Yumi Kasai

C-terminal truncations in human 3'-5' DNA exonuclease TREX1 cause autosomal dominant retinal vasculopathy with cerebral leukodystrophy

Autosomal dominant retinal vasculopathy with cerebral leukodystrophy is a microvascular endotheliopathy with middle-age onset. In nine families, we identified heterozygous C-terminal frameshift mutations in TREX1, which encodes a 3′-5′ exonuclease. These truncated proteins retain exonuclease activity but lose normal perinuclear localization. These data have implications for the maintenance of vascular integrity in the degenerative cerebral microangiopathies leading to stroke and dementias.

Novel MEK1 Mutation Identified by Mutational Analysis of Epidermal Growth Factor Receptor Signaling Pathway Genes in Lung Adenocarcinoma

Genetic lesions affecting a number of kinases and other elements within the epidermal growth factor receptor (EGFR) signaling pathway have been implicated in the pathogenesis of human non-small-cell lung cancer (NSCLC). We performed mutational profiling of a large cohort of lung adenocarcinomas to uncover other potential somatic mutations in genes of this pathway that could contribute to lung tumorigenesis. We have identified in 2 of 207 primary lung tumors a somatic activating mutation in exon 2 of MEK1 (i.e., mitogen-activated protein kinase kinase 1 or MAP2K1) that substitutes asparagine for lysine at amino acid 57 (K57N) in the nonkinase portion of the kinase. Neither of these two tumors harbored known mutations in other genes encoding components of the EGFR signaling pathway (i.e., EGFR, HER2, KRAS, PIK3CA, and BRAF). Expression of mutant, but not wild-type, MEK1 leads to constitutive activity of extracellular signal-regulated kinase (ERK)-1/2 in human 293T cells and to growth factor-independent proliferation of murine Ba/F3 cells. A selective MEK inhibitor, AZD6244, inhibits mutant-induced ERK activity in 293T cells and growth of mutant-bearing Ba/F3 cells. We also screened 85 NSCLC cell lines for MEK1 exon 2 mutations; one line (NCI-H1437) harbors a Q56P substitution, a known transformation-competent allele of MEK1 originally identified in rat fibroblasts, and is sensitive to treatment with AZD6244. MEK1 mutants have not previously been reported in lung cancer and may provide a target for effective therapy in a small subset of patients with lung adenocarcinoma.

Massive parallel sequencing uncovers actionable FGFR2–PPHLN1 fusion and ARAF mutations in intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (iCCA) is a fatal bile duct cancer with dismal prognosis and limited therapeutic options. By performing RNA- and exome-sequencing analyses, we report a novel fusion event, FGFR2-PPHLN1 (16%), and damaging mutations in the ARAF oncogene (11%). Here we demonstrate that the chromosomal translocation t(10;12)(q26;q12) leading to FGFR2-PPHLN1 fusion possesses transforming and oncogenic activity, which is successfully inhibited by a selective FGFR2 inhibitor in vitro. Among the ARAF mutations, N217I and G322S lead to activation of the pathway and N217I shows oncogenic potential in vitro. Screening of a cohort of 107 iCCA patients reveals that FGFR2 fusions represent the most recurrent targetable alteration (45%, 17/107), while they are rarely present in other primary liver tumours (0/100 of hepatocellular carcinoma (HCC); 1/21 of mixed iCCA-HCC). Taken together, around 70% of iCCA patients harbour at least one actionable molecular alteration (FGFR2 fusions, IDH1/2, ARAF, KRAS, BRAF and FGF19) that is amenable for therapeutic targeting.

Mutational Analysis of EGFR and Related Signaling Pathway Genes in Lung Adenocarcinomas Identifies a Novel Somatic Kinase Domain Mutation in FGFR4

Background Fifty percent of lung adenocarcinomas harbor somatic mutations in six genes that encode proteins in the EGFR signaling pathway, i.e., EGFR, HER2/ERBB2, HER4/ERBB4, PIK3CA, BRAF, and KRAS. We performed mutational profiling of a large cohort of lung adenocarcinomas to uncover other potential somatic mutations in genes of this signaling pathway that could contribute to lung tumorigenesis. Methodology/Principal Findings We analyzed genomic DNA from a total of 261 resected, clinically annotated non-small cell lung cancer (NSCLC) specimens. The coding sequences of 39 genes were screened for somatic mutations via high-throughput dideoxynucleotide sequencing of PCR-amplified gene products. Mutations were considered to be somatic only if they were found in an independent tumor-derived PCR product but not in matched normal tissue. Sequencing of 9MB of tumor sequence identified 239 putative genetic variants. We further examined 22 variants found in RAS family genes and 135 variants localized to exons encoding the kinase domain of respective proteins. We identified a total of 37 non-synonymous somatic mutations; 36 were found collectively in EGFR, KRAS, BRAF, and PIK3CA. One somatic mutation was a previously unreported mutation in the kinase domain (exon 16) of FGFR4 (Glu681Lys), identified in 1 of 158 tumors. The FGFR4 mutation is analogous to a reported tumor-specific somatic mutation in ERBB2 and is located in the same exon as a previously reported kinase domain mutation in FGFR4 (Pro712Thr) in a lung adenocarcinoma cell line. Conclusions/Significance This study is one of the first comprehensive mutational analyses of major genes in a specific signaling pathway in a sizeable cohort of lung adenocarcinomas. Our results suggest the majority of gain-of-function mutations within kinase genes in the EGFR signaling pathway have already been identified. Our findings also implicate FGFR4 in the pathogenesis of a subset of lung adenocarcinomas.

Integrated Genomic Analysis Implicates Haploinsufficiency of Multiple Chromosome 5q31.2 Genes in De Novo Myelodysplastic Syndromes Pathogenesis

Deletions spanning chromosome 5q31.2 are among the most common recurring cytogenetic abnormalities detectable in myelodysplastic syndromes (MDS). Prior genomic studies have suggested that haploinsufficiency of multiple 5q31.2 genes may contribute to MDS pathogenesis. However, this hypothesis has never been formally tested. Therefore, we designed this study to systematically and comprehensively evaluate all 28 chromosome 5q31.2 genes and directly test whether haploinsufficiency of a single 5q31.2 gene may result from a heterozygous nucleotide mutation or microdeletion. We selected paired tumor (bone marrow) and germline (skin) DNA samples from 46 de novo MDS patients (37 without a cytogenetic 5q31.2 deletion) and performed total exonic gene resequencing (479 amplicons) and array comparative genomic hybridization (CGH). We found no somatic nucleotide changes in the 46 MDS samples, and no cytogenetically silent 5q31.2 deletions in 20/20 samples analyzed by array CGH. Twelve novel single nucleotide polymorphisms were discovered. The mRNA levels of 7 genes in the commonly deleted interval were reduced by 50% in CD34+ cells from del(5q) MDS samples, and no gene showed complete loss of expression. Taken together, these data show that small deletions and/or point mutations in individual 5q31.2 genes are not common events in MDS, and implicate haploinsufficiency of multiple genes as the relevant genetic consequence of this common deletion.

Low Frequency of telomerase Rna Mutations Among Children With Aplastic Anemia or Myelodysplastic Syndrome

Mutations in TERC, the RNA component of telomerase, result in autosomal dominant dyskeratosis congenita (DC), a rare bone marrow failure syndrome. TERC mutations have been detected in a subset of patients previously diagnosed with aplastic anemia and myelodysplastic syndrome (MDS), and these TERC mutations are clinically relevant as patients with DC respond poorly to conventional therapies. We aimed to determine the frequency of TERC mutations in pediatric patients with aplastic anemia and MDS who required a hematopoietic stem cell transplant. We obtained 284 blood samples from the National Donor Marrow Program Research Sample Repository from children and adolescents with bone marrow failure who underwent an unrelated stem cell transplant. We screened these samples for mutations in the TERC gene using direct DNA sequencing. We found 2 patients with sequence alterations in TERC. We identified a 2 base pair deletion (−240delCT) in a 4-year-old child with MDS and a single nucleotide alteration (−99→CG) in a 1-year-old child with juvenile myelomonocytic leukemia. Screening for TERC gene mutations is unlikely to diagnose occult DC in children with severe bone marrow failure who require a hematopoietic stem cell transplant.

The Roberts syndrome/SC phocomelia spectrum—A case report of an adult with review of the literature†

Roberts syndrome (RBS) (OMIM #268300) is a rare autosomal recessive disorder characterized by tetraphocomelia (symmetrical limb reduction), craniofacial anomalies, growth retardation, mental retardation, cardiac and renal abnormalities. The syndrome is caused by mutations in the ESCO2 (establishment of cohesion 1 homolog 2) (Entrez 609353) gene, which is located at 8p21.1, and encodes a protein essential in establishing sister chromatid cohesion during S phase. SC phocomelia (SC) (OMIM #269000), has less severe symmetric limb reduction, flexion contractures of various joints, minor facial anomalies, growth retardation and occasionally, mental retardation. These two syndromes can be considered part of a spectrum, with RBS at the most severe range in which severely affected infants may be stillborn or die in the post‐natal period, while individuals with SC phocomelia represent the milder end of the spectrum and typically survive to adulthood. In both presentations, karyotype investigations characteristically reveal premature centromere separation (PCS), otherwise known as heterochromatin repulsion or puffing. There is little literature about the follow‐up of adults with the spectrum of RBS/SC phocomelia or their recommended management. We report on an adult presentation of RBS/SC phocomelia spectrum disorder with a history of major cardiac malformation in childhood, normal limbs on physical examination, mild facial anomalies, mild learning difficulties, and PCS. Molecular studies of ESCO2 have confirmed the diagnosis. A literature review, focussing on adult manifestations of this condition and a discussion of follow‐up guidelines are presented.

Absence of Mutations in the Epidermal Growth Factor Receptor (EGFR) Kinase Domain in Patients with Mesothelioma

To the Editor: Malignant mesothelioma, a debilitating and often fatal malignancy occurs most commonly in patients with a history of exposure to asbestos. Majority of patients with pleural malignant mesothelioma present with unresectable disease. Epidermal Growth Factor Receptor (EGFR) is one of erbB family of receptor tyrosine kinases (TK) known to play a critical role in the cell proliferation and survival in malignant neoplasms. Several studies have reported an increased expression of EGFR in mesothelioma. EGFR-expressing mesothelioma cell lines seemed to be sensitive to treatment with EGFR tyrosine kinase inhibitors.1 After these observations, the Cancer and Leukemia Study Group B conducted a phase II trial of gefitinib, an EGFR TK inhibitor in 43 previously untreated patients with mesothelioma.2 Of the 28 samples tested for EGFR expression, 27 (97%) had an overexpression of EGFR. Despite EGFR overexpression, gefitinib was not active in malignant mesothelioma. EGFR expression is not an independent prognostic factor in patients with mesothelioma.2,3 There is a striking discordance between EGFR expression and lack of response to EGFR TK inhibitors in malignant mesothelioma. Mutations in exons 18–21 of the EGFR are associated with response to EGFR TK inhibitors in non-small cell lung cancer. There are only limited data on the prevalence of EGFR TK mutations in mesothelioma. We sought to investigate the presence of any EGFR mutations in our patients with mesothelioma. A total of 32 patients with a histologically confirmed diagnosis of mesothelioma seen at the Washington University School of Medicine were included in this study. DNA samples were obtained from paraffin-embedded tissue blocks containing representative malignant cells. High-throughput (96well plate) bidirectional dideoxynucleotide sequencing of polymerase chain reactionamplified gene products was performed at the Genome Sequencing Center (Washington University in St. Louis) as per standard protocol http://genome.wustl. edu/activity/med_seq/protocols.cgi. Exonic regions 18 –21, including the exon/intron boundaries of EGFR (Entrez GeneID 2065) were amplified by polymerase chain reaction. The sequence traces were assembled and scanned for variations from the reference sequence through the use of PolyPhred and PolyScan. All detailed protocols and primer sequences are available through the Washington University School of Medicine GSC website (http://genome.wustl. edu/platforms.cgi?id 7). After sequencing the DNA from the 32 mesothelioma samples, we found no evidence of mutations in our set of patients with mesothelioma. Our findings are consistent with the three earlier published studies.3–5 We further confirm the findings that although increased expression of EGFR is common in patients with mesothelioma, mutations of the EGFR kinase region commonly occurring in non-small cell lung cancer seem to be uncommon in mesothelioma. Lack of activating mutations in the EGFR TK domain may explain the inactivity of EGFR tyrosine kinase inhibitors in mesothelioma.

Comprehensive Genetic Variant Discovery in the Surfactant Protein B Gene

Completely penetrant mutations in the surfactant protein B gene (SFTPB) and >75% reduction of SFTPB expression disrupt pulmonary surfactant function and cause neonatal respiratory distress syndrome. To inform studies of genetic regulation of SFTPB expression, we created a catalogue of SFTPB variants by comprehensive resequencing from an unselected, population-based cohort (n = 1,116). We found an excess of low-frequency variation [81 SNPs and five small insertion/deletions (in/dels)]. Despite its small genomic size (9.7 kb), SFTPB was characterized by weak linkage disequilibrium (LD) and high haplotype diversity. Using the HapMap Yoruban and European populations, we identified a recombination hot spot that spans SFTPB, was not detectable in our focused resequencing data, and accounts for weak LD. Using homology-based software tools, we discovered no definitively damaging exonic variants. We conclude that excess low-frequency variation, intragenic recombination and lack of common disruptive exonic variants favor complete resequencing as the optimal approach for genetic association studies to identify regulatory SFTPB variants that cause neonatal respiratory distress syndrome in genetically diverse populations.

Insights into beta cell regeneration for diabetes via integration of molecular landscapes in human insulinomas

Although diabetes results in part from a deficiency of normal pancreatic beta cells, inducing human beta cells to regenerate is difficult. Reasoning that insulinomas hold the “genomic recipe” for beta cell expansion, we surveyed 38 human insulinomas to obtain insights into therapeutic pathways for beta cell regeneration. An integrative analysis of whole-exome and RNA-sequencing data was employed to extensively characterize the genomic and molecular landscape of insulinomas relative to normal beta cells. Here, we show at the pathway level that the majority of the insulinomas display mutations, copy number variants and/or dysregulation of epigenetic modifying genes, most prominently in the polycomb and trithorax families. Importantly, these processes are coupled to co-expression network modules associated with cell proliferation, revealing candidates for inducing beta cell regeneration. Validation of key computational predictions supports the concept that understanding the molecular complexity of insulinoma may be a valuable approach to diabetes drug discovery.Diabetes results in part from a deficiency of functional pancreatic beta cells. Here, the authors study the genomic and epigenetic landscapes of human insulinomas to gain insight into possible pathways for therapeutic beta cell regeneration, highlighting epigenetic genes and pathways.