Correct Genotype Research Articles

P077 : IMPROVED SBTENGINE BATCH ANALYSIS MODULE

Aim HLA typing by Sanger Sequencing is a high-resolution typing method which depend on the correct analysis of the sequence traces. Using a combination of Core sequencing and Group Specific Sequencing reactions (GSSPs) results in allele level typing in the vast majority of samples. However, the reliability of the results depend on the sequence quality and the accuracy of the manual inspection and editing. SBTengine® is a software package which optimizes the SBT analysis workflow. CWD lists and genotype expected frequencies are applied to focus on the crucial steps of the analysis. GSSP advisor and GPS predictor are tools used to minimize the amount of work needed to come to the required resolution. However, an accurate manual inspection and editing are still required. Methods To reduce the manual inspection and editing work, we developed a module for batch analysis, which analyzes Sanger SBT data to come to an HLA typing result. The analysis includes an extensive evaluation of the sequence quality at every position in the context of the other traces of a sample. This means that the results are based on the combined quality of all traces. The module also optimizes the result in the context of the expected frequencies of the genotyping results. The quality of the traces directly influences the possibilities of batch analysis, which is a balance between acceptance of some data which turns out to result in inaccuracy of determining the typing results, or rejection, which results in rejection of a sample for automated typing. Result Here we will present an update on the batch analysis feature. The results of the batch analysis depend on the basic quality of the traces. With good quality data, in the majority of cases the correct genotype is identified, which makes the module applicable for registry typing. A small percentage of samples may not come to the correct genotype, although it is expected that this concerns only minor subtypes. For donor-recipient typing, the module can perform the first analysis, which always has to be followed by an intensive evaluation by the lab supervisor. Conclusion In conclusion: The batch analysis leads to a reduction of editing, and offers a method for increased efficiency of Sanger SBT data analysis. E.H. Rozemuller: Employee; Company/Organization; GenDx. Stock Shareholder; Company/Organization; GenDx. J. Geerligs: Employee; Company/Organization; GenDx. M.T. Penning: Employee; Company/Organization; GenDx. W. Mulder: Employee; Company/Organization; GenDx. Stock Shareholder; Company/Organization; GenDx.

Pepper, chili (Capsicum annuum).

Pepper is a recalcitrant plant for Agrobacterium-mediated genetic transformation. Several obstacles to genetic transformation remain such as extremely low transformation rates; the choice of correct genotype is critical; and there is a high frequency of false positives due to direct shoot formation. Here, we report a useful protocol with a suitable selection method. The most important aspect of the pepper transformation protocol is selecting shoots growing from the callus, which is referred to as callus-mediated shoot formation. This protocol is a reproducible and reliable system for pepper transformation.

Possible incorrect genotyping of heterozygous factor V Leiden and Prothrombin 20210 gene mutations by the GeneXpert assay

BackgroundThe GeneXpert analyzer is a hands-off system for the detection of Factor V Leiden and of Prothrombin G20210A (GPRO) gene thrombophilic mutations. Although the system is efficient and easy to use, we report the rare possibility of incorrect genotyping. Methods1648 samples were evaluated using the GeneXpert HemosIL Factor II and Factor V assay: 1319 were freshly analyzed while 329 were frozen, thawed and diluted with saline prior to analysis to avoid clogging of the instrument syringe. ResultsTwo samples, both heterozygous, one for the factor V Leiden and the other for the GPRO gene, were incorrectly genotyped as homozygous for the relative mutation. Inspection of the Ct values and amplification curves and genotyping with PCR revealed the correct genotype as heterozygous for factor V Leiden and GPRO mutation. DiscussionThe GeneXpert HemosIL Factor II and Factor V assay is an automated, fast genotyping assay requiring almost no sample manipulation, advantageous characteristics if compared with other PCR-based methods. However, an inattentive use of it can generate incorrect diagnosis. A careful handling of the sample, in particular correct dilution of frozen/thawed samples before analysis, and the inspection of the amplification curves and Ct values are required to avoid artifacts.

Demonstrating a multi-drug resistant Mycobacterium tuberculosis amplification microarray.

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Behavioral signatures related to genetic disorders in autism

BackgroundAutism spectrum disorder (ASD) is well recognized to be genetically heterogeneous. It is assumed that the genetic risk factors give rise to a broad spectrum of indistinguishable behavioral presentations.MethodsWe tested this assumption by analyzing the Autism Diagnostic Interview-Revised (ADI-R) symptom profiles in samples comprising six genetic disorders that carry an increased risk for ASD (22q11.2 deletion, Down’s syndrome, Prader-Willi, supernumerary marker chromosome 15, tuberous sclerosis complex and Klinefelter syndrome; total n = 322 cases, groups ranging in sample sizes from 21 to 90 cases). We mined the data to test the existence and specificity of ADI-R profiles using a multiclass extension of support vector machine (SVM) learning. We subsequently applied the SVM genetic disorder algorithm on idiopathic ASD profiles from the Autism Genetics Resource Exchange (AGRE).ResultsGenetic disorders were associated with behavioral specificity, indicated by the accuracy and certainty of SVM predictions; one-by-one genetic disorder stratifications were highly accurate leading to 63% accuracy of correct genotype prediction when all six genetic disorder groups were analyzed simultaneously. Application of the SVM algorithm to AGRE cases indicated that the algorithm could detect similarity of genetic behavioral signatures in idiopathic ASD subjects. Also, affected sib pairs in the AGRE were behaviorally more similar when they had been allocated to the same genetic disorder group.ConclusionsOur findings provide evidence for genotype-phenotype correlations in relation to autistic symptomatology. SVM algorithms may be used to stratify idiopathic cases of ASD according to behavioral signature patterns associated with genetic disorders. Together, the results suggest a new approach for disentangling the heterogeneity of ASD.

Insulin signaling promotes germline proliferation in C. elegans

There were errors published in Development137, 671-680.We recently discovered that the genotypes of several strains that we reported as daf-16(mu86) actually carry the daf-16(m26) allele. Correct genotypes for strains listed in Tables S1 and S3 and those used in experiments reported in Fig. 3C and Fig. S2C and related text and legends are:The conclusions of the paper are not altered by these errors. The authors apologise to readers for these mistakes.

Overexpression Of Leukemia Associated CblY371H Mutation In Transgenic Mice Causes Dosage Dependent Embryonic Lethality

Juvenile Myelomonocytic Leukemia (JMML) is a devastating childhood cancer which is rapidly fatal with infiltration of myeloid cells into multiple organs (Hess, Zutter, Castleberry, & Emanuel, 1996). Based on the observation that uniparental disomy was found in the chromosomal region 11q in JMML patient samples, about 15% of patient samples were found to contain a mutation in c-Cbl (Loh et al., 2009). Moreover, mutant Cbl was also found to be a tumor suppressor gene where a germline mutation results in the predisposition for developing JMML (Niemeyer et al., 2010). The c-Cbl gene encodes a multifunctional adaptor protein which contains an N-terminal tyrosine-kinase binding (TKB) domain, a RING finger motif which contain E3 ligase activity, and a C-terminal ubiquitin-associated domain. Previous mutations in myeloid malignancies have been described where mutations occur in the RING finger domain or the linker domain (Caligiuri et al., 2007; Dunbar et al., 2008). Interestingly, a hotspot for mutations at residue 371 exists in JMML patients where 1/3 of the detected mutations are a tyrosine to histidine substitution, Y371H (Loh et al., 2009). This residue belongs in the linker region of the CBL protein, and it was previously observed that Y371 mediates the binding of c-Cbl to the p85 subunit of PI3 kinase (Blaydes et al., 2001). In vitro, CblY371H mutation does indeed destroy its ligase function resulting in prolonged signaling through the Ras pathway only when the endogenous c-Cbl gene was silenced (Niemeyer et al., 2010). However, how mutant Cbl gives rise to JMML and how it acts in concert with other genes in the pathogenesis of JMML requires further study.To address these questions, we tested the oncogenicity of the CblY371H mutation in transgenic mice. Of the 6 founder transgenic mice, we chose L5 line for further analysis because it had the highest level of expression. As expected, overexpression of CblY371H by itself in wild type mice had no phenotype since inactivation of wild type Cbl allele is seen in patients. We, then, generated transgenic mice which were heterozygous for both the BAC transgene and the cbl null mutation (CblY371H; Cbl+/-) and then bred them to Cbl heterozygous knockout mice (Cbl+/-). Because homozygous knockout mice have reduced fertility, we preferentially utilized heterozygous mice for the breeding (El Chami et al., 2005). Interestingly, out of the first 118 progeny genotyped where 1/8 of the pups is expected to be the desired genotype (CblY371H;Cbl-/-), no pups were of the desired genotype suggesting embryonic lethality. Because mice which lack both c-cbl and cbl-b are embryonic lethal, we reasoned that high expression that is achieved from the L5 line was causing a dominant negative affect on cbl-b function resulting in an embryonic lethal phenotype. We then tested if a lower level of the transgene expression would result in viable pups. We used the K5 line which was the second highest expressing transgenic line. We were now able to generate the desired genotype (CblY371H; Cbl-/-) although at less than expected numbers. Thus far, we have generated 4 mice which have the correct genotype which are being monitored for disease. We also monitored the heterozygous mice that carried the transgene (CblY371H; Cbl+/-) which were also asymptomatic without any obvious phenotype. Further studies are being done to characterize the hematopoietic phenotype in these mice by isolating fetal liver cells followed by transplantation into lethally irradiated mice and will be discussed. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Optimization of a Molecular Genetic Assay for the Sensitive Detection of KRAS Mutations in Colorectal Carcinoma

Activating mutations of codons 12 and 13 of the KRAS gene occur frequently in colorectal carcinomas. Clinical trialshave shown patients with metastatic colorectal cancer benefit from treatment with tyrosine kinase inhibitors, eg.Vectibix (panitumumab), used to block EGFR overexpression if point mutations in codons 12 or 13 of the KRAS geneare absent. The DxS KRAS mutation kit, mandated by Health Canada as a companion diagnostic, doubled in price to$200-$500 per patient tested, making it less attractive for use by clinical laboratories. Using 25 cases of colorectalcancer previously analyzed by an external laboratory, we validated and compared DxS (Amplification Refractory)and a less costly method, SNaPshot (Primer Extension), for KRAS mutation analysis as well as two extractionmethods, Qiagen FFPE and PicoPure. We demonstrated that DNA purified using the Qiagen FFPE kit produced moreconsistent results. KRAS mutation status was obtained for all samples assayed and were compared with results fromthe external laboratory. Both DxS and SNaPshot met the required analytic sensitivity of 1% mutant to wild-typebackground. One sample was discordant by both assays with those previously reported due to either the assaysor sampling from a different location in the tumour tissue. SNaPshot detected a Gly13Asp variant as an artefactin addition to the correct genotype in four samples. DNA quality from the fixed tissues and PCR amplificationconditions were investigated as the cause. After further validation and refinement of the amplification conditions,the SNaPshot method has now been adopted for clinical use.

Modeling one complete versus triplicate analyses in low template DNA typing

There are generally two strategies for low template DNA typing: the complete strategy, which uses all available DNA in a single PCR and subsequent typing, and the consensus strategy, in which the biological sample is divided into two or more aliquots and the genotype profile is determined by consensus from these "replicates." In this study, the consensus and complete strategies are compared by a statistical approach in terms of the accuracy of obtaining the correct genotype at a single locus for single source samples. Logistic models were employed to describe the allele drop-out and drop-in events. The parameters of the models were estimated with empirical or hypothetical data. The probabilities of obtaining the true genotype and the chances to observe drop-out and drop-in alleles were estimated and compared for both strategies. Consistent with a previous experimental study, this study found that, with relatively high input DNA (e.g., ≥ 100 pg), the complete strategy performs better than the consensus strategy to obtain the true genotype and the complete strategy will display less dropped out alleles. The consensus strategy had less drop-in alleles for ≤ 100 pg DNA samples. Moreover, the limitations of the logistic models were discussed. Ideal models with better fit of empirical data approximating casework conditions were proposed for future studies.

‘Stochastic’ effects at balanced mixtures: A calibration study

DNA mixtures are challenging not only at low template DNA level but also at highly balanced quantitative ratio. In this latter case, interpretation may be complicated by the joint action of combinatorial uncertainty and stochastic effects of the PCR. We explore this particular and so far little noticed aspect of mixture interpretation by first providing a complete quantitative combinatorial analysis of the two-person mixture model (2PM) at highly balanced ratio of contributors, and then by carrying out a calibration study of the 2PM model on good quality experimental mixtures. The calibration tests provided the evidence for the existence of irregular distribution of peak heights, that can misguide the correct genotype assignment at high template ratios too. Repeating the experiment, performing Bayesian analysis to the whole evidence and developing a careful joint prediction of all plausible genotype datasets is highly mandatory in these cases, prior to set evidentiary LRs and use them in court.

A protocol to improve genotyping of problematic microsatellite loci of Trypanosoma brucei gambiense from body fluids

Microsatellite genotyping of Trypanosoma brucei gambiense, the causative agent of human African trypanosomiasis or sleeping sickness, and population genetics tools, are useful for inferring population parameters such as population size and dispersal. Amplifying parasite DNA directly from body fluids (i.e., blood, lymph or cerebrospinal fluid) allows avoiding costly and tedious isolation phases. It is however associated to increased frequencies of amplification failures (allelic dropouts and/or null alleles) at some loci. In this paper, we present a study focused on three T. brucei gambiense microsatellite loci suspected to present amplification problems when amplified from body fluids sampled in Guinean sleeping sickness foci. We checked for the real nature of blank and apparent homozygous genotypes of parasite DNA directly amplified from body fluids and tested the effect of three different DNA quantities of trypanosomes. Our results show that some initially blank and homozygous genotypes happen to be actual heterozygous genotypes. In Guinea, lymph from the cervical nymph nodes, known to contain the highest concentrations of parasites, appeared to provide the best amplification results. Simply repeating the PCR may be enough to retrieve the correct genotype, but we also show that increasing initial DNA content provides better results while undertaking first amplification. We finally propose an optimal protocol for amplifying trypanosome’s DNA directly from body fluids that should be adapted to local characteristics and/or constraints.

HBV Subgenotypes D1, D2, D-del! Are ‘Old‘ Genotyping Methods Interpreted Correctly?

HBV Subgenotypes D1, D2, D-del! Are ‘Old‘ Genotyping Methods Interpreted Correctly?

Quantifying single nucleotide variant detection sensitivity in exome sequencing

BackgroundThe targeted capture and sequencing of genomic regions has rapidly demonstrated its utility in genetic studies. Inherent in this technology is considerable heterogeneity of target coverage and this is expected to systematically impact our sensitivity to detect genuine polymorphisms. To fully interpret the polymorphisms identified in a genetic study it is often essential to both detect polymorphisms and to understand where and with what probability real polymorphisms may have been missed.ResultsUsing down-sampling of 30 deeply sequenced exomes and a set of gold-standard single nucleotide variant (SNV) genotype calls for each sample, we developed an empirical model relating the read depth at a polymorphic site to the probability of calling the correct genotype at that site. We find that measured sensitivity in SNV detection is substantially worse than that predicted from the naive expectation of sampling from a binomial. This calibrated model allows us to produce single nucleotide resolution SNV sensitivity estimates which can be merged to give summary sensitivity measures for any arbitrary partition of the target sequences (nucleotide, exon, gene, pathway, exome). These metrics are directly comparable between platforms and can be combined between samples to give “power estimates” for an entire study. We estimate a local read depth of 13X is required to detect the alleles and genotype of a heterozygous SNV 95% of the time, but only 3X for a homozygous SNV. At a mean on-target read depth of 20X, commonly used for rare disease exome sequencing studies, we predict 5–15% of heterozygous and 1–4% of homozygous SNVs in the targeted regions will be missed.ConclusionsNon-reference alleles in the heterozygote state have a high chance of being missed when commonly applied read coverage thresholds are used despite the widely held assumption that there is good polymorphism detection at these coverage levels. Such alleles are likely to be of functional importance in population based studies of rare diseases, somatic mutations in cancer and explaining the “missing heritability” of quantitative traits.

Results of the First Italian External Quality Assurance Scheme for Somatic EGFR Mutation Testing in Non–Small-Cell Lung Cancer

The Italian Association of Medical Oncology (AIOM) and the Italian Society of Pathology and Cytology organized an external quality assessment (EQA) scheme for EGFR mutation testing in non-small-cell lung cancer. Ten specimens, including three small biopsies with known epidermal growth factor receptor (EGFR) mutation status, were validated in three referral laboratories and provided to 47 participating centers. The participants were requested to perform mutational analysis, using their usual method, and to submit results within a 4-week time frame. According to a predefined scoring system, two points were assigned to correct genotype and zero points to false-negative or false-positive results. The threshold to pass the EQA was set at higher than 18 of 20 points. Two rounds were preplanned. All participating centers submitted the results within the time frame. Polymerase chain reaction (PCR)/sequencing was the main methodology used (n = 37 laboratories), although a few centers did use pyrosequencing (n = 8) or real-time PCR (n = 2). A significant number of analytical errors were observed (n = 20), with a high frequency of false-positive results (n = 16). The lower scores were obtained for the small biopsies. Fourteen of 47 centers (30%) that did not pass the first round, having a score less than or equal to 18 points, used PCR/sequencing, whereas 10 of 10 laboratories, using pyrosequencing or real-time PCR, passed the first round. Eight laboratories passed the second round. Overall, 41of 47 centers (87%) passed the EQA. The results of the EQA for EGFR testing in non-small-cell lung cancer suggest that good quality EGFR mutational analysis is performed in Italian laboratories, although differences between testing methods were observed, especially for small biopsies.

Genes, pseudogenes and like genes: The case of 21-hydroxylase in Italian population

BackgroundsRecently, we have considered two new findings in genetics of 21-hydroxylase deficiency with great interested: the existence of rare RCCX trimodular haplotypes, where the CYP21A2 like-gene downstream of the TNXA gene carries from one to six pseudogene mutations, and population specific allelic frequencies of wild-type CYP21A2 loci in the CYP21A1P pseudogene. Both these events represent a further complication in CYP21A2 genetics. Therefore, the choice of the molecular protocol becomes a crucial point when genetic analysis is required. In this regard, we must consider that the literature is still lacking consistent data on the Italian population. For this reason, we report genetic results obtained on 375 healthy individuals of Italian origin. MethodsDifferent genetic protocols were compared and novel molecular strategies were performed. ResultsIn our group, only two known haplotypes were identified. In addition, specific allelic frequencies of CYP21A2 wild-type loci in the pseudogene have been established. ConclusionsBased on our results, we can affirm that the employment of different molecular methods is necessary to ensure a correct CYP21A2 genotyping. In order to avoid mistakes both in patient diagnosis and/or in risk evaluation of the relatives, each case should be investigated in function of a careful clinical evaluation and the molecular test should be performed in specialized centres.

A New Microarray Substrate for Ultra-Sensitive Genotyping of KRAS and BRAF Gene Variants in Colorectal Cancer

Molecular diagnostics of human cancers may increase accuracy in prognosis, facilitate the selection of the optimal therapeutic regimen, improve patient outcome, reduce costs of treatment and favour development of personalized approaches to patient care. Moreover sensitivity and specificity are fundamental characteristics of any diagnostic method. We developed a highly sensitive microarray for the detection of common KRAS and BRAF oncogenic mutations. In colorectal cancer, KRAS and BRAF mutations have been shown to identify a cluster of patients that does not respond to anti-EGFR therapies; the identification of these mutations is therefore clinically extremely important. To verify the technical characteristics of the microarray system for the correct identification of the KRAS mutational status at the two hotspot codons 12 and 13 and of the BRAFV600E mutation in colorectal tumor, we selected 75 samples previously characterized by conventional and CO-amplification at Lower Denaturation temperature-PCR (COLD-PCR) followed by High Resolution Melting analysis and direct sequencing. Among these samples, 60 were collected during surgery and immediately steeped in RNAlater while the 15 remainders were formalin-fixed and paraffin-embedded (FFPE) tissues. The detection limit of the proposed method was different for the 7 KRAS mutations tested and for the V600E BRAF mutation. In particular, the microarray system has been able to detect a minimum of about 0.01% of mutated alleles in a background of wild-type DNA. A blind validation displayed complete concordance of results. The excellent agreement of the results showed that the new microarray substrate is highly specific in assigning the correct genotype without any enrichment strategy.

Saturated linkage map construction in Rubus idaeus using genotyping by sequencing and genome-independent imputation

BackgroundRapid development of highly saturated genetic maps aids molecular breeding, which can accelerate gain per breeding cycle in woody perennial plants such as Rubus idaeus (red raspberry). Recently, robust genotyping methods based on high-throughput sequencing were developed, which provide high marker density, but result in some genotype errors and a large number of missing genotype values. Imputation can reduce the number of missing values and can correct genotyping errors, but current methods of imputation require a reference genome and thus are not an option for most species.ResultsGenotyping by Sequencing (GBS) was used to produce highly saturated maps for a R. idaeus pseudo-testcross progeny. While low coverage and high variance in sequencing resulted in a large number of missing values for some individuals, a novel method of imputation based on maximum likelihood marker ordering from initial marker segregation overcame the challenge of missing values, and made map construction computationally tractable. The two resulting parental maps contained 4521 and 2391 molecular markers spanning 462.7 and 376.6 cM respectively over seven linkage groups. Detection of precise genomic regions with segregation distortion was possible because of map saturation. Microsatellites (SSRs) linked these results to published maps for cross-validation and map comparison.ConclusionsGBS together with genome-independent imputation provides a rapid method for genetic map construction in any pseudo-testcross progeny. Our method of imputation estimates the correct genotype call of missing values and corrects genotyping errors that lead to inflated map size and reduced precision in marker placement. Comparison of SSRs to published R. idaeus maps showed that the linkage maps constructed with GBS and our method of imputation were robust, and marker positioning reliable. The high marker density allowed identification of genomic regions with segregation distortion in R. idaeus, which may help to identify deleterious alleles that are the basis of inbreeding depression in the species.

Tumor Development, Growth Characteristics and Spectrum of Genetic Aberrations in the TH-MYCN Mouse Model of Neuroblastoma

BackgroundThe TH-MYCN transgenic neuroblastoma model, with targeted MYCN expression to the developing neural crest, has been used to study neuroblastoma development and evaluate novel targeted tumor therapies.MethodsWe followed tumor development in 395 TH-MYCN (129X1/SvJ) mice (125 negative, 206 hemizygous and 64 homozygous mice) by abdominal palpations up to 40 weeks of age. DNA sequencing of MYCN in the original plasmid construct and mouse genomic DNA was done to verify the accuracy. Copy number analysis with Affymetrix® Mouse Diversity Genotyping Arrays was used to characterize acquired genetic aberrations.ResultsDNA sequencing confirmed presence of human MYCN cDNA in genomic TH-MYCN DNA corresponding to the original plasmid construct. Tumor incidence and growth correlated significantly to transgene status with event-free survival for hemizygous mice at 50%, and 0% for homozygous mice. Hemizygous mice developed tumors at 5.6–19 weeks (median 9.1) and homozygous mice at 4.0–6.9 weeks (5.4). The mean treatment window, time from palpable tumor to sacrifice, for hemizygous and homozygous mice was 15 and 5.2 days, respectively. Hemizygous mice developing tumors as early as homozygous mice had a longer treatment window. Age at tumor development did not influence treatment window for hemizygous mice, whereas treatment window in homozygous mice decreased significantly with increasing age. Seven out of 10 analysed tumors had a flat DNA profile with neither segmental nor numerical chromosomal aberrations. Only three tumors from hemizygous mice showed acquired genetic features with one or more numerical aberrations. Of these, one event corresponded to gain on the mouse equivalent of human chromosome 17.ConclusionHemizygous and homozygous TH-MYCN mice have significantly different neuroblastoma incidence, tumor growth characteristics and treatment windows but overlap in age at tumor development making correct early genotyping essential to evaluate therapeutic interventions. Contrasting previous studies, our data show that TH-MYCN tumors have few genetic aberrations.

A combined proteomic and genetic analysis of the highly variable platelet proteome: From plasmatic proteins and SNPs

High biological variation in protein expression represents a major challenge in clinical proteomics. In a study based on 2D-DIGE, we found that the standardised abundance of only a few proteins varied by more than 50%. While some of the highest variable proteins in platelets of 52 healthy elderly were of plasmatic origin, such as albumin or haptoglobin, absence of several other high-abundant plasma proteins strongly suggests that plasma-derived proteins represent an integral part of the platelet proteome. Amongst the highly variable platelet-derived proteins, two spots were both identified as GSTO1 and assigned to either the wild-type or mutant isoform of SNP A140D. Remarkably, when the spots were considered within the respective genotype groups, their CV decreased to about the median variation. Albeit 2D-DIGE allowed correct genotyping, two individuals seemed to be GSTO1*A140 deficient. Probing 2D-Western blots with novel mAb, however, detected A140 protein as additional spot at pH 8.1, caused by the SNPs E155del and E208K. In contrast to previous studies, we show that GSTO1 protein is expressed in vivo, despite the deletion E155. Our data indicate that incorporation of exogenous proteins and genetic polymorphisms of endogenous proteins represent the main source of extreme biological variation in the platelet proteome.

Analysis of cDNA Molecules is Not Suitable for the Molecular Diagnosis of Mucopolysaccharidosis Type I

Nonsense-mediated decay (NMD) is a mechanism of the recognition and degradation of messenger RNA containing a premature stop codon. Nonsense mutations are the main mutations that lead to Mucopolysaccharidosis type I. To determine the effect of NMD on correct genotyping based on cDNA sequencing, we standardized the sequencing from alpha-L-iduronidase gene cDNA molecules and validated this process for a group of patients whose mutations had been previously identified by DNA analysis. Although the whole gene could be amplified in 5 polymerase chain reactions, cDNA proved unsuitable for molecular analysis as patients bearing splice site and nonsense mutations were not genotyped.