Evidence for multi-copy Mega-NUMTs in the human genome.

The maternal mode of mitochondrial DNA (mtDNA) inheritance is central to human genetics. Recently, evidence for bi-parental inheritance of mtDNA was claimed for individuals of three pedigrees that suffered mitochondrial disorders. We sequenced mtDNA using both direct Sanger and Massively Parallel Sequencing in several tissues of eleven maternally related and other affiliated healthy individuals of a family pedigree and observed mixed mitotypes in eight individuals. Cells without nuclear DNA, i.e. thrombocytes and hair shafts, only showed the mitotype of haplogroup (hg) V. Skin biopsies were prepared to generate ρ° cells void of mtDNA, sequencing of which resulted in a hg U4c1 mitotype. The position of the Mega-NUMT sequence was determined by fluorescence in situ hybridization and two different quantitative PCR assays were used to determine the number of contributing mtDNA copies. Thus, evidence for the presence of repetitive, full mitogenome Mega-NUMTs matching haplogroup U4c1 in various tissues of eight maternally related individuals was provided. Multi-copy Mega-NUMTs mimic mixtures of mtDNA that cannot be experimentally avoided and thus may appear in diverse fields of mtDNA research and diagnostics. We demonstrate that hair shaft mtDNA sequencing provides a simple but reliable approach to exclude NUMTs as source of misleading results.

Searching for blood in Chinese lacquerware: zhu xie hui.

The study gives an overview of the tests and analyses undertaken in the past 20 years to establish the presence of blood in the foundation layers of Chinese lacquer artefacts and also shows the development of analytical methods over that period. When undertaking the conservation of lacquer objects it is crucial to know the type of binding medium as this influences the selection of any consolidants that may be required in the treatment. Microchemical tests to identify blood using benzidine and luminol, various chromatographic and mass spectrometric techniques and DNA analyses were assessed on selected Chinese lacquer objects, and the results gained are summarized.

Chimerism in DNA of buccal swabs from recipients after allogeneic hematopoietic stem cell transplantations: implications for forensic DNA testing.

We attempted to quantitatively determine the chimeric state in a total of 162 buccal swabs from 77 adult recipients aged 19-74 (median 50 years) after allogeneic hematopoietic cell transplantation by estimating the chimeric recipient/donor DNA ratios through analysis of 15 autosomal short tandem repeat markers. From each individual between one and nine, buccal swabs were taken at known time intervals after transplantation, ranging from 17 to 3,361 days (median 394 days). In buccal cells, the determined recipient/donor DNA ratios turned out to be highly variable between individuals and also within an individual. Relative donor chimerism levels (%Ch) between 0 and 100 % were detected with maximal frequencies between 10 and 30 %. Blood was always found to show the donor's genotype while hair samples in all cases gave the recipient's genotype. We examine chimerism levels with respect to age, gender, and posttransplantation period and discuss the results in the context of forensic identity testing.

The LASSIE MPS panel: Predicting externally visible traits in dogs for forensic purposes.

Predicting the outward appearance of dogs via their DNA, also known as Canine DNA Phenotyping, is a young, emerging field of research in forensic genetics. The few previous studies published in this respect were restricted to the consecutive analysis of single DNA markers, a process that is time- and sample-consuming and therefore not a viable option for limited forensic specimens. Here, we report on the development and evaluation of a Massively Parallel Sequencing (MPS) based molecular genetic assay, the LASSIE MPS Panel. This panel aims to predict externally visible as well as skeletal traits, which include coat color, coat pattern, coat structure, tail morphology, skull shape, ear shape, eye color and body size from DNA using 44 genetic markers in a single molecular genetic assay. A biostatistical naïve Bayes classification approach was applied to identify the most informative marker combinations for predicting phenotypes. Overall, the predictive performance was characterized by a very high classification success for some of the trait categories, and high to moderate success for others. The performance of the developed predictive framework was further evaluated using blind samples from three randomly selected dog individuals, whose appearance was well predicted.

Morphological and Tissue Characterization with 3D Reconstruction of a 350-Year-Old Austrian Ardea purpurea Glacier Mummy.

Glaciers are dwindling archives, releasing animal mummies preserved in the ice for centuries due to climate changes. As preservation varies, residual soft tissues may differently expand the biological information content of such mummies. DNA studies have proven the possibility of extracting and analyzing DNA preserved in skeletal residuals and sediments for hundreds or thousands of years. Paleoradiology is the method of choice as a non-destructive tool for analyzing mummies, including micro-computed tomography (micro-CT) and magnetic resonance imaging (MRI). Together with radiocarbon dating, histo-anatomical analyses, and DNA sequencing, these techniques were employed to identify a 350-year-old Austrian Ardea purpurea glacier mummy from the Ötztal Alps. Combining these techniques proved to be a robust methodological concept for collecting inaccessible information regarding the structural organization of the mummy. The variety of methodological approaches resulted in a distinct picture of the morphological patterns of the glacier animal mummy. The BLAST search in GenBank resulted in a 100% and 98.7% match in the cytb gene sequence with two entries of the species Purple heron (Ardea purpurea; Accession number KJ941160.1 and KJ190948.1) and a 98% match with the same species for the 16 s sequence (KJ190948.1), which was confirmed by the anatomic characteristics deduced from micro-CT and MRI.

The toll-like receptor 2 (TLR2) -196 to -174 del/ins polymorphism affects viral loads and susceptibility to hepatocellular carcinoma in chronic hepatitis C.

Chronic hepatitis C virus (HCV) infection is a major risk factor for hepatocellular carcinoma (HCC). HCV proteins core and NS3 can bind to toll-like receptor 2 (TLR2) and trigger inflammatory responses. Polymorphisms in the TLR2 gene predispose to various forms of malignancy but have not been studied in HCV-associated HCC. Here, we investigated whether single nucleotide polymorphisms (SNPs), rs4696480, rs5743708, rs5743704 and the -196 to -174 del/ins polymorphism of the TLR2 gene affect the risk for HCC in chronic hepatitis C. The study involved 189 and 192 HCV genotype 1 infected patients with and without HCC, respectively, as well as 347 healthy controls. TLR2 alleles were determined by hybridization probe assays and allele-specific short fragment polymerase chain reaction on a LightCycler system. All TLR2 polymorphisms matched the Hardy-Weinberg equilibrium in each study group. Although TLR2 SNPs showed no effect, the frequency of the TLR2 -196 to -174 del allele was significantly higher in patients with HCV-associated HCC (22.5%) than in HCV-infected patients without HCC (15.6%, p = 0.016) and healthy controls (15.3%, p = 0.003). HCV-infected carriers of a TLR2 -196 to -174 del allele had significantly higher HCV viral loads than TLR2 -196 to -174 ins/ins homozygous patients (p = 0.031). Finally, in carriers of the TLR2 -196 to -174 del allele, stimulation of monocytes resulted in significantly lower TLR2 expression levels and interleukin-8 (IL-8) induction than in individuals with the TLR2 -196 to -174 ins/ins genotype (p < 0.05). Our data suggest the TLR2 -196 to -174 del allele to affect HCV viral loads and to increase the risk for HCC in HCV genotype1-infected patients.

The CXCL1 rs4074 A allele is associated with enhanced CXCL1 responses to TLR2 ligands and predisposes to cirrhosis in HCV genotype 1-infected Caucasian patients.

BACKGROUND & AIMS: CXCL1 is a ligand for CXC chemokine-receptor 2 expressed on hepatic stellate cells (HSC). Thus, CXCL1 might contribute to HSC activation and fibrogenesis. Here, we investigated whether the CXCL1 rs4074 polymorphism affects CXCL1 expression and progression of chronic hepatitis C virus (HCV) infection towards cirrhosis. METHODS: The study involved 237 patients with chronic HCV genotype 1 infection (75 with cirrhosis) and 342 healthy controls. The CXCL1 rs4074 polymorphism was determined by a LightSNiP assay on the LightCycler system. CXCL1 serum levels and induction in response to HCV proteins were studied by ELISA. RESULTS: Distributions of CXCL1 genotypes (GG/GA/AA) matched the Hardy-Weinberg equilibrium in all subgroups (HCV-associated cirrhosis: 29.3%/54.7%/16.0%; non-cirrhotic HCV infection: 45.1%/44.4%/10.5%, healthy controls: 46.2%/40.9%/12.9%). HCV-infected cirrhotic patients had a significantly greater CXCL1 rs4074 A allele frequency (43.3%) than patients without cirrhosis (32.7%, OR=1.573, p=0.03) and healthy controls (33.3%, OR=1.529, p=0.02). In vitro carriers of the A allele produced greater amounts of CXCL1 in response to TLR2-ligands including HCV core and NS3, and HCV-infected carriers of the CXCL1 rs4074 A allele had higher CXCL1 serum levels than those with the G/G genotype. Moreover, multivariate Cox-regression analysis confirmed age and the presence of a CXCL1 rs4074 A allele as risk factors for cirrhosis. CONCLUSIONS: Enhanced production of CXCL1 in response to HCV antigens in carriers of the rs4074 A allele together with its increased frequency in cirrhotic patients with hepatitis C suggest the CXCL1 rs4074 A allele as a genetic risk factor for cirrhosis progression in hepatitis C.

Post hoc deconvolution of human mitochondrial DNA mixtures by EMMA 2 using fine-tuned Phylotree nomenclature.

In this paper we present a new algorithm for splitting (partial) human mitogenomes into components with high similarity to haplogroup motifs of Phylotree. The algorithm reads a (partial) mitogenome coded by the differences to the reference (rCRS) and outputs the estimated haplogroups of the putative components. The algorithm requires no special information on the raw data of the sequencing process and is therefore suited for the post hoc analysis of mixtures of any sequencing technology. The software EMMA 2 implementing the algorithm will be made available via the EMPOP (https://empop.online) database and extends the nine years old software EMMA for haplogrouping single mitogenomes to mixtures with at most three components.

Towards Forensic DNA Phenotyping for Predicting Visible Traits in Dogs.

The popularity of dogs as human companions explains why these pets regularly come into focus in forensic cases such as bite attacks or accidents. Canine evidence, e.g., dog hairs, can also act as a link between the victim and suspect in a crime case due to the close contact between dogs and their owners. In line with human DNA identification, dog individualization from crime scene evidence is mainly based on the analysis of short tandem repeat (STR) markers. However, when the DNA profile does not match a reference, additional information regarding the appearance of the dog may provide substantial intelligence value. Key features of the dog's appearance, such as the body size and coat colour are well-recognizable and easy to describe even to non-dog experts, including most investigating officers and eyewitnesses. Therefore, it is reasonable to complement eyewitnesses' testimonies with externally visible traits predicted from associated canine DNA samples. Here, the feasibility and suitability of canine DNA phenotyping is explored from scratch in the form of a proof of concept study. To predict the overall appearance of an unknown dog from its DNA as accurately as possible, the following six traits were chosen: (1) coat colour, (2) coat pattern, (3) coat structure, (4) body size, (5) ear shape, and (6) tail length. A total of 21 genetic markers known for high predicting values for these traits were selected from previously published datasets, comprising 15 SNPs and six INDELS. Three of them belonged to SINE insertions. The experiments were designed in three phases. In the first two stages, the performance of the markers was tested on DNA samples from dogs with well-documented physical characteristics from different breeds. The final blind test, including dogs with initially withheld appearance information, showed that the majority of the selected markers allowed to develop composite sketches, providing a realistic impression of the tested dogs. We regard this study as the first attempt to evaluate the possibilities and limitations of forensic canine DNA phenotyping.

Evaluation of DNA Extraction Methods Developed for Forensic and Ancient DNA Applications Using Bone Samples of Different Age.

The efficient extraction of DNA from challenging samples, such as bones, is critical for the success of downstream genotyping analysis in molecular genetic disciplines. Even though the ancient DNA community has developed several protocols targeting small DNA fragments that are typically present in decomposed or old specimens, only recently forensic geneticists have started to adopt those protocols. Here, we compare an ancient DNA extraction protocol (Dabney) with a bone extraction method (Loreille) typically used in forensics. Real-time quantitative PCR and forensically representative typing methods including fragment size analysis and sequencing were used to assess protocol performance. We used four bone samples of different age in replicates to study the effects of both extraction methods. Our results confirm Loreille's overall increased gain of DNA when enough tissue is available and Dabney's improved efficiency for retrieving shorter DNA fragments that is beneficial when highly degraded DNA is present. The results suggest that the choice of extraction method needs to be based on available sample, degradation state, and targeted genotyping method. We modified the Dabney protocol by pooling parallel lysates prior to purification to study gain and performance in single tube typing assays and found that up to six parallel lysates lead to an almost linear gain of extracted DNA. These data are promising for further forensic investigations as the adapted Dabney protocol combines increased sensitivity for degraded DNA with necessary total DNA amount for forensic applications.

Forensic characterization and statistical considerations of the CaDNAP 13-STR panel in 1,184 domestic dogs from Germany, Austria, and Switzerland.

Crime scene samples originating from domestic dogs such as hair, blood, or saliva can be probative as possible transfer evidence in human crime and in dog attack cases. In the majority of such cases canine DNA identification using short tandem repeat (STR) analysis is the method of choice, which demands, among others, a systematic survey of allele frequency data in the relevant dog populations. A set of 13 highly polymorphic canine STR markers was used to analyze samples of 1,184 dogs (including 967 purebred dogs) from the so-called DACH countries (Germany, Austria, Switzerland). This CaDNAP 13-STR panel has previously been validated for canine identification in a forensic context. Here, we present robust estimates of allele frequencies, which are essential to assess the weight of the evidence by estimating the probability of a matching DNA profile within the dog population under question, e.g. in the form of a random match probability (RMP). The geographical provenance of the tested dogs showed a negligible influence on the observed genotype variation. Therefore, we combined the STR data from all three countries into a single dog population sample (DPS). In contrast, pronounced genetic differentiation between dog breeds was found by principal component analysis and sub-structure analysis with the STRUCTURE software. These findings entailed the need to account for the effects of DPS breed composition on allele frequency estimates. A possible strategy, which was favored here, relies on collecting a DPS that is guided by the breed composition of the relevant dog population. In total, dogs from 166 different breeds were included in our DPS, 64 of them including at least 5 individuals (n = 771 dogs). Sampling reflected the abundance of breeds in the DACH countries with the following being the most common ones: German Shepherds (population frequency: 14.3%), Dachshunds (5.9%), Labrador Retrievers (3.9%), and Golden Retrievers (3.2%). The pedigree listing of the purebred dogs in our DPS ranked German Shepherds (DPS frequency 8.5%) first, followed by Labrador Retrievers (3.9%), Golden Retrievers (3%), and Dachshunds (2.5%). RMP values based on overall allele frequencies and accounting for substructure using FST between breeds ranged between 10-13 and 10-14 and represent a conservative approach of RMP assessment.

Dog breed affiliation with a forensically validated canine STR set.

We tested a panel of 13 highly polymorphic canine short tandem repeat (STR) markers for dog breed assignment using 392 dog samples from the 23 most popular breeds in Austria, Germany, and Switzerland. This STR panel had originally been selected for canine identification. The dog breeds sampled in this study featured a population frequency ≥1% and accounted for nearly 57% of the entire pedigree dog population in these three countries. Breed selection was based on a survey comprising records for nearly 1.9 million purebred dogs belonging to more than 500 different breeds. To derive breed membership from STR genotypes, a range of algorithms were used. These methods included discriminant analysis of principal components (DAPC), STRUCTURE, GeneClass2, and the adegenet package for R. STRUCTURE analyses suggested 21 distinct genetic clusters. Differentiation between most breeds was clearly discernable. Fourteen of 23 breeds (61%) exhibited maximum mean cluster membership proportions of more than 0.70 with a highest value of 0.90 found for Cavalier King Charles Spaniels. Dogs of only 6 breeds (26%) failed to consistently show only one major cluster. The DAPC method yielded the best assignment results in all 23 declared breeds with 97.5% assignment success. The frequency-based assignment test also provided a high success rate of 87%. These results indicate the potential viability of dog breed prediction using a well-established and sensitive set of 13 canine STR markers intended for forensic routine use.

Hairy matters: MtDNA quantity and sequence variation along and among human head hairs.

Hairs from the same donor have been found to differ in mtDNA sequence within and among themselves and from other tissues, which impacts interpretation of results obtained in a forensic setting. However, little is known on the magnitude of this phenomenon and published data on systematic studies are scarce. We addressed this issue by generating mtDNA control region (CR) profiles of >450 hair fragments from 21 donors by Sanger-type sequencing (STS). To mirror forensic scenarios, we compared hair haplotypes from the same donors to each other, to the corresponding buccal swab reference haplotypes and analyzed several fragments of individual hairs. We also investigated the effects of hair color, donor sex and age, mtDNA haplogroup and chemical treatment on mtDNA quantity, amplification success and variation. We observed a wide range of individual CR sequence variation. The reference haplotype was the only or most common (≥75%) hair haplotype for most donors. However, in two individuals, the reference haplotype was only found in about a third of the investigated hairs, mainly due to differences at highly variable positions. Similarly, most hairs revealed the reference haplotype along their entire length, however, about a fifth of the hairs contained up to 71% of segments with deviant haplotypes, independent of the longitudinal position. Variation affected numerous positions, typically restricted to the individual hair and in most cases heteroplasmic, but also fixed (i.e. homoplasmic) substitutions were observed. While existing forensic mtDNA interpretation guidelines were found still sufficient for all comparisons to reference haplotypes, some comparisons between hairs from the same donor could yield false exclusions when those guidelines are strictly followed. This study pinpoints the special care required when interpreting mtDNA results from hair in forensic casework.

Hepatocyte-specific Keap1 deletion reduces liver steatosis but not inflammation during non-alcoholic steatohepatitis development.

Generation of reactive oxygen species (ROS) in response to fatty acids accumulation has been classically proposed as a possible "second hit" triggering progression from simple steatosis to non-alcoholic steatohepatitis (NASH). In this study we challenged hepatocyte-specific Keap1 knockout mice (Keap1(Δhepa)) and littermate Cre- controls (Keap1(fx/fx)) with two different diet models of NASH in order to evaluate the effects of the anti-oxidant transcription factor Nrf2 over-activation on hepatic metabolism and disease progression. After 4 weeks of MCD diet the liver/body weight ratio of Keap1(Δhepa) mice was significantly higher compared to littermate controls with no differences in total body weight. Strikingly, liver histology revealed a dramatic reduction of lipid droplets confirmed by a decreased content of intra-hepatic triglycerides in Keap1(Δhepa) compared to controls. In parallel to reduced expression of genes involved in lipid droplet formation, protein expression of Liver X Receptor (LXRα/ß) and Peroxisome proliferator-activated receptor α (PPARα) was significantly decreased. In contrast, genes involved in mitochondrial lipid catabolism were markedly up-regulated in Keap1(Δhepa) livers. A similar phenotype characterized by inhibition of lipogenesis in favor of increased mitochondrial catabolic activity was also observed after 13 weeks of western diet administration. MCD-induced apoptosis was significantly dampened in Keap1(Δhepa) compared to Keap1(fx/fx) as detected by TUNEL, cleaved caspase-3 and Bcl-2 protein expression analyses. However, no differences in inflammatory F4/80- and CD11b-positive cells and pro-fibrogenic genes were detected between the two groups. Although hepatic lack of Keap1 did not ameliorate inflammation, the resulting constitutive Nrf2 over-activation in hepatocytes strongly reduced hepatic steatosis via enhanced lipid catabolism and repressed de novo lipogenesis during murine NASH development.

Molecular genetic analysis on the remains of the Dark Countess: Revisiting the French Royal family.

The "Dark Counts" were a mysterious couple that appeared in the Thuringian village Eishausen in 1807. After living in self imposed solitude for 30 years the woman died and was buried under the name Sophia Botta. Her companion, who presented himself as Vavel de Versay, died in 1845 and was later identified as Leonardus Cornelius van der Valck, secretary of the Dutch embassy in Paris. Their lifestyle led to speculations that she was the true princess Marie Thérèse Charlotte of France, daughter of Louis XVI and Marie Antoinette. According to these speculations she was substituted by another young woman on a voyage from Paris to Vienna. Molecular genetic analyses were set out to test the remains attributed to the Dark Countess. Mitochondrial DNA testing brought concordant results determined in two forensic laboratories (Innsbruck, Austria and Freiburg, Germany) on parallel samples of the remains. The results were in exclusion to both, the mitochondrial lineage earlier reported for the French Royal family and the mitochondrial haplotype observed in a living descendant of the Royal family.

A farnesoid X receptor polymorphism predisposes to spontaneous bacterial peritonitis.

BACKGROUND: In mice, the farnesoid X receptor is involved in bacterial translocation, which can result in spontaneous bacterial peritonitis in patients with cirrhosis. We investigated if polymorphisms in the farnesoid X receptor gene influence the risk for spontaneous bacterial peritonitis. METHODS: Laboratory and clinical data of 293 cirrhotic patients with ascites and 226 healthy controls were prospectively collected. The rs56163822, rs11110390 and rs12313471 polymorphisms of the farnesoid X receptor were determined. RESULTS: 115 (39%) patients had spontaneous bacterial peritonitis. Distribution of all farnesoid X receptor genotypes matched the Hardy-Weinberg equilibrium. Patients with spontaneous bacterial peritonitis had a higher frequency of the rs56163822 GT genotype (7.0%) than patients without (1.7%, OR=4.4, p=0.02). This genotype was confirmed as predictor of spontaneous bacterial peritonitis by binary logistic regression analysis (OR=6.8, p=0.018). CONCLUSION: The farnesoid X receptor rs56163822 GT genotype increases the risk for spontaneous bacterial peritonitis in cirrhotic patients with ascites.

Influence of the CXCL1 rs4074 A allele on alcohol induced cirrhosis and HCC in patients of European descent.

BACKGROUND AND AIMS: CXCL1 (CXC chemokine-ligand-1) is a ligand for CXC chemokine receptor 2 expressed on hepatic stellate cells (HSC). Thus, CXCL1 might contribute to HSC activation and fibrogenesis. In the present study, we investigated the influence of the CXCL1 rs4074 polymorphism on the occurrence of alcohol induced liver cirrhosis and hepatocellular carcinoma (HCC). METHODS: The study involved 458 patients with alcoholic cirrhosis (170 with HCC), 115 alcoholics without liver disease and 342 healthy controls. All subjects were genotyped for the CXCL1 rs4074 polymorphism and CXCL1 serum levels of 132 patients were measured. In vitro CXCL1 secretion in TLR-transfected cell lines were studied by ELISA. RESULTS: Distribution of the CXCL1 genotypes (GG/GA/AA) was 159/219/80 in patients with alcoholic cirrhosis, 52/44/19 in alcoholic controls and 158/140/44 in healthy controls. Patients with alcohol-induced cirrhosis were significantly more often carriers of the CXCL1 rs4074 A allele (65.3%) than alcoholics without liver disease (54.8%, OR=1.55; 95%CI=1.025-2.350; p=0.04) and healthy controls (53.8%, OR=1.62; 95%CI=1.212-2.151; p=0.001). Accordingly, the frequency of the CXCL1 rs4074 A allele was significantly higher in the cirrhotic patients than in the subjects without cirrhosis (41.4% vs. 33.9%, OR=1.38, 95% CI:1.14-1.66, p=0.001). Furthermore cirrhotic carriers of the CXCL1 rs4074 A allele had significantly higher CXCL1 serum levels than carriers of the GG genotype. In contrast to sera from healthy controls, sera from patients with alcoholic cirrhosis induced CXCL1 secretion in TLR2- (p=0.016) and TLR4- (p=0.008) transfected HEK293 cells. This finding indicates that sera from patients with alcoholic cirrhosis contain soluble ligands that can induce CXCL1 production via stimulation of TLRs. CONCLUSION: The enhanced CXCL1 serum levels in carriers of the rs4074 A allele together with their increased frequency in patients with alcohol induced cirrhosis suggest the CXCL1 rs4074 A allele as a genetic risk factor for alcoholic cirrhosis.

Detection of IGF2BP3, HOXB7, and NEK2 mRNA expression in brush cytology specimens as a new diagnostic tool in patients with biliary strictures.

INTRODUCTION: It is a challenging task to distinguish between benign and malignant lesions in patients with biliary strictures. Here we analyze whether determination of target gene mRNA levels in intraductal brush cytology specimens may be used to improve the diagnosis of bile duct carcinoma. MATERIALS AND METHODS: Brush cytology specimens from 119 patients with biliary strictures (malignant: n = 72; benign: n = 47) were analyzed in a retrospective cohort study. mRNA of IGF-II mRNA-binding protein 3 (IGF2BP3), homeobox B7 (HOXB7), Forkhead box M1 (FOXM1), kinesin family member 2C (KIF2C) and serine/threonine kinase NEK2 was determined by semi-quantitative RT-PCR using the ΔCt method. RESULTS: IGF2BP3 (p<0.0001), HOXB7 (p<0.0001), and NEK2 (p<0.0001) mRNA expression levels were significantly increased in patients with cholangiocarcinoma or pancreatic cancer. Median ΔCt values differed by 3.5 cycles (IGF2BP3), 2.8 cycles (HOXB7) and 1.3 cycles (NEK2) corresponding to 11-fold, 7-fold and 2.5-fold increased mRNA levels in malignant versus benign samples. Sensitivity to detect biliary cancer was 76.4% for IGF2BP3 (80.9% specificity); 72.2% for HOXB7 (78.7% specificity) and 65.3% for NEK2 (72.3% specificity), whereas routine cytology reached only 43.1% sensitivity (85.4% specificity). Diagnostic precision was further improved, when all three molecular markers were assessed in combination (77.8% sensitivity, 87.2% specificity) and achieved 87.5% sensitivity and 87.2% specificity when molecular markers were combined with routine cytology. CONCLUSIONS: Our data suggest that measuring IGF2BP3, HOXB7 and NEK2 mRNA levels by RT-PCR in addition to cytology has the potential to improve detection of malignant biliary disorders from brush cytology specimens.

The PNPLA3 rs738409 148M/M genotype is a risk factor for liver cancer in alcoholic cirrhosis but shows no or weak association in hepatitis C cirrhosis.

BACKGROUND: An isoleucine>methionine mutation at position 148 in the PNPLA3 gene (p.I148M, rs738409) has recently been identified as a susceptibility factor for liver damage in steatohepatitis. Here, we studied whether the PNPLA3 rs738409 polymorphism also affects predisposition to hepatocellular carcinoma (HCC). METHODS: We compared distributions of PNPLA3 genotypes in 80 and 81 Caucasian patients with alcoholic and hepatitis C virus (HCV)-associated HCC to 80 and 81 age- and sex-matched patients with alcohol-related and HCV-related cirrhosis without HCC, respectively. PNPLA3 genotypes in 190 healthy individuals from the same population served as reference. Potential confounders obesity, diabetes, HCV genotype and HBV co-infection were controlled by univariate and multivariate logistic regression with forward variable selection. RESULTS: PNPLA3 genotypes were in Hardy-Weinberg equilibrium for all study groups. The frequency of the 148M allele was significantly (p<0.001) increased in alcoholic cirrhosis with (53.7%) and without HCC (36.2%) but was not different between healthy controls (22.9%) and patients with cirrhosis (25.3%; p = 0.545) and HCC (30.2%; p = 0.071) due to hepatitis C. HCC risk was highest in 148M/M homozygous patients with alcoholic liver disease (odds ratio (OR) 16.8 versus healthy controls; 95% confidence interval (CI) 6.68-42.43, p<0.001). Finally, multivariate regression confirmed 148M/M homozygosity (OR 2.8; 95%-CI: 1.24-6.42; p = 0.013) as HCC risk factor in alcoholic cirrhosis. In HCV-related cirrhosis only HCV genotype 1 was confirmed as a HCC risk factor (OR 4.2; 95%-CI: 1.50-11.52; p = 0.006). CONCLUSION: The PNPLA3 148M variant is a prominent risk factor for HCC in patients with alcoholic cirrhosis, while its effects are negligible in patients with cirrhosis due to HCV. This polymorphism provides an useful tool to identify individuals with particularly high HCC risk in patients with alcoholic liver disease that should be taken into account in future HCC prevention studies.

Mini-midi-mito: adapting the amplification and sequencing strategy of mtDNA to the degradation state of crime scene samples.

The degradation state of some biological traces recovered from the crime scene requires the amplification of very short fragments to attain a useful mitochondrial (mt)DNA sequence. We have previously introduced two mini-multiplex assays that amplify 10 overlapping control region (CR) fragments in two separate multiplex PCRs, which brought successful CR consensus sequences from even highly degraded DNA extracts. This procedure requires a total of 20 sequencing reactions per sample, which is laborious and cost intensive. For only moderately degraded samples that we encounter more frequently with typical mtDNA casework material, we developed two new multiplex assays that use a subset of the mini-amplicon primers but embrace larger fragments (midis) and require only 10 sequencing reactions to build a double-stranded CR consensus sequence. We used a preceding mtDNA quantitation step by real-time PCR with two different target fragments (143 and 283 bp) that roughly correspond to the average fragment sizes of the different multiplex approaches to estimate size-dependent mtDNA quantities and to aid the choice of the appropriate PCR multiplexes with respect to quality of the results and required costs.