High risk of development of renal cell tumor in end-stage kidney disease: the role of microenvironment.

End-stage renal disease (ESRD) and acquired cystic renal disease (ACRD) are associated with high risk of development of renal cell tumors (RCT) displaying unusual phenotype and genotype. The underlying molecular mechanism is not yet known. To explore the molecular microenvironment, we have established the expression profile of ESRD/ACRD kidneys. RNA extracted from normal and ESRD/ACRD kidneys and distinct types of RCT was subjected to Affymetrix HG U133 micro array analysis. A gene expression signature indicated cancer-related biological processes in the remodeling of ESRD/ACRD kidneys. Quantitative RT-PCR studies confirmed a specific gene signature including a functional group of inflammatory cytokines and also cytokeratins associated with stem cell characteristics of epithelial cells. Several of the signature genes including the SCEL were expressed in ESRD/ACRD-associated papillary RCT as well. Immunohistological analysis confirmed the expression of CXCL8 and its receptor CXCR2 as well as the expression of SCEL in hyperplastic tubular, cystic, and papillary structures and RCTs in ESRD/ACRD kidney. Our data indicates that ESRD/ACRD is a novel disease and the inflammatory microenvironment altered plasticity, and stem cell characteristics of epithelial cells may be associated with the high risk of tumor development.

mRNA-based skin identification for forensic applications.

Although the identification of human skin cells is of important relevance in many forensic cases, there is currently no reliable method available. Here, we present a highly specific and sensitive messenger RNA (mRNA) approach for skin identification, meeting the key requirements in forensic analyses. We examined 11 candidate genes with skin-specific expression, as ascertained from expression databases and the literature, as well as five candidate reference genes ascertained from previous studies, in skin samples and in other forensically relevant tissues. We identified mRNA transcripts from three genes CDSN, LOR and KRT9, showing strong over-expression in skin samples relative to samples from forensic body fluids, making them suitable markers for skin identification. Out of the candidate reference genes tested, only ACTB showed similarly high expression in skin and body-fluid samples, providing a suitable reference marker for quantitative real-time PCR (qPCR) analysis of skin. Analyses of palmar and thumbprint skin samples indicate that our qPCR approach for the three skin-targeted mRNA markers, as well as the reference mRNA marker ACTB, is highly sensitive, allowing successful detection of minute amounts of skin material including full, half and quarter thumbprints, albeit with decreased success in decreasing print material. Furthermore, thumbprints stored for 6.5 months provided similar results relative to freshly analysed samples implying reasonable time-wise stability of the three skin-targeted mRNAs as well as the ACTB reference mRNA. Our study represents the first attempt towards reliable mRNA-based skin identification in forensic applications with particular relevance for future trace/touched object analyses in forensic case work. Although the approach for skin identification introduced here can be informative when applied on its own, we recommend for increased reliability the integration of (one or more of) the skin-targeted mRNA markers presented here into multiplex assays additionally including mRNA markers targeting alternative cell types expected in forensic samples.

Forensic pregnancy diagnostics with placental mRNA markers.

Current methods for pregnancy diagnostics are based on immunodetection of pregnancy-specific proteins and in a forensic context suffer from sensitivity and specificity issues. Here, we applied reverse transcriptase polymerase chain reaction (RT-PCR) technology to 11 genes previously reported with placental mRNA circulating in maternal blood. We found two genes, hPL and betahCG, with pregnancy-specific expression in whole blood samples. RT-PCR detection of hPL was positive in all samples tested throughout the pregnancy, whereas betahCG was detectable until half of the second trimester but not at later gestation ages. For hPL, in vitro stability of the transcript was demonstrated until 2 months of age, and the hPL-specific RT-PCR assay applied was highly sensitive with reliable detection from down to 0.25 cm(2) dried bloodstain. We therefore suggest hPL-specific RT-PCR as a new molecular tool for forensic pregnancy diagnostics from dried blood stains. Moreover, our results indicate that the time-wise reverse expression of hPL and betahCG during pregnancy may allow an RT-PCR-based estimation of the gestational age from blood stains, adding to the value of forensic pregnancy diagnosis for crime scene investigations.

MicroRNA markers for forensic body fluid identification obtained from microarray screening and quantitative RT-PCR confirmation.

MicroRNAs (miRNAs) are non-protein coding molecules with important regulatory functions; many have tissue-specific expression patterns. Their very small size in principle makes them less prone to degradation processes, unlike messenger RNAs (mRNAs), which were previously proposed as molecular tools for forensic body fluid identification. To identify suitable miRNA markers for forensic body fluid identification, we first screened total RNA samples derived from saliva, semen, vaginal secretion, and venous and menstrual blood for the expression of 718 human miRNAs using a microarray platform. All body fluids could be easily distinguished from each other on the basis of complete array-based miRNA expression profiles. Results from quantitative reverse transcription PCR (RT-PCR; TaqMan) assays for microarray candidate markers confirmed strong over-expression in the targeting body fluid of several miRNAs for venous blood and several others for semen. However, no candidate markers from array experiments for other body fluids such as saliva, vaginal secretion, or menstrual blood could be confirmed by RT-PCR. Time-wise degradation of venous blood and semen stains for at least 1 year under lab conditions did not significantly affect the detection sensitivity of the identified miRNA markers. The detection limit of the TaqMan assays tested for selected venous blood and semen miRNA markers required only subpicogram amounts of total RNA per single RT-PCR test, which is considerably less than usually needed for reliable mRNA RT-PCR detection. We therefore propose the application of several stable miRNA markers for the forensic identification of blood stains and several others for semen stain identification, using commercially available TaqMan assays. Additional work remains necessary in search for suitable miRNA markers for other forensically relevant body fluids.

Three genetic developmental stages of papillary renal cell tumors: duplication of chromosome 1q marks fatal progression.

Papillary renal cell tumors (RCT) make up a cytomorphologically and biologically heterogeneous group of kidney cancers including renal cell adenomas (RCA) and renal cell carcinomas (RCC). To find genetic markers landmarking the tumor progression, we have evaluated the genetic alterations obtained by karyotyping, chromosomal and array-CGH and compared with the cytological characteristics and biological behavior of 60 papillary RCTs. Based on the genetic and clinical data, we have separated 3 groups of tumors and proposed 3 genetically defined developmental stages of papillary RCTs. Papillary RCAs are characterized by combined trisomy of chromosomes 7 and 17, whereas papillary RCCs displayed additional trisomies of 3q, 8q, 12q, 16q and 20q. In addition to the genetic changes occurring in the second group, the third group of tumors was characterized by 1q gain and 6q, 8p, 9p and 14q losses. Kaplan-Meier analysis revealed a significant association between chromosome 1q gain and deadly outcome of the disease. The cytomorphological variation and size of tumors in the second and third groups did not correlate with the clinical outcome. Therefore, we suggest that our genetic classification system landmarking papillary RCA, papillary RCC without and with progression offer a better system to characterize the tumor biology of clinical significance than a cellular/morphological classification.

New markers for old stains: stable mRNA markers for blood and saliva identification from up to 16-year-old stains.

In forensic science, the unequivocal identification of the cellular origin of crime scene samples used for DNA profiling can provide crucial information for crime scene reconstruction. We have previously shown that various mRNA markers from genes with expression patterns specific for blood and saliva can be established from whole-genome expression analysis of time-wise degraded samples and were stable enough to specifically identify blood and saliva stains up to 180 days of age. Here, we showed that nine blood-specific and five saliva-specific mRNA markers can be amplified successfully and reliably in much older blood (13-16 years) and saliva (2-6 years) stains, respectively, suggesting their suitability for tissue identification in forensic case work. Moreover, our findings imply that forensic RNA testing can be reliable and robust if degraded samples are considered in the marker ascertainment procedure, with promising expectations beyond tissue identification purposes.

Introducing novel type of human DNA markers for forensic tissue identification: DNA copy number variation allows the detection of blood and semen.

Establishing the cellular or tissue-type origin of human biological traces found at crimes scenes is forensically relevant, as it allows evaluating the crime relevance of such traces and enables reconstructing the sequence of crime events. Messenger RNA and micro RNA markers are useful for forensic tissue identification, but provide challenges for linking RNA-identified cell/tissue types with DNA-identified trace donors, especially in mixed traces. DNA methylation markers overcome this problem, but provide technical challenges due to the DNA treatment required by most analysis methods. Here we introduce a novel type of DNA markers for forensic tissue identification analysed without prior DNA treatment, namely copy number variation (CNV). Using genome-wide CNV screening followed-up by targeted qPCR confirmation, and using qPCR analysis of additional CNV-like candidate DNA markers, in samples of several individuals from all commonly encountered forensically-relevant tissue types, we identified DNA markers specific for blood and semen, respectively. Preliminary forensic validation testing demonstrates that the developed qPCR assays are highly sensitive - delivering positive results down to picogram level of input DNA, specific, and can cope well with degraded DNA, providing suitable prerequisites for forensic applications. Moreover, we exemplified that using the CNV qPCR products as input material for subsequent forensic STR analysis delivered full STR profiles, opening-up new avenues of using the same DNA aliquot for both forensic purposes, tissue and individual identification. Provided additional forensic validation studies, we envision the application of these novel DNA markers for forensic tissue identification in future forensic casework. Such CNV markers are particularly useful for tissue identification in old/cold cases, where aged/old DNA extracts are available that contain no RNA and are not suitable for DNA methylation analysis due to limited DNA quantity and quality.

Differential expression of a new isoform of DLG2 in renal oncocytoma.

BACKGROUND: Renal oncocytoma, a benign tumour of the kidney, may pose a differential diagnostic problem due to overlapping phenotype with chromophobe renal cell carcinoma or other types of renal cell tumours. Therefore, identification of molecular markers would be of great value for molecular diagnostics of this tumour type. METHODS: In the current study we applied various techniques, including Affymetrix microarray hybridization and semiquantitative RT-PCR, to identify genes expressed differentially in renal oncocytomas. Subsequently, we used RACE and Northern blot hybridization to characterize the potential candidates for molecular diagnosis. RESULTS: We have identified new isoform of DLG2 gene, which contains 3'-end exons of the known DLG2 gene along with the hypothetical gene FLJ37266. The new isoform is specifically upregulated in renal oncocytoma, whereas the known DLG2 gene is downregulated in this type of kidney tumour. CONCLUSION: The new isoform of DLG2 is the promising candidate gene for molecular differential diagnostics of renal oncocytoma.

Human age estimation from blood using mRNA, DNA methylation, DNA rearrangement, and telomere length.

Establishing the age of unknown persons, or persons with unknown age, can provide important leads in police investigations, disaster victim identification, fraud cases, and in other legal affairs. Previous methods mostly relied on morphological features available from teeth or skeletal parts. The development of molecular methods for age estimation allowing to use human specimens that possess no morphological age information, such as bloodstains, is extremely valuable as this type of samples is commonly found at crime scenes. Recently, we introduced a DNA-based approach for human age estimation from blood based on the quantification of T-cell specific DNA rearrangements (sjTRECs), which achieves accurate assignment of blood DNA samples to one of four 20-year-interval age categories. Aiming at improving the accuracy of molecular age estimation from blood, we investigated different types of biomarkers. We started out by systematic genome-wide surveys for new age-informative mRNA and DNA methylation markers in blood from the same young and old individuals using microarray technologies. The obtained candidate markers were validated in independent samples covering a wide age range using alternative technologies together with previously proposed DNA methylation, sjTREC, and telomere length markers. Cross-validated multiple regression analysis was applied for estimating and validating the age predictive power of various sets of biomarkers within and across different marker types. We found that DNA methylation markers outperformed mRNA, sjTREC, and telomere length in age predictive power. The best performing model included 8 DNA methylation markers derived from 3 CpG islands reaching a high level of accuracy (cross-validated R(2)=0.88, SE±6.97 years, mean absolute deviation 5.07 years). However, our data also suggest that mRNA markers can provide independent age information: a model using a combined set of 5 DNA methylation markers and one mRNA marker could provide similarly high accuracy (cross-validated R(2)=0.86, SE±7.62 years, mean absolute deviation 4.60 years). Overall, our study provides new and confirms previously suggested molecular biomarkers for age estimation from blood. Moreover, our comparative study design revealed that DNA methylation markers are superior for this purpose over other types of molecular biomarkers tested. While the new and some previous findings are highly promising, before molecular age estimation can eventually meet forensic practice, the proposed biomarkers should be tested further in larger sets of blood samples from both healthy and unhealthy individuals, and markers and genotyping methods shall be validated to meet forensic standards.

Homozygous losses detected by array comparative genomic hybridization in multiplex urothelial carcinomas of the bladder.

Urothelial carcinomas (UCs) may present at first as a solitary or multifocal neoplasm. We applied high resolution array comparative genomic hybridization to 24 solitary and 32 multiplex UCs and used the hidden Markov model algorithm to identify the copy number changes at the probe level. Copy number losses and homozygous deletions at the chromosome 9p region affecting the CDKN2A and MTAP genes were the most frequent alterations in both groups of tumors. We have delineated two new tumor suppressor gene regions at chromosome 9p that harbor the PTPRD and BNC2 genes. Copy number losses at chromosomal regions 2q, 8p, and 18p occurred preferentially in solitary UCs, whereas multiplex UCs displayed loss of large chromosomal regions at 9q, 10q, 11q, 18q, and 21q. Homozygous deletions harboring loci of cell adhesion genes such as claudins, desmocollins, and desmogleins were seen exclusively in multiplex UCs. Amplifications occurred only in invasive G3 UCs irrespective of staging. Our study suggests that solitary and multiplex UCs may have divergent genetic pathways. The biallelic inactivation of cellular adhesion genes by homozygous deletions in multiplex UCs may explain the frequent intravesical spreading of tumor cells. .

A multiplex (m)RNA-profiling system for the forensic identification of body fluids and contact traces.

In current forensic practice, information about the possible biological origin of forensic traces is mostly determined using protein-based presumptive testing. Recently, messenger RNA-profiling has emerged as an alternative strategy to examine the biological origin. Here we describe the development of a single multiplex mRNA-based system for the discrimination of the most common forensic body fluids as well as skin cells. A DNA/RNA co-isolation protocol was established that results in DNA yields equivalent to our standard in-house validated DNA extraction procedure which uses silica-based columns. An endpoint RT-PCR assay was developed that simultaneously amplifies 19 (m)RNA markers. This multiplex assay analyses three housekeeping, three blood, two saliva, two semen, two menstrual secretion, two vaginal mucosa, three general mucosa and two skin markers. The assay has good sensitivity as full RNA profiles for blood, semen and saliva were obtained when using ≥0.05 µL body fluid starting material whereas full DNA profiles were obtained with ≥0.1 µL. We investigated the specificity of the markers by analysing 15 different sets of each type of body fluid and skin with each set consisting of 8 individuals. Since skin markers have not been incorporated in multiplex endpoint PCR assays previously, we analysed these markers in more detail. Interestingly, both skin markers gave a positive result in samplings of the hands, feet, back and lips but negative in tongue samplings. Positive identification (regarding both DNA- and RNA-profiling) was obtained for specimens stored for many years, e.g. blood (28 years-old), semen (28 years-old), saliva (6 years-old), skin (10 years-old) and menstrual secretion (4 years-old). The described approach of combined DNA- and RNA-profiling of body fluids and contact traces assists in the interpretation of forensic stains by providing information about not only the donor(s) that contributed to the stain but also by indicating which cell types are present.

Gene expression profiling of chromophobe renal cell carcinomas and renal oncocytomas by Affymetrix GeneChip using pooled and individual tumours.

Due to overlapping morphology, malignant chromophobe renal cell carcinomas (RCC) and benign renal oncocytomas (RO) may pose a diagnostic problem. In the present study, we have applied different algorithms to evaluate the data sets obtained by hybridisation of pooled and also individual samples of renal cell tumours (RCT) onto two different gene expression platforms. The two approaches revealed high similarities in the gene expression profiles of chromophobe RCCs and ROs but also some differences. After identifying the differentially expressed genes by statistic analyses, the candidate genes were further selected by a real time and normal RT-PCR and their products were analysed by immunohistochemistry. We have identified CD82 and S100A1 as valuable markers for chromophobe RCC as well as AQP6 for ROs. However, these genes are expressed at the protein level in other types of RCTs as well albeit at a low frequency and low intensity. As none of the selected genes marks exclusively one type of RCTs, for the differential diagnosis of chromophobe RCCs and ROs, a set of markers such as CD82, S100A1 and AQP6 as well as some others would be an option in routine histological laboratories.

Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples.

Human body fluids such as blood and saliva represent the most common source of biological material found at a crime scene. Reliable tissue identification in forensic science can reveal significant insights into crime scene reconstruction and can thus contribute toward solving crimes. Limitations of existing presumptive tests for body fluid identification in forensics, which are usually based on chemoluminescence or protein analysis, are expected to be overcome by RNA-based methods, provided that stable RNA markers with tissue-specific expression patterns are available. To generate sets of stable RNA markers for reliable identification of blood and saliva stains we (1) performed whole-genome gene expression analyses on a series of time-wise degraded blood and saliva stain samples using the Affymetrix U133 plus2 GeneChip, (2) consulted expression databases to obtain additional information on tissue specificity, and (3) confirmed expression patterns of the most promising candidate genes by quantitative real-time polymerase chain reaction including additional forensically relevant tissues such as semen and vaginal secretion. Overall, we identified nine stable mRNA markers for blood and five stable mRNA markers for saliva detection showing tissue-specific expression signals in stains aged up to 180 days of age, expectedly older. Although, all of the markers were able to differentiate blood/saliva from semen samples, none of them could differentiate vaginal secretion because of the complex nature of vaginal secretion and the biological similarity of buccal and vaginal mucosa. We propose the use of these 14 stable mRNA markers for identification of blood and saliva stains in future forensic practice.

LPIN2 is associated with type 2 diabetes, glucose metabolism, and body composition.

OBJECTIVE: To identify the type 2 diabetes gene located at chromosome 18p11. RESEARCH DESIGN AND METHODS: We investigated the region in a young genetically isolated population by genotyping 34 single nucleotide polymorphisms (SNPs) in 78 case subjects and 101 control subjects. Two SNPs were selected and followed up in two cohorts. The first cohort came from a general Dutch population. In this cohort, association with type 2 diabetes was investigated using 616 type 2 diabetic case subjects and 2,890 control subjects; association with oral glucose tolerance test data was performed in 361 normoglycemic people. Association with fat distribution was studied in the second replication cohort, consisting of 836 people from the genetically isolated population. RESULTS: At the initial step, we found that the common C allele of SNP rs3745012 was associated with type 2 diabetes (odds ratio 2.01, P = 0.03). This SNP is located at the 3' untranslated region of the LPIN2 gene, which is a plausible candidate for type 2 diabetes and obesity. In the cohort from the general Dutch population, we demonstrated that rs3745012 interacts with BMI in determination of type 2 diabetes: whereas in subjects with high BMI, the common C allele is associated with type 2 diabetes, the same allele exhibits a neutral or protective effect in lean subjects (P = 0.05 overall effect, P = 0.02 interaction). Most remarkably, rs3745012 strongly affected composite insulin sensitivity index (P = 0.006 for overall effect, P = 0.004 for interaction). In the second replication cohort, we found that the allele C of rs3745012 increases trunk-to-legs fat mass ratio (P = 0.001) and may affect other fat-related measurements. CONCLUSIONS: rs3745012 SNP of the LPIN2 gene is associated with type 2 diabetes and fat distribution.