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Abstract Background Whole genome amplification (WGA) and laser assisted micro-dissection represent two recently developed technologies that can greatly advance biological and medical research. WGA allows the analysis of multiple genomic loci from a single genome and has been performed on single cells from cell suspensions and from enzymatically-digested tissues. Laser micro-dissection makes it possible to isolate specific single cells from heterogeneous tissues. Results Here we applied for the first time WGA on laser micro-dissected single cells from stained tissue sections, and developed a protocol for sequentially performing the two procedures. The combined procedure allows correlating the cell's genome with its natural morphology and precise anatomical position. From each cell we amplified 122 genomic and mitochondrial loci. In cells obtained from fresh tissue sections, 64.5% of alleles successfully amplified to ~700000 copies each, and mitochondrial DNA was amplified successfully in all cells. Multiplex PCR amplification and analysis of cells from pre-stored sections yielded significantly poorer results. Sequencing and capillary electrophoresis of WGA products allowed detection of slippage mutations in microsatellites (MS), and point mutations in P53. Conclusion Comprehensive genomic analysis of single cells from stained tissue sections opens new research opportunities for cell lineage and depth analyses, genome-wide mutation surveys, and other single cell assays.

Aims: Layer-specific left ventricular (LV) strain alterations have been suggested as a specific finding in Fabry disease (FD). Our study aimed to assess the diagnostic value of layer-specific radial strain (RS) indices compared to the established LV regional strain pattern in cardiac amyloidosis (CA) and FD, i.e. apical sparing and posterolateral strain deficiency (PLSD)., Methods and Results: We retrospectively analysed the global, subendocardial, subepicardial LV radial strain, the corresponding strain gradient, as well as the regional and global longitudinal strain. The diagnostic accuracy of the diverse LV strain analyses was comparatively assessed using receiver operating characteristic curve and multivariable regression analyses. In 40 FD and 76 CA patients, CA featured more reduced layer strain values [global RS -12.3 (-15.6 to -9.6) in CA vs. -16.7 (-20.0 to -13.6) in FD; P < 0.001; subendocardial RS -22.3 (-27.4 to -15.9) vs. -28.3 (-31.8 to -23.6), P < 0.001; subepicardial RS -6.6 (-8.6 to -4.7) in CA vs. -8.9 (-11.7 to - 6.5) in FD; P < 0.001]. Global radial and longitudinal strain held an area under the curve (AUC) of 0.75 (0.66-0.84) and AUC 0.73 (0.63-0.83). While the apical sparing and PLSD strain pattern showed the highest accuracy as single parameters [AUC 0.87 (0.79-0.95) and 0.81 (0.72-0.89), P < 0.001], the combination of subendocardial RS and the apical sparing pattern featured the highest diagnostic accuracy [AUC 0.92 (0.87-0.97)]., Conclusion: Combining radial strain-derived parameters to the established strain pattern apical sparing and PLSD improve the diagnostic accuracy in the echocardiographic assessment in suspected storage disease., Competing Interests: Conflict of interest: none declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Background: Cardiac amyloidosis (CA) and Fabry disease (FD) cause myocardial damage but may also affect the valvular and subvalvular apparatus. We aimed to evaluate the diagnostic accuracy of new echocardiographic indices including mitral valve thickness and papillary muscle (PM) hypertrophy to differentiate CA and FD., Methods: In patients with confirmed CA and FD, a detailed assessment of valvular function, mitral valve leaflet thickness and PM area as well as PM left ventricular area ratio (PM/LV-ratio) was performed in offline analyses. Receiver operating characteristic curve analyses were conducted to determine the diagnostic accuracy of mitral valve thickness, PM hypertrophy, and PM/LV-ratio to distinguish CA from FD., Results: We retrospectively analyzed a cohort of 129 patients (FD n = 49, CA n = 80). CA patients showed significantly more thickened mitral valve leaflets (4.1 ± 1.3 mm vs. 2.9 ± 1.1 mm, p < 0.001) and a higher PM area [4.0 (3.1-4.6) mm 2 vs. 2.8 (2.1-4.6) mm 2 , p = 0.009] with a comparable PM/LV-ratio in both groups. Mitral valve thickness showed the highest diagnostic accuracy to discriminate CA [AUC 0.77 (95% CI 0.67-0.87)]. The prevalence of aortic, tricuspid, and pulmonary valve regurgitation was significantly higher in CA (aortic regurgitation ≥ II° 13% vs. 4%, tricuspid regurgitation≥ II° 19% vs. 8%, p < 0.001)., Conclusion: Our results suggest that the assessment of mitral valve thickness may be a new useful echocardiographic parameter to differentiate CA and FD, whereas papillary muscle hypertrophy and PM/LV-ratio showed a limited diagnostic performance to discriminate CA. German clinical trials registry: DRKS00027403., Competing Interests: Declaration of Competing Interest Isabel Mattig is participant in the BIH Charité Clinician Scientist Program funded by the Charité – Universitätsmedizin Berlin and the Berlin Institute of Health at Charité (BIH). She received research grants from Pfizer Pharmaceuticals and Sanofi, lecture fee and financial reimbursement for advisory board activities from Sanofi outside the submitted work. SSp achieved financial reimbursement for lectures from Pfizer Pharmaceuticals. KH achieved financial reimbursement for consulting, advisory board activities and travel support by Akcea Therapeuticals Inc., Alnylam Pharmaceuticals Inc., Swedish Orphan Biovitrum, and Pfizer Pharmaceuticals, research funding by Alnylam Pharmaceuticals Inc., and Pfizer Pharmaceuticals as well as research funding by the foundation Charité (BIH clinical fellow). AB received lecture fee from Pfizer Pharmaceuticals., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Small cell lung cancer (SCLC) is an aggressive malignancy composed of distinct transcriptional subtypes, but implementing subtyping in the clinic has remained challenging, particularly due to limited tissue availability. Given the known epigenetic regulation of critical SCLC transcriptional programs, we hypothesized that subtype-specific patterns of DNA methylation could be detected in tumor or blood from SCLC patients. Using genomic-wide reduced-representation bisulfite sequencing (RRBS) in two cohorts totaling 179 SCLC patients and using machine learning approaches, we report a highly accurate DNA methylation-based classifier (SCLC-DMC) that can distinguish SCLC subtypes. We further adjust the classifier for circulating-free DNA (cfDNA) to subtype SCLC from plasma. Using the cfDNA classifier (cfDMC), we demonstrate that SCLC phenotypes can evolve during disease progression, highlighting the need for longitudinal tracking of SCLC during clinical treatment. These data establish that tumor and cfDNA methylation can be used to identify SCLC subtypes and might guide precision SCLC therapy., Competing Interests: Declaration of interests S.H., C.M.G., L.A.B., and J.V.H. own intellectual property on the classification of SCLC from DNA methylation and gene expression. D.F., A.W., A.S., and C.A.S. are full time employees of Nucleix and own stocks and stock options of Nucleix. Furthermore, S.H. reports consulting fees from Guardant Health, AstraZeneca, Boehringer Ingelheim, and Qiagen. C.M.G. is a member of the advisory board at Jazz Pharmaceuticals, AstraZeneca, and Bristol Myers Squibb and served as speaker for AstraZeneca and BeiGene. P.R. received travel support from AstraZeneca, BMS, and MSD. E.A. reports consulting fees from Eli Lilly, AstraZeneca, BMS, Boehringer Ingelheim, Takeda, Roche, and MSD, speaker’s fees from AstraZeneca, BMS, Boehringer Ingelheim, Roche, and MSD, research funding from Roche and AstraZeneca and travel support from AstraZeneca and Takeda. P.H. reports research grants from Thermo Fisher Scientific and Biocartis, and speakers’ fees from AstraZeneca, Roche, Novartis, Bristol-Myers Squibb, Pfizer, Bayer, Illumina, Biocartis, Thermo Fisher Scientific, AbbVie, Amgen, Janssen, Eli Lilly, Daiichi Sankyo, Pierre Fabre, and Guardant. V.H. reports speakers’ fees from BMS. C.M.L. reports personal fees from Amgen, Arrivent, AstraZeneca, Blueprints Medicine, Cepheid, D2G Oncology, Daiichi Sankyo, Eli Lilly, EMD Serono, Foundation Medicine, Genentech, Janssen, Medscape, Novartis, Pfizer, Puma, Syros, and Takeda. N.V. receives consulting fees from Sanofi, Regeneron, Oncocyte, and Eli Lilly, and research funding from Mirati. M.B.N. receives royalties and licensing fees from Spectrum Pharmaceuticals. I.H. received personal as well as institutional funding from Nucleix. J.Z. served on advisory board for AstraZeneca and Geneplus and received speaker’s fees from BMS, Geneplus, OrigMed, Innovent and grants from Merck, Johnson and Johnson. L.A.B received consulting fees and research funding from AstraZeneca, GenMab, Sierra Oncology, research funding from ToleroPharmaceuticals and served as advisor or consultant for PharmaMar, AbbVie, Bristol-Myers Squibb, Alethia, Merck, Pfizer, Jazz Pharmaceuticals, Genentech, and Debiopharm Group. J.V.H. served as advisor for AstraZeneca, EMD Serono, Boehringer-Ingelheim, Catalyst, Genentech, GlaxoSmithKline, Guardant Health, Foundation medicine, Hengrui Therapeutics, Eli Lilly, Novartis, Spectrum, Sanofi, Takeda, Mirati Therapeutics, BMS, BrightPath Biotherapeutics, Janssen Global Services, Nexus Health Systems, Pneuma Respiratory, Kairos Venture Investments, Roche, Leads Biolabs, RefleXion, Chugai Pharmaceuticals, received research support from AstraZeneca, GlaxoSmithKline, Spectrum as well as royalties and licensing fees from Spectrum., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Echocardiographic differentiation of cardiac amyloidosis (CA) and Fabry disease (FD) is often challenging using standard echocardiographic parameters. We retrospectively analyzed the diagnostic accuracy of right heart and left atrial strain parameters to discriminate CA from FD using receiver operating characteristic curve analyses and logistic regression models. A total of 47 FD and 88 CA patients with left ventricular wall thickening were analyzed. The comparison of both cardiomyopathies revealed significantly reduced global and free wall longitudinal right ventricular strain (RVS; global RVS: CA - 13 ± 4%, n = 67, vs. FD - 18 ± 4%, n = 39, p < 0.001) as well as right atrial strain (RAS; reservoir RAS: CA 12 ± 8%, n = 70, vs. FD 26 ± 9%, n = 40, p < 0.001) and left atrial strain (LAS) in CA patients. Individually, global RVS as well as phasic LAS and RAS showed the highest diagnostic accuracy to distinguish CA and FD. The best diagnostic accuracy was achieved by combining the age, basal RV diameter, global RVS, and reservoir and conduit RAS (area under the curve 0.96 [95% CI 0.90-1.00]). Differential echocardiographic diagnostic work-up of patients with suspected CA or FD can be improved by integrating structural and functional parameters of the right heart and the left atrium.Trial registration: DRKS00027403., (© 2024. The Author(s).)

Background: In most cases of transcatheter valve embolization and migration (TVEM), the embolized valve remains in the aorta after implantation of a second valve into the aortic root. There is little data on potential late complications such as valve thrombosis or aortic wall alterations by embolized valves., Aims: The aim of this study was to analyze the incidence of TVEM in a large cohort of patients undergoing transcatheter aortic valve implantation (TAVI) and to examine embolized valves by computed tomography (CT) late after TAVI., Methods: The patient database of our center was screened for cases of TVEM between July 2009 and July 2021. To identify risk factors, TVEM cases were compared to a cohort of 200 consecutive TAVI cases. Out of 35 surviving TVEM patients, ten patients underwent follow-up by echocardiography and CT., Results: 54 TVEM occurred in 3757 TAVI procedures, 46 cases were managed percutaneously. Horizontal aorta (odds ratio [OR] 7.51, 95% confidence interval [CI] 3.4-16.6, p < 0.001), implantation of a self-expanding valve (OR 4.63, 95% CI 2.2-9.7, p < 0.01) and a left ventricular ejection fraction < 40% (OR 2.94, 95% CI 1.1-7.3, p = 0.016) were identified as risk factors for TVEM. CT scans were performed on average 26.3 months after TAVI (range 2-84 months) and detected hypoattenuated leaflet thickening (HALT) in two patients as well as parts of the stent frame protruding into the aortic wall in three patients., Conclusion: TVEM represents a rare complication of TAVI. Follow up-CT detected no pathological findings requiring intervention., Competing Interests: HD, KS, ML, DL, UL, and MS were received financial research support and speakers’ fees from Abbott, Edwards LifeSciences and Medtronic. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Frumkin, Pietron, Kind, Brand, Knebel, Laule, Leistner, Landmesser, Krackhardt, Sherif, Sündermann, Grubitzsch, Lembcke, Niehues, Stangl and Dreger.)

Background: In [ 99m Tc]Tc-DPD scintigraphy for myocardial ATTR amyloidosis, planar images 3 hour p.i. and SPECT/CT acquisition in L-mode are recommended. This study investigated if earlier planar images (1 hour p.i.) are beneficial and if SPECT/CT acquisition should be preferred in H-mode (180° detector angle) or L-mode (90°)., Methods: In SPECT/CT phantom measurements (NaI cameras, N = 2; CZT, N = 1), peak contrast recovery (CRpeak) was derived from sphere inserts or myocardial insert (cardiac phantom; signal-to-background ratio [SBR], 10:1 or 5:1). In 25 positive and 38 negative patients (reference: endomyocardial biopsy or clinical diagnosis), Perugini scores and heart-to-contralateral (H/CL) count ratios were derived from planar images 1 hour and 3 hour p.i., Results: In phantom measurements, accuracy of myocardial CRpeak at SBR 10:1 (H-mode, 0.95-0.99) and reproducibility at 5:1 (H-mode, 1.02-1.14) was comparable for H-mode and L-mode. However, L-mode showed higher variability of background counts and sphere CRpeak throughout the field of view than H-mode. In patients, sensitivity/specificity were ≥ 95% for H/CL ratios at both time points and visual scoring 3 hour. At 1 hour, visual scores showed specificity of 89% and reduced reader's confidence., Conclusions: Early DPD images provided no additional value for visual scoring or H/CL ratios. In SPECT/CT, H-mode is preferred over L-mode, especially if quantification is applied apart from the myocardium., (© 2021. The Author(s).)

Background: "Classical" echocardiographic signs of Fabry cardiomyopathy (FC), such as left ventricular hypertrophy (LVH), posterolateral strain impairment (PLSI), and papillary muscle hypertrophy may be of limited diagnostic accuracy in clinical practice. Our aim was to evaluate the diagnostic value of left atrial (LA) strain impairment compared to "classical" echocardiographic findings to discriminate FC., Methods: In standard echocardiographic assessments, we retrospectively analyzed the diagnostic value of the "classical" red flags of FC as well as LA strain in 20 FC patients and in 20 subjects with other causes of LVH. Receiver operating characteristic (ROC) curve analysis was performed to assess the respective diagnostic accuracy., Results: FC was confirmed in 20 patients by genetic testing. In the LVH group, 12 patients were classified by biopsy to have hypertrophic cardiomyopathy, two had hypertensive heart disease, and six LVH combined with borderline myocarditis. Global and regional left ventricular (LV) strain was not significantly different between groups while LA strain was significantly impaired in FC (Left atrial reservoir strain (LASr) 19.1%±8.4 in FC and 25.6%±8.9 in LVH, p = 0.009; left atrial conduction strain (LAScd) -8.4%±4.9 in FC and -15.9%±8.4 in LVH, p < 0.01). LAScd, with an area under the curve (AUC) of .81 (95% confidence interval [CI] .66-.96) showed the highest diagnostic accuracy to discriminate FC. The PLSI pattern showed an AUC of .49, quantification of papillary muscle hypertrophy an AUC of .47., Conclusion: Adding LA strain analysis to a comprehensive echocardiographic work-up of unclear LVH may be helpful to identify FC as a possible cause., (© 2021 The Authors. Echocardiography published by Wiley Periodicals LLC.)

Aims: Traditional echocardiographic parameters for the assessment of suspected cardiac amyloidosis (CA) are of limited diagnostic accuracy. We sought to explore differences and the discriminative value of phasic left atrial strain (LAS) reductions and of regional longitudinal left ventricular (LV) strain alterations (relative apical sparing; RELAPS) in CA and other causes of LV wall thickening (LVH)., Methods and Results: We included 54 patients with unclear LVH (mean septal diastolic wall thickness 17.8 ± 3.5 mm); CA was bioptically confirmed in 35 patients (8 mATTR, 6 wtATTR, 20 AL, and 1 AA amyloidosis) and LVH in 19 subjects. We analysed RELAPS as well as LA reservoir (LASr), conduit (LAScd), and contraction strain (LASct) using 2D speckle tracking echocardiography (EchoPAC software, GE). RELAPS was higher (1.37 ± 0.94 vs. 0.86 ± 0.29, P < 0.007), whereas atrial mechanics were significantly reduced in CA (LASr, LAScd, and LASct: 9.7 ± 5.2%, -6.5 ± 3.5%, and -5.0 ± 4.1% in CA; and 22.7 ± 7.8%, -13.9 ± 5.2%, and -13.0 ± 5.5% in LVH, respectively; P < 0.001 each). With an area under the curve (AUC) of 0.91 [95% confidence interval (CI) 0.82-0.99], LASr showed a higher diagnostic accuracy in discriminating CA than RELAPS (AUC 0.74, 95% CI 0.59-0.88). LASr and LAScd remained significantly associated with CA in a multivariate regression model., Conclusion: Phasic LAS was significantly reduced in patients with CA and showed a higher diagnostic accuracy in discriminating CA than RELAPS. The additional assessment of phasic LAS may be useful to rule in the possible diagnosis of CA in patients with unclear LVH., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

Background: Aortic stenosis is a common finding in cardiac amyloidosis (CA). Younger patients often remain asymptomatic. If unrecognized, this can lead to serious complications such as heart failure. Progression of aortic stenosis can be accelerated in patients with chronic kidney disease and need for dialysis. Perioperative risk in these patients is often high due to the underlying systemic disease., Case Summary: A 40-year-old Caucasian man with known AA amyloidosis, highly active Ankylosing Spondylitis and need for chronic dialysis due to end-stage chronic renal failure presented for echocardiographic routine exam without reporting any cardiac symptoms. At the last visit 4 years ago, a normal heart valve function was noted and no echocardiographic follow-up was performed in the following. Now, rapid progression with severe aortic valve and mitral valve stenosis was stated and the patient underwent combined aortic and mitral surgical valve replacement following discussion in the multidisciplinary cardiology meeting. Macroscopic examination of the valves revealed significant calcification and histological examination showed the high presence of amyloid by Congo-red staining and immunohistological staining for AA-Amyloid. Both valve prosthetic devices showed normal function as well as a normal left ventricular ejection fraction in initial post-operative transoesophageal echocardiography. After prolonged and complicated post-operative course in the intensive care unit the patient died 3 months after surgery due to intractable multiorgan failure in combined severe abdominal septic and cardiogenic shock., Discussion: Concomitant CA and chronic dialysis can accelerate the onset of severe aortic valve stenosis. Young patients, as in this case, often stay asymptomatic, perioperative risk increases with duration of chronic dialysis and severity of valve stenosis. This increases the need for regular short-term echocardiographic examinations even in clinical stable patients., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Infective endocarditis is a severe, mostly bacterial disease characterized by high mortality and morbidity. Both diagnosis and therapy are difficult, and mortality rates have not improved over decades. We review diagnostic and management problems, the disease's modern face in the era of widespread cardiac implants and valve interventions, and current diagnostic and therapeutic recommendations., Competing Interests: Disclosure The authors report no conflicts of interest in this work., (© Georg Thieme Verlag KG Stuttgart · New York.)

BRCA1 mutation is associated with carcinogenesis, especially of breast tissue. Telomere maintenance is crucial for malignant transformation. Being a part of the DNA repair machinery, BRCA1 may be implicated in telomere biology. We explored the role of BRCA1 in telomere maintenance in lymphocytes of BRCA1/2 mutation carriers and in in vitro system by knocking down its expression in non-malignant breast epithelial cells.The results in both systems were similar. BRCA1/2 mutation caused perturbation of telomere homeostasis, shortening of the single stranded telomere overhang and increased the intercellular telomere length variability as well as the number of telomere free chromosomal ends and telomeric circles. These changes resulted in an increased DNA damage status. Telomerase activity, inducibility and expression remained unchanged. BRCA1 mutation resulted also in changes in the binding of shelterin proteins to telomeres. DNMT-1 levels were markedly reduced both in the carriers and in in vitro system. The methylation pattern of the sub-telomeric regions in carriers suggested hypomethylation in chromosome 10. The expression of a distinct set of genes was also changed, some of which may relate to pre-disposition to malignancy.These results show that BRCA gene products have a role in telomere length homeostasis. It is plausible that these perturbations contribute to malignant transformation in BRCA mutants.

Determining whether the source tissue of biological material is semen is important in confirming sexual assaults, which account for a considerable percentage of crime cases. The gold standard for confirming the presence of semen is microscopic identification of sperm cells, however, this method is labor intensive and operator-dependent. Protein-based immunologic assays, such as PSA, are highly sensitive and relatively fast, but suffer from low specificity in some situations. In addition, proteins are less stable than DNA under most environmental insults. Recently, forensic tissue identification advanced with the development of several approaches based on mRNA and miRNA for identification of various body fluids. Herein is described DNA source identifier (DSI)-semen, a DNA-based assay that determines whether the source tissue of a sample is semen based on detection of semen-specific methylation patterns in five genomic loci. The assay is comprised of a simple single tube biochemical procedure, similar to DNA profiling, followed by automatic software analysis, yielding the identification (semen/non-semen) accompanied by a statistical confidence level. Three additional internal control loci are used to ascertain the reliability of the results. The assay, which aims to replace microscopic examination, can easily be integrated by forensic laboratories and is automatable. The kit was tested on 135 samples of semen, saliva, venous blood, menstrual blood, urine, and vaginal swabs and the identification of semen vs. non-semen was correct in all cases. In order to test the assay's applicability in "real-life" situations, 33 actual casework samples from the forensic biological lab of the Israeli police were analyzed, and the results were compared with microscopic examination performed by Israeli police personnel. There was complete concordance between both analyses except for one sample, in which the assay identified semen whereas no sperm was seen in the microscope. This sample likely represents true semen because sperm cells were detected from an adjacent sample from the same garment, therefore in this case the assay appears to be more sensitive than the microscopic examination. These results demonstrate that this assay is a bona fide confirmatory test for semen., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Identifying the source tissue of biological material found at crime scenes can be very informative in a number of cases. Despite their usefulness, current visual, catalytic, enzymatic, and immunologic tests for presumptive and confirmatory tissue identification are applicable only to a subset of samples, might suffer limitations such as low specificity, lack of sensitivity, and are substantially impacted by environmental insults. Moreover these assays are incompatible and thus cannot be multiplexed. Thus they are less amenable to automation. In addition their results are operator-dependent. A better alternative approach is tissue identification based on messenger RNA (mRNA) or microRNA (miRNA); however, RNA is not as stable as DNA, and requires the use of non-standard procedures by forensic laboratories. Herein a DNA-based assay is described that enables tissue identification based on detection of tissue-specific methylation patterns. DNA samples are subjected to digestion by a methylation-sensitive restriction endonuclease followed by multiplex amplification of specific genomic targets with fluorescent-labeled primers, capillary electrophoresis of amplification products, and automatic signal analysis by dedicated software, yielding the source tissue of the sample. The single tube assay was designed for easy integration by forensic laboratories (as the assay utilizes the same platforms as current forensic STR profiling). The system is fully automatable, provides operator-independent results, and allows combining tissue identification with profiling in a single procedure. The assay was tested on 50 DNA samples from blood, saliva, semen, and skin epidermis, and the source tissue was successfully identified in all cases. Detection of semen and DNA profiling were combined into one assay and the ability to detect mixtures of semen and saliva in various ratios was demonstrated. The assay correctly detected semen in all samples where it was present, and the calculated percentage of semen was comparable to the fraction of semen in the samples. The results demonstrate that methylation-based tissue identification is more than a proof-of-concept. The methodology holds promise as another viable forensic DNA analysis tool for characterization of biological materials., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Over the past twenty years, DNA analysis has revolutionized forensic science, and has become a dominant tool in law enforcement. Today, DNA evidence is key to the conviction or exoneration of suspects of various types of crime, from theft to rape and murder. However, the disturbing possibility that DNA evidence can be faked has been overlooked. It turns out that standard molecular biology techniques such as PCR, molecular cloning, and recently developed whole genome amplification (WGA), enable anyone with basic equipment and know-how to produce practically unlimited amounts of in vitro synthesized (artificial) DNA with any desired genetic profile. This artificial DNA can then be applied to surfaces of objects or incorporated into genuine human tissues and planted in crime scenes. Here we show that the current forensic procedure fails to distinguish between such samples of blood, saliva, and touched surfaces with artificial DNA, and corresponding samples with in vivo generated (natural) DNA. Furthermore, genotyping of both artificial and natural samples with Profiler Plus((R)) yielded full profiles with no anomalies. In order to effectively deal with this problem, we developed an authentication assay, which distinguishes between natural and artificial DNA based on methylation analysis of a set of genomic loci: in natural DNA, some loci are methylated and others are unmethylated, while in artificial DNA all loci are unmethylated. The assay was tested on natural and artificial samples of blood, saliva, and touched surfaces, with complete success. Adopting an authentication assay for casework samples as part of the forensic procedure is necessary for maintaining the high credibility of DNA evidence in the judiciary system.

Revealing the lineage relations among cancer cells can shed light on tumor growth patterns and metastasis formation, yet cell lineages have been difficult to come by in the absence of a suitable method. We previously developed a method for reconstructing cell lineage trees from genomic variability caused by somatic mutations. Here, we apply the method to cancer and reconstruct, for the first time, a lineage tree of neoplastic and adjacent normal cells obtained by laser microdissection from tissue sections of a mouse lymphoma. Analysis of the reconstructed tree reveals that the tumor initiated from a single founder cell, approximately 5 months before diagnosis, that the tumor grew in a physically coherent manner, and that the average number of cell divisions accumulated in cancerous cells was almost twice than in adjacent normal lung epithelial cells but slightly less than the expected figure for normal B lymphocytes. The cells were also genotyped at the TP53 locus, and neoplastic cells were found to share a common mutation, which was most likely present in a heterozygous state. Our work shows that the ability to obtain data regarding the physical appearance, precise anatomic position, genotypic profile, and lineage position of single cells may be useful for investigating cancer development, progression, and interaction with the microenvironment.

The depth of a cell of a multicellular organism is the number of cell divisions it underwent since the zygote, and knowing this basic cell property would help address fundamental problems in several areas of biology. At present, the depths of the vast majority of human and mouse cell types are unknown. Here, we show a method for estimating the depth of a cell by analyzing somatic mutations in its microsatellites, and provide to our knowledge for the first time reliable depth estimates for several cells types in mice. According to our estimates, the average depth of oocytes is 29, consistent with previous estimates. The average depth of B cells ranges from 34 to 79, linearly related to the mouse age, suggesting a rate of one cell division per day. In contrast, various types of adult stem cells underwent on average fewer cell divisions, supporting the notion that adult stem cells are relatively quiescent. Our method for depth estimation opens a window for revealing tissue turnover rates in animals, including humans, which has important implications for our knowledge of the body under physiological and pathological conditions.

The cell lineage tree of a multicellular organism represents its history of cell divisions from the very first cell, the zygote. A new method for high-resolution reconstruction of parts of such cell lineage trees was recently developed based on phylogenetic analysis of somatic mutations accumulated during normal development of an organism. In this study we apply this method in mice to reconstruct the lineage trees of distinct cell types. We address for the first time basic questions in developmental biology of higher organisms, namely what is the correlation between the lineage relation among cells and their (1) function, (2) physical proximity and (3) anatomical proximity. We analyzed B-cells, kidney-, mesenchymal- and hematopoietic-stem cells, as well as satellite cells, which are adult skeletal muscle stem cells isolated from their niche on the muscle fibers (myofibers) from various skeletal muscles. Our results demonstrate that all analyzed cell types are intermingled in the lineage tree, indicating that none of these cell types are single exclusive clones. We also show a significant correlation between the physical proximity of satellite cells within muscles and their lineage. Furthermore, we show that satellite cells obtained from a single myofiber are significantly clustered in the lineage tree, reflecting their common developmental origin. Lineage analysis based on somatic mutations enables performing high resolution reconstruction of lineage trees in mice and humans, which can provide fundamental insights to many aspects of their development and tissue maintenance.

Background: Whole genome amplification (WGA) and laser assisted micro-dissection represent two recently developed technologies that can greatly advance biological and medical research. WGA allows the analysis of multiple genomic loci from a single genome and has been performed on single cells from cell suspensions and from enzymatically-digested tissues. Laser micro-dissection makes it possible to isolate specific single cells from heterogeneous tissues., Results: Here we applied for the first time WGA on laser micro-dissected single cells from stained tissue sections, and developed a protocol for sequentially performing the two procedures. The combined procedure allows correlating the cell's genome with its natural morphology and precise anatomical position. From each cell we amplified 122 genomic and mitochondrial loci. In cells obtained from fresh tissue sections, 64.5% of alleles successfully amplified to approximately 700000 copies each, and mitochondrial DNA was amplified successfully in all cells. Multiplex PCR amplification and analysis of cells from pre-stored sections yielded significantly poorer results. Sequencing and capillary electrophoresis of WGA products allowed detection of slippage mutations in microsatellites (MS), and point mutations in P53., Conclusion: Comprehensive genomic analysis of single cells from stained tissue sections opens new research opportunities for cell lineage and depth analyses, genome-wide mutation surveys, and other single cell assays.

What is the lineage relation among the cells of an organism? The answer is sought by developmental biology, immunology, stem cell research, brain research, and cancer research, yet complete cell lineage trees have been reconstructed only for simple organisms such as Caenorhabditis elegans. We discovered that somatic mutations accumulated during normal development of a higher organism implicitly encode its entire cell lineage tree with very high precision. Our mathematical analysis of known mutation rates in microsatellites (MSs) shows that the entire cell lineage tree of a human embryo, or a mouse, in which no cell is a descendent of more than 40 divisions, can be reconstructed from information on somatic MS mutations alone with no errors, with probability greater than 99.95%. Analyzing all approximately 1.5 million MSs of each cell of an organism may not be practical at present, but we also show that in a genetically unstable organism, analyzing only a few hundred MSs may suffice to reconstruct portions of its cell lineage tree. We demonstrate the utility of the approach by reconstructing cell lineage trees from DNA samples of a human cell line displaying MS instability. Our discovery and its associated procedure, which we have automated, may point the way to a future "Human Cell Lineage Project" that would aim to resolve fundamental open questions in biology and medicine by reconstructing ever larger portions of the human cell lineage tree.

Activation of the tumor necrosis factor R1/Fas receptor results in the cleavage of cytosolic BID to truncated tBID. tBID translocates to the mitochondria to induce the oligomerization of BAX or BAK, resulting in the release of cytochrome c (Cyt c). Here we demonstrate that in tumor necrosis factor alpha-activated FL5.12 cells, tBID becomes part of a 45-kDa cross-linkable mitochondrial complex that does not include BAX or BAK. Using fluorescence resonance energy transfer analysis and co-immunoprecipitation, we demonstrate that tBID-tBID interactions occur in the mitochondria of living cells. Cross-linking experiments using a tBID-GST chimera indicated that tBID forms homotrimers in the mitochondrial membrane. To test the functional consequence of tBID oligomerization, we expressed a chimeric FKBP-tBID molecule. Enforced dimerization of FKBP-tBID by the bivalent ligand FK1012 resulted in Cyt c release, caspase activation, and apoptosis. Surprisingly, enforced dimerization of tBID did not result in the dimerization of either BAX or BAK. Moreover, a tBID BH3 mutant (G94E), which does not interact with or induce the dimerization of either BAX or BAK, formed the 45-kDa complex and induced both Cyt c release and apoptosis. Thus, tBID oligomerization may represent an alternative mechanism for inducing mitochondrial dysfunction and apoptosis.

Malnutrition is prevalent in end-stage renal disease (ESRD) patients treated with hemodialysis (HD) and peritoneal dialysis (PD). In addition, there is increased incidence of morbidity in this group. Evaluation of nutritional status is important. Application of body composition in the ESRD population to evaluate body compartments and to assess nutritional health has become more common in clinical practice. Neutron activation analysis (NAA) may provide data on metabolically active tissue by quantification of total body potassium (TBK) for body cell mass and assessment of protein by total body nitrogen (TBN). This method may be able to detect changes in body composition before clinical signs of malnutrition are apparent. Ten HD (5 male and 5 female) and 10 PD patients (7 male and 3 female) were evaluated by NAA, TBK, and isotope dilution. Female PD patients had an increased total body water (TBW) and increased intracellular water compared to HD females. Albumin was lower in PD women. There was no significant difference between PD men and laboratory controls in TBW, extracellular water, and TBN. The clinical application of body composition methods for evaluation of dialysis patients by serial assessment and for development of a bedside tool needs further study.