Volker Endris

Mutations in the SHANK2 synaptic scaffolding gene in autism spectrum disorder and mental retardation

Using microarrays, we identified de novo copy number variations in the SHANK2 synaptic scaffolding gene in two unrelated individuals with autism-spectrum disorder (ASD) and mental retardation. DNA sequencing of SHANK2 in 396 individuals with ASD, 184 individuals with mental retardation and 659 unaffected individuals (controls) revealed additional variants that were specific to ASD and mental retardation cases, including a de novo nonsense mutation and seven rare inherited changes. Our findings further link common genes between ASD and intellectual disability.

SHANK1 Deletions in Males with Autism Spectrum Disorder

Recent studies have highlighted the involvement of rare (<1% frequency) copy-number variations and point mutations in the genetic etiology of autism spectrum disorder (ASD); these variants particularly affect genes involved in the neuronal synaptic complex. The SHANK gene family consists of three members (SHANK1, SHANK2, and SHANK3), which encode scaffolding proteins required for the proper formation and function of neuronal synapses. Although SHANK2 and SHANK3 mutations have been implicated in ASD and intellectual disability, the involvement of SHANK1 is unknown. Here, we assess microarray data from 1,158 Canadian and 456 European individuals with ASD to discover microdeletions at the SHANK1 locus on chromosome 19. We identify a hemizygous SHANK1 deletion that segregates in a four-generation family in which male carriers--but not female carriers--have ASD with higher functioning. A de novo SHANK1 deletion was also detected in an unrelated male individual with ASD with higher functioning, and no equivalent SHANK1 mutations were found in >15,000 controls (p = 0.009). The discovery of apparent reduced penetrance of ASD in females bearing inherited autosomal SHANK1 deletions provides a possible contributory model for the male gender bias in autism. The data are also informative for clinical-genetics interpretations of both inherited and sporadic forms of ASD involving SHANK1.

The novel Rho-GTPase activating gene MEGAP/ srGAP3 has a putative role in severe mental retardation

In the last few years, several genes involved in X-specific mental retardation (MR) have been identified by using genetic analysis. Although it is likely that additional genes responsible for idiopathic MR are also localized on the autosomes, cloning and characterization of such genes have been elusive so far. Here, we report the isolation of a previously uncharacterized gene, MEGAP, which is disrupted and functionally inactivated by a translocation breakpoint in a patient who shares some characteristic clinical features, such as hypotonia and severe MR, with the 3p− syndrome. By fluorescence in situ hybridization and loss of heterozygosity analysis, we demonstrated that this gene resides on chromosome 3p25 and is deleted in 3p− patients that present MR. MEGAP/srGAP3 mRNA is predominantly and highly expressed in fetal and adult brain, specifically in the neurons of the hippocampus and cortex, structures known to play a pivotal role in higher cognitive function, learning, and memory. We describe several MEGAP/srGAP3 transcript isoforms and show that MEGAP/srGAP3a and -b represent functional GTPase-activating proteins (GAP) by an in vitro GAP assay. MEGAP/srGAP3 has recently been shown to be part of the Slit-Robo pathway regulating neuronal migration and axonal branching, highlighting the important role of MEGAP/srGAP3 in mental development. We propose that haploinsufficiency of MEGAP/srGAP3 leads to the abnormal development of neuronal structures that are important for normal cognitive function.

Molecular Diagnostic Profiling of Lung Cancer Specimens with a Semiconductor-Based Massive Parallel Sequencing Approach Feasibility, Costs, and Performance Compared with Conventional Sequencing

In the context of personalized oncology, screening for somatic tumor mutations is crucial for prediction of an individual patients response to therapy. Massive parallel sequencing (MPS) has been suggested for routine diagnostics, but this technology has not been sufficiently evaluated with respect to feasibility, reliability, and cost effectiveness with routine diagnostic formalin-fixed, paraffin-embedded material. We performed ultradeep targeted semiconductor-based MPS (190 amplicons covering hotspot mutations in 46 genes) in a variety of formalin-fixed, paraffin-embedded diagnostic samples of lung adenocarcinoma tissue with known EGFR mutations (n = 28). The samples reflected the typical spectrum of tissue material for diagnostics, including small biopsies and samples with low tumor-cell content. Using MPS, we successfully sequenced all samples, with a mean read depth of 2947 reads per amplicon. High-quality sequence reads were obtained from samples containing ≥10% tumor material. In all but one sample, variant calling identified the same EGFR mutations as were detected by conventional Sanger sequencing. Moreover, we identified 43 additional mutations in 17 genes and detected amplifications in the EGFR and ERBB2 genes. MPS performance was reliable and independent of the type of material, as well as of the fixation and extraction methods, but was influenced by tumor-cell content and the degree of DNA degradation. Using sample multiplexing, focused MPS approached diagnostically acceptable cost rates.

Dynamic expression of the Slit‐Robo GTPase activating protein genes during development of the murine nervous system

We investigated the expression of the three known Slit‐Robo GTPase activating protein (srGAP) genes in the developing murine nervous system using in situ hybridization. The three genes are expressed during embryonic and early postnatal development in the murine nervous system, showing a distinct pattern of expression in the olfactory system, the eye, forebrain and midbrain structures, the cerebellum, the spinal cord, and dorsal root ganglia, which we discuss in relation to Slit‐Robo expression patterns and signaling pathways. We also report srGAP2 expression in zones of neuronal differentiation and srGAP3 in ventricular zones of neurogenesis in many different tissues of the central nervous system (CNS). Compared to srGAP2 and srGAP3, the onset of srGAP1 expression is later in most CNS tissues. We propose that these differences in expression point to functional differences between these three genes in the development of neural tissues. J. Comp. Neurol. 513:224–236, 2009.

Homozygous loss of CHRNA7 on chromosome 15q13.3 causes severe encephalopathy with seizures and hypotonia.

Homozygous Loss of CHRNA7 on Chromosome 15q13.3 Causes Severe Encephalopathy With Seizures and Hypotonia Volker Endris, Karl Hackmann, Teresa M. Neuhann, Ute Grasshoff, Michael Bonin, Ulrich Haug, Gabriele Hahn, Jens C. Schallner, Evelin Schr€ock, Sigrid Tinschert, Gudrun Rappold, and Ute Moog* Department of Molecular Human Genetics, Institute of Human Genetics, Heidelberg University, Heidelberg, Germany Institute of Clinical Genetics, Technical University Dresden, Dresden, Germany Institute of Human Genetics, Medical Genetics T€ubingen, T€ubingen, Germany Center for Child Neurology and Social Pediatrics Maulbronn, Maulbronn, Germany Children’s Hospital, Medical Faculty Carl Gustav Carus, University of Technology, Dresden, Germany Institute of Diagnostic Radiology, Department of Pediatric Radiology, Medical Faculty Carl Gustav Carus, University of Technology, Dresden, Germany Department of Human Genetics, Institute of Human Genetics, Heidelberg University, Heidelberg, Germany

High-throughput diagnostic profiling of clinically actionable gene fusions in lung cancer.

Molecular profiling of non‐small cell lung cancers (NSCLC) has a strong impact on clinical decision making and current oncological therapies. Besides detection of activating mutations in EGFR, analysis of ALK and ROS1 gene rearrangements has come into focus for targeted therapies. Targeted massive parallel sequencing (MPS) has been established for routine diagnostic profiling of the most prevalent oncogenic mutations in NSCLC, but not for the detection of gene rearrangements yet. Here, we present and evaluate an MPS‐based panel sequencing approach which simultaneously detects ALK, ROS1, and RET fusions as well as somatic mutations in a single multiplex assay using formalin‐fixed paraffin‐embedded (FFPE) tissue. To this end, we first evaluated sensitivity and specificity of the fusion assay retrospectively by employing it to a set of 50 NSCLC with known gene fusions (n = 35) and with no gene fusions (n = 15). The sensitivity and specificity of the MPS assay for the detection of known fusions was 100%. In a second prospective phase, we implemented the approach of parallel mutation and gene fusion detection in our routine diagnostic workflow to assess performance of the test in a diagnostic outreach setting. Our prospective screening of 109 NSCLC samples revealed four gene fusions all of which were confirmed by FISH. In conclusion, our approach facilitates simultaneous high‐throughput detection of gene fusions and somatic mutations in NSCLC samples and is able to replace conventional methods.

Srgap3−/− mice present a neurodevelopmental disorder with schizophrenia-related intermediate phenotypes

Mutations in the SRGAP3 gene residing on chromosome 3p25 have previously been associated with intellectual disability. Genome‐wide association studies have also revealed SRGAP3, together with genes from the same cellular network, as risk genes for schizophrenia. SRGAP3 regulates cytoskeletal dynamics through the RHO protein RAC1. RHO proteins are known to be involved in cytoskeletal reorganization during brain development to control processes such as synaptic plasticity. To elucidate the importance of SRGAP3 in brain development, we generated Srgap3‐knockout mice. Ten percent of these mice developed a hydrocephalus and died before adulthood. Surviving mice showed various neuroanatomical changes, including enlarged lateral ventricles, white matter tracts, and dendritic spines together with molecular changes, including an increased basal activity of RAC1. Srgap3–/– mice additionally exhibited a complex behavioral phenotype. Behavioral studies revealed an impaired spontaneous alternation and social behavior, while long‐term memory was unchanged. The animals also had tics. Lower locomotor activity was observed in male Srgap3–/– only. Srgap3–/– mice showed increased methylphenidate stimulation in males and an impaired prepulse inhibition in females. Together, the results show neurodevelopmental aberration in Srgap3–/– mice, with many of the observed phenotypes matching several schizophrenia‐related intermediate phenotypes. Mutations of SRGAP3 may thus contribute to various neurodevelopmental disorders.—Waltereit, R., Leimer, U., von Bohlen und Halbach, O., Panke, J., Hölter, S. M., Garrett, L., Wittig, K., Schneider, M., Schmitt, C., Calzada‐Wack, J., Neff, F., Becker, L., Prehn, C., Kutscherjawy, S., Endris, V., Bacon, C., Fuchs, H., Gailus‐Durner, V., Berger, S., Schönig, K., Adamski, J., Klopstock, T., Esposito, I., Wurst, W., Hrabě de Angelis, M., Rappold, G., Wieland, T., Bartsch, D. Srgap3–/– mice present a neurodevelopmental disorder with schizophrenia‐related intermediate phenotypes. FASEB J. 26, 4418–4428 (2012). www.fasebj.org

Distribution of MED12 mutations in fibroadenomas and phyllodes tumors of the breast—implications for tumor biology and pathological diagnosis

Somatic mutations in exon 2 of MED12 have been described in benign and malignant smooth muscle cell tumors suggesting a functional role in these neoplasms. Recently fibroadenomas of the breast were also reported to harbor MED12 mutations. Hence, we explored MED12 mutations in fibroepithelial tumors of the breast, histological subtypes of fibroadenomas and phyllodes tumors, to validate and extend previous efforts. Using conventional Sanger sequencing, we profiled 39 cases of fibroepithelial breast tumors comprising classic histological subtypes of fibroadenomas as well as benign and malignant phyllodes tumors for mutations in exon 2 of MED12. MED12 mutations were detected in 60% of all tumor samples with the majority being missense mutations affecting codon 44. Additionally, we report novel in‐frame deletions that have not been described previously. Sixty‐two percent of the fibroadenomas harbored mutated MED12 with intracanalicular fibroadenomas being the most frequently mutated histological subtype (82%). Of note, 8/11 of benign phyllodes tumors had MED12 mutations while only 1/5 of malignant phyllodes tumors showed mutations in exon 2 of MED12. In conclusion, we confirm the frequent occurrence of MED12 mutations in fibroadenomas, provide evidence that most intracanalicular fibroadenomas closely resembling benign phyllodes as well as benign phyllodes tumors harbor MED12 mutations, and conclude that MED12 mutations in malignant phyllodes tumors appear to be relatively rare.

Mutations in POLE and survival of colorectal cancer patients - link to disease stage and treatment

Recent molecular profiling studies reported a new class of ultramutated colorectal cancers (CRCs), which are caused by exonuclease domain mutations (EDMs) in DNA polymerase ϵ (POLE). Data on the clinical implications of these findings as to whether these mutations define a unique CRC entity with distinct clinical outcome are lacking. We performed Sanger sequencing of the POLE exonuclease domain in 431 well‐characterized patients with microsatellite stable (MSS) CRCs of a population‐based patient cohort. Mutation data were analyzed for associations with major epidemiological, clinical, genetic, and pathological parameters including overall survival (OS) and disease‐specific survival (DSS). In 373 of 431 MSS CRC, all exons of the exonuclease domain were analyzable. Fifty‐four mutations were identified in 46 of these samples (12.3%). Besides already reported EDMs, we detected many new mutations in exons 13 and 14 (corresponding to amino acids 410–491) as well as in exon 9 and exon 11 (corresponding to aa 268–303 and aa 341–369). However, we did not see any significant associations of EDMs with clinicopathological parameters, including sex, age, tumor location and tumor stage, CIMP, KRAS, and BRAF mutations. While with a median follow‐up time of 5.0 years, survival analysis of the whole cohort revealed nonsignificantly different adjusted hazard ratios (HRs) of 1.35 (95% CI: 0.82–2.25) and 1.44 (0.81–2.58) for OS and DSS indicating slightly impaired survival of patients with EDMs, subgroup analysis for patients with stage III/IV disease receiving chemotherapy revealed a statistically significantly increased adjusted HR (1.87; 95%CI: 1.02–3.44). In conclusion, POLE EDMs do not appear to define an entirely new clinically distinct disease entity in CRC but may have prognostic or predictive implications in CRC subgroups, whose significance remains to be investigated in future studies.