Peter Nuernberg

Induced pluripotent stem cell-derived neuronal cells from a sporadic Alzheimer’s disease donor as a model for investigating AD-associated gene regulatory networks

BackgroundAlzheimer’s disease (AD) is a complex, irreversible neurodegenerative disorder. At present there are neither reliable markers to diagnose AD at an early stage nor therapy. To investigate underlying disease mechanisms, induced pluripotent stem cells (iPSCs) allow the generation of patient-derived neuronal cells in a dish.ResultsIn this study, employing iPS technology, we derived and characterized iPSCs from dermal fibroblasts of an 82-year-old female patient affected by sporadic AD. The AD-iPSCs were differentiated into neuronal cells, in order to generate disease-specific protein association networks modeling the molecular pathology on the transcriptome level of AD, to analyse the reflection of the disease phenotype in gene expression in AD-iPS neuronal cells, in particular in the ubiquitin-proteasome system (UPS), and to address expression of typical AD proteins.We detected the expression of p-tau and GSK3B, a physiological kinase of tau, in neuronal cells derived from AD-iPSCs. Treatment of neuronal cells differentiated from AD-iPSCs with an inhibitor of γ-secretase resulted in the down-regulation of p-tau. Transcriptome analysis of AD-iPS derived neuronal cells revealed significant changes in the expression of genes associated with AD and with the constitutive as well as the inducible subunits of the proteasome complex. The neuronal cells expressed numerous genes associated with sub-regions within the brain thus suggesting the usefulness of our in-vitro model. Moreover, an AD-related protein interaction network composed of APP and GSK3B among others could be generated using neuronal cells differentiated from two AD-iPS cell lines.ConclusionsOur study demonstrates how an iPSC-based model system could represent (i) a tool to study the underlying molecular basis of sporadic AD, (ii) a platform for drug screening and toxicology studies which might unveil novel therapeutic avenues for this debilitating neuronal disorder.

Heterogeneous Mechanisms of Primary and Acquired Resistance to Third-Generation EGFR Inhibitors.

Purpose: To identify novel mechanisms of resistance to third-generation EGFR inhibitors in patients with lung adenocarcinoma that progressed under therapy with either AZD9291 or rociletinib (CO-1686). Experimental Design: We analyzed tumor biopsies from seven patients obtained before, during, and/or after treatment with AZD9291 or rociletinib (CO-1686). Targeted sequencing and FISH analyses were performed, and the relevance of candidate genes was functionally assessed in in vitro models. Results: We found recurrent amplification of either MET or ERBB2 in tumors that were resistant or developed resistance to third-generation EGFR inhibitors and show that ERBB2 and MET activation can confer resistance to these compounds. Furthermore, we identified a KRASG12S mutation in a patient with acquired resistance to AZD9291 as a potential driver of acquired resistance. Finally, we show that dual inhibition of EGFR/MEK might be a viable strategy to overcome resistance in EGFR-mutant cells expressing mutant KRAS. Conclusions: Our data suggest that heterogeneous mechanisms of resistance can drive primary and acquired resistance to third-generation EGFR inhibitors and provide a rationale for potential combination strategies. Clin Cancer Res; 22(19); 4837–47. ©2016 AACR.

Genetic variation in dopaminergic activity is associated with the risk for psychiatric side effects of levetiracetam

Purpose:  Levetiracetam (LEV) is a highly effective antiepileptic agent. A clinically relevant psychiatric complication of LEV treatment, however, is the provocation of irritability and aggression. Recent behavioral research indicates that personality traits may predispose to these side effects. To assess the genetic basis of the adverse psychotropic profile of LEV, a candidate gene‐based two‐stage association study was conducted.

Mapping of a new locus for congenital anomalies of the kidney and urinary tract on chromosome 8q24

BACKGROUND Congenital anomalies of the kidney and urinary tract (CAKUT) account for the majority of end-stage renal disease in children (50%). Previous studies have mapped autosomal dominant loci for CAKUT. We here report a genome-wide search for linkage in a large pedigree of Somalian descent containing eight affected individuals with a non-syndromic form of CAKUT. METHODS Clinical data and blood samples were obtained from a Somalian family with eight individuals with CAKUT including high-grade vesicoureteral reflux and unilateral renal agenesis. Total genome search for linkage was performed using a 50K SNP Affymetric DNA microarray. As neither parent is affected, the results of the SNP array were analysed under recessive models of inheritance, with and without the assumption of consanguinity. RESULTS Using the non-consanguineous recessive model, a new gene locus (CAKUT1) for CAKUT was mapped to chromosome 8q24 with a significant maximum parametric Logarithm of the ODDs (LOD) score (LOD(max)) of 4.2. Recombinations were observed in two patients defining a critical genetic interval of 2.5 Mb physical distance flanked by markers SNP_A-1740062 and SNP_A-1653225. CONCLUSION We have thus identified a new non-syndromic recessive gene locus for CAKUT (CAKUT1) on chromosome 8q24. The identification of the disease-causing gene will provide further insights into the pathogenesis of urinary tract malformations and mechanisms of renal development.

Label-Free Protein-RNA Interactome Analysis Identifies Khsrp Signaling Downstream of the p38/Mk2 Kinase Complex as a Critical Modulator of Cell Cycle Progression

Growing evidence suggests a key role for RNA binding proteins (RBPs) in genome stability programs. Additionally, recent developments in RNA sequencing technologies, as well as mass-spectrometry techniques, have greatly expanded our knowledge on protein-RNA interactions. We here use full transcriptome sequencing and label-free LC/MS/MS to identify global changes in protein-RNA interactions in response to etoposide-induced genotoxic stress. We show that RBPs have distinct binding patterns in response to genotoxic stress and that inactivation of the RBP regulator module, p38/MK2, can affect the entire spectrum of protein-RNA interactions that take place in response to stress. In addition to validating the role of known RBPs like Srsf1, Srsf2, Elavl1 in the genotoxic stress response, we add a new collection of RBPs to the DNA damage response. We identify Khsrp as a highly regulated RBP in response to genotoxic stress and further validate its role as a driver of the G1/S transition through the suppression of Cdkn1aP21 transcripts. Finally, we identify KHSRP as an indicator of overall survival, as well as disease free survival in glioblastoma multiforme.

Deep ion sequencing of amplicon adapter ligated libraries: a novel tool in molecular diagnostics of formalin fixed and paraffin embedded tissues

Due to the advanced progress in personalised therapy concepts for non-small cell lung cancer (NSCLC), we applied the ion semiconductor sequencing (ISS) approach to molecular diagnosis of NSCLC, analysing a set of therapy relevant gene loci. DNA from macrodissected tumour samples of formalin fixed biopsies was used for PCR amplification of EGFR exons 18, 19, 21 and KRAS exon 1. A total of 128 PCR products were analysed by conventional termination sequencing as well as by ISS. Sensitivity of ISS was additionally determined using 100–10 000 copies of reference mutants. All somatic mutations detected by direct Sanger sequencing were also identified by ISS. No additional mutants were detected. Running samples with limited copies of mutated alleles revealed high sensitivity, detecting less than 10% (2500 copies) mutants in a human wild type background. In conclusion, multiplexed mutation analyses by ISS is an efficient technology that can easily be linked to existing PCR approaches in molecular pathology.

Epidemiological, clinical and biochemical characterization of the p.(Ala359Asp) SMPD1 variant causing Niemann–Pick disease type B

Niemann–Pick disease type B (NPDB) is a rare, inherited lysosomal storage disorder that occurs due to variants in the sphingomyelin phosphodiesterase 1 (SMPD1) gene and the resultant deficiency of acid sphingomyelinase (ASM) activity. While numerous variants causing NPDB have been described, only a small number have been studied in any detail. Herein, we describe the frequency of the p.(Ala359Asp) variant in the healthy Chilean population, and determine the haplotype background of homozygous patients to establish if this variant originated from a common founder. Genomic DNA samples from 1691 healthy individuals were analyzed for the p.(Ala359Asp) variant. The frequency of p.(Ala359Asp) was found to be 1/105.7, predicting a disease incidence of 1/44 960 in Chile, higher than the incidence estimated by the number of confirmed NPDB cases. We also describe the clinical characteristics of 13 patients homozygous for p.(Ala359Asp) and all of them had moderate to severe NPDB disease. In addition, a conserved haplotype and shared 280 Kb region around the SMPD1 gene was observed in the patients analyzed, indicating that the variant originated from a common ancestor. The haplotype frequency and mitochondrial DNA analysis suggest an Amerindian origin for the variant. To assess the effect of the p.(Ala359Asp) variant, we transfected cells with the ASM-p.(Ala359Asp) cDNA and the activity was only 4.2% compared with the wild-type cDNA, definitively demonstrating the causative effect of the variant on ASM function. Information on common variants such as p.(Ala359Asp) is essential to guide the successful implementation for future therapies and benefit to patients.

Single molecule real time sequencing in ADTKD- MUC1 allows complete assembly of the VNTR and exact positioning of causative mutations

Recently, the Mucin-1 (MUC1) gene has been identified as a causal gene of autosomal dominant tubulointerstitial kidney disease (ADTKD). Most causative mutations are buried within a GC-rich 60 basepair variable number of tandem repeat (VNTR), which escapes identification by massive parallel sequencing methods due to the complexity of the VNTR. We established long read single molecule real time sequencing (SMRT) targeted to the MUC1-VNTR as an alternative strategy to the snapshot assay. Our approach allows complete VNTR assembly, thereby enabling the detection of all variants residing within the VNTR and simultaneous determination of VNTR length. We present high resolution data on the VNTR architecture for a cohort of snapshot positive (n = 9) and negative (n = 7) ADTKD families. By SMRT sequencing we could confirm the diagnosis in all previously tested cases, reconstruct both VNTR alleles and determine the exact position of the causative variant in eight of nine families. This study demonstrates that precise positioning of the causative mutation(s) and identification of other coding and noncoding sequence variants in ADTKD-MUC1 is feasible. SMRT sequencing could provide a powerful tool to uncover potential factors encoded within the VNTR that associate with intra- and interfamilial phenotype variability of MUC1 related kidney disease.

Beijing clades of Mycobacterium tuberculosis are associated with differential survival in HIV-negative Russian patients

We conducted a prospective study to establish factors associated with survival in tuberculosis patients in Russia including social, clinical and pathogen-related genetic parameters. Specifically we wished to determine whether different strains/clades of the Beijing lineage exerted a differential effect of survival. HIV-negative culture-confirmed cases were recruited during 2008-2010 across Samara Oblast and censored in December 2011. Molecular characterization was performed by a combination of spoligotyping, multilocus VNTR typing and whole genome sequencing (WGS). We analyzed 2602 strains and detected a high prevalence of Beijing family (n=1933; 74%) represented largely by two highly homogenous dominant clades A (n=794) and B (n=402) and non-A/non-B (n=737). Multivariable analysis of 1366 patients with full clinical and genotyping data showed that multi- and extensive drug resistance (HR=1.86; 95%CI: 1.52, 2.28 and HR=2.19; 95%CI: 1.55, 3.11) had the largest impact on survival. In addition older age, extensive lung damage, shortness of breath, treatment in the past and alcohol abuse reduced survival time. After adjustment for clinical and demographic predictors there was evidence that clades A and B combined were associated with poorer survival than other Beijing strains (HR=0.48; 95%CI 0.34, 0.67). All other pathogen-related factors (polymorphisms in genes plcA, plcB, plcC, lipR, dosT and pks15/1) had no effect on survival. In conclusion, drug resistance exerted the greatest effect on survival of TB patients. Nevertheless we provide evidence for the independent biological effect on survival of different Beijing family strains even within the same defined geographical population. Better understanding of the role of different strain factors in active disease and their influence on outcome is essential.

A novel MYO6 splice site mutation causes autosomal dominant sensorineural hearing loss type DFNA22 with a favourable outcome after cochlear implantation.

Mutations in MYO6 encoding an atypical myosin motor protein important for inner ear hair cell function have been associated with autosomal recessive (DFNB37) and autosomal dominant (DFNA22) types of hearing loss in a few families worldwide. After genome-wide linkage analysis, we identified a novel MYO6 mutation at the splice acceptor site of exon 7 (c.554-1G>A) in an extended German family with autosomal dominant postlingual non-syndromic hearing impairment. Analysis of blood-derived cDNA revealed different aberrantly spliced mRNAs caused by the mutation, which are predicted to severely interfere with protein function. Two of the family members underwent cochlear implantation at ages 53 and 65. Here, we present detailed clinical data of this family which suggest a favourable outcome of cochlear implantation in hearing-impaired individuals with a MYO6 mutation.