK and Bcl-2 inhibitors (ibrutinib and venetoclax, respectively) whose CLL transformed after prior ibrutinib. Significant reduction of tumor burden was observed in both cases with one complete response and one mixed response.Conclusion: An increased expression of PD-1, Akt/mTOR and Bcl-2 gene signature was first observed in RT patients after prior ibrutinib therapy. PD-1 overexpression in the tumor B-cells of RT and progressive CLL patients likely regulate AKT/mtOR to upregulate Bcl-2. Targeting both BTK and Bcl-2 pathways in addition to PD-1 blockade appear to be a promising strategy to treat these aggressive diseases. DisclosuresParikh:Gilead: Honoraria; Janssen: Research Funding; Abbvie: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding. Kenderian:Novartis: Patents & Royalties; Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding. Ansell:Takeda: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Celldex: Research Funding; Merck & Co: Research Funding; Regeneron: Research Funding; LAM Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Research Funding; Pfizer: Research Funding. Kay:Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees. Ding:Merck: Research Funding.

Solid pediatric tumors : A brief survey of the rarity cabinet

Solid tumors in childhood are extremely rare entities, which are usually treated in specialized centers. Diagnosis and therapy are carried out according to a joint European protocol, whereby the pathological evaluation and therapy are carried out according to international guidelines. For the correct diagnosis and/or therapy of most tumors, analysis of specific genetic changes is mandatory; therefore, tumors have to be adequately sampled for parallel genetic analysis during the pathological work-up. A second opinion reference of the histopathological assessment is part of the international guidelines. Neuroblastomas, congenital mesoblastic nephromas and rhabdoid tumors are examples of solid tumors in childhood that are not restricted to one organ and occur exclusively during childhood.

The problem of molecular-genetic identification of sweat and grease deposits in the human fingerprints

The objective of the present experimental molecular-genetic study of DNA contained in of human fingerprints was to establish the relationship between the reference genetic profiles and the genotypes of the individuals leaving their fingerprints on a smooth metal object. The biological material for the purpose of the investigation was sampled at different time intervals. The were taken using a scotch tape and used to obtain the complete genetic profile immediately after the fingerprints had been left as well as within the next 24 hours and one week. It proved impossible to identify the complete genetic profile one month after the fingerprints had been left. The alleles not typical for reference samples were identified within one week after swabbing the material from the metal surface. The results of the sudy can be explained by the decrease of the concentration of the initial DNA-matrix in the samples due to its degradation in the course of time. It is concluded that the parallel genetic analysis is needed if reliable evidence of identity of the profiles of interest or its absence is to be obtained.

A microfluidic device integrated with multichamber polymerase chain reaction and multichannel separation for genetic analysis

This work describes a microfluidic device integrated with multichamber polymerase chain reaction (PCR) and multichannel separation for parallel genetic analysis. The microdevice consists of three functional units: temperature control, multiple PCR (four chambers PCR), and multiple channel separation (four separation channels, each channel connected to a PCR chamber). Platinum (Pt)/titanium (Ti) microheater was used to ensure homogeneous temperature field, and Pt-chip sensor was used for temperature monitoring. The interface between chip-PCR and chip separation was simplified by connecting the PCR chamber with separation channel directly. After chip-PCR, PCR products were introduced into parallel separation channels for subsequent separation/detection by applying an electric field automatically. This microdevice was successfully applied for detection of pathogens including hepatitis B virus (HBV) and Mycobacterium tuberculosis (MTB), and genotyping of human leucocyte antigen (HLA)-B27 as well, demonstrating the feasibility of the integrated microdevice for parallel genetic analysis.

Integrative analyses of genetic variation in enzyme activities of primary carbohydrate metabolism reveal distinct modes of regulation in Arabidopsis thaliana

Multiparallel QTL analysis of 15 Arabidopsis primary carbohydrate metabolism enzymes reveals that traits affecting primary metabolism are often correlated.

Multichannel PCR-CE Microdevice for Genetic Analysis

We have developed a fully integrated multichannel polymerase chain reaction-capillary electrophoresis (PCR-CE) microdevice with nanoliter reactor volumes for highly parallel genetic analyses. Resistance temperature detectors and heaters made out of Ti/Pt are integrated on the microchip using a scalable radial design to provide precise temperature control of the four parallel PCR-CE reactor systems. Heating rates of >15 degrees C s(-1) and cooling rates of >10 degrees C s(-1) allow cycle times of 50 s and 30 complete PCR cycles in <27 min. PDMS membrane valves control and localize PCR reagents in the 380-nL reactors. By directly integrating PCR reactors with the CE separation system, efficient coupling of amplification with separation is achieved. The microdevice demonstrates good amplification uniformity and sensitivity down to 10 initial template copies in the 380-nL reactor (approximately 43 aM) with signal-to-noise ratio greater than 10. Parallel PCR-CE multiplex amplification and genetic analyses of four different samples with (1) both M13mp18 control template and E. coli K12 cells, (2) only M13mp18 template, (3) only E. coli K12 cells, and (4) negative control are completed in less than 30 min in a single run.

ProbeMaker: an extensible framework for design of sets of oligonucleotide probes

BackgroundProcedures for genetic analyses based on oligonucleotide probes are powerful tools that can allow highly parallel investigations of genetic material. Such procedures require the design of large sets of probes using application-specific design constraints.ResultsProbeMaker is a software framework for computer-assisted design and analysis of sets of oligonucleotide probe sequences. The tool assists in the design of probes for sets of target sequences, incorporating sequence motifs for purposes such as amplification, visualization, or identification. An extension system allows the framework to be equipped with application-specific components for evaluation of probe sequences, and provides the possibility to include support for importing sequence data from a variety of file formats.ConclusionProbeMaker is a suitable tool for many different oligonucleotide design and analysis tasks, including the design of probe sets for various types of parallel genetic analyses, experimental validation of design parameters, and in silico testing of probe sequence evaluation algorithms.

Microfabricated 384-lane capillary array electrophoresis bioanalyzer for ultrahigh-throughput genetic analysis.

A microfabricated 384-lane capillary array electrophoresis device is developed and utilized for massively parallel genetic analysis. The 384 capillary lanes, arrayed radially about the center of a 200-mm-diameter glass substrate sandwich, are constructed using scalable microfabrication techniques derived from the semiconductor industry. Samples are loaded into reservoirs on the perimeter of the wafer, separated on the 8-cm-long poly(dimethylacrylamide) gel-filled channels, and detected with a four-color rotary confocal fluorescence scanner. The performance and throughput of this bioanalyzer are demonstrated by simultaneous genotyping 384 individuals for the common hemochromatosis-linked H63D mutation in the human HFE gene in only 325 s. This lab-on-a-chip device thoroughly exploits the power of microfabrication to produce high-density capillary electrophoresis arrays and to use them for high-throughput bioanalysis.

Comparative Mapping of Canine and Human Proximal Xq and Genetic Analysis of Canine X-Linked Severe Combined Immunodeficiency

Parallel genetic analysis of animal and human genetic diseases can facilitate the identification and characterization of the causative gene defects. For example, canine X-linked severe combined immunodeficiency (SCID) is characterized by clinical, pathological, and immunological manifestations similar to the most common form of human SCID. To derive a canine syntenic map including genes that in humans are located in proximal Xq, near human X-linked SCID, poly(TG) polymorphisms were identified at the canine phosphoglycerate kinase (PGK) and choroideremia (CHM) loci. These plus a polymorphic poly(CAG) sequence in exon 1 of the canine androgen receptor gene (AR) were used to genotype members of the colony informative for X-linked SCID. No recombinations among SCIDX1, AR, PGK, or CHM were observed. Fluorescence in situ hybridization localized PGK and CHM to proximal Xq in the dog, in the same chromosomal location occupied by the human genes. Somatic cell hybrid analysis and methylation differences at AR demonstrated that female dogs carrying X-linked SCID have the same lymphocyte-limited skewed X-chromosome inactivation patterns as human carriers. These genetic and phenotypic findings provide evidence that mutations in the same gene, now identified as the gamma chain of the IL-2 receptor, cause canine and human X-linked SCID. This approach is an efficient method for comparative gene mapping and disease identification.