Mutations In Different Genes Research Articles

Hepatocellular adenoma management: advances but still a long way to go.

Hepatocellular adenomas (HCAs) are composed of four molecular subgroups: mutations inactivating the HNF1A gene; the inflammatory phenotype with mutations of different genes leading to STAT3 activation; the activation of β-catenin by mutations in exon 3; among β-HCA, half display both inflammatory and β-catenin-activated phenotypes; and the unclassified tumors. The identification of these subtypes by MRI and immunohistochemistry on tissue is considered as a major criterion to manage patients. Of particular relevance is the identification of the β-catenin-mutated group due to its high risk of malignant transformation. In spite of this progress, the classification has not gained recognition among surgeons. It is hoped that by working as a multidisciplinary team, including surgeons, radiologists, pathologists and molecular biologists, patients will be managed more rationally. In this article, we will present known and new data, well accepted and that which is still controversial. The progress made in the field of HCA in the last 12 years, whether in epidemiology, diagnosis (clinical, pathology, imaging) or management, is related in one way or another to molecular advances.

The Epidemiology of Nonsurgical Hypoparathyroidism in Denmark: A Nationwide Case Finding Study

Nonsurgical hypoparathyroidism (HypoPT) is a rare disorder most often caused by mutations in different genes. It is characterized by hypocalcaemia with inappropriately low PTH levels. Knowledge about this group of patients, including their mortality and morbidity, is very sparse. The aim was to identify all patients diagnosed with nonsurgical HypoPT in Denmark and assess their mortality and risk of complications. Through registers and review of individual patient hospital charts we identified all patients diagnosed with nonsurgical HypoPT in Denmark between 1977 and 2012. We assessed their mortality and morbidity by comparing them with a group of age- and gender-matched population-based controls. We identified a total of 180 patients with nonsurgical HypoPT among whom 123 (68%) were alive at the date of follow-up (prevalence of 2.3/100,000 inhabitants). Compared with controls, mortality was not increased, but patients had a significantly increased risk of renal insufficiency (hazard ratio [HR] 6.01), cardiovascular diseases (HR 1.91), neuropsychiatric complications (HR 2.45), infections (HR 1.94), seizures (HR 10.05), cataract (HR 4.21), and fractures at the upper extremities (HR 1.93). In contrast patients had significantly reduced risk of malignant diseases (HR 0.44). In conclusion, nonsurgical HypoPT is a rare disease associated with a number of complications that should be considered when taking care of these patients.

Overlap of familial Mediterranean fever and hyper-IgD syndrome in an Arabic kindred.

Hyperimmunoglobulinemia D Syndrome (HIDS) has rarely been reported in Arabs. Moreover, the simultaneous presence of mutations in MEFV and MVK segregating in the same family is exceptional. We report an Arabic girl presenting since the age of 8-years with two patterns of recurrent episodes of fever, and associated with a spectrum of clinical features suggestive of overlap between familial Mediterranean fever (FMF) and HIDS. Her 19-year old brother presented since the age of 1 year with prolonged episodes of fever and was diagnosed with HIDS at the age of 7 years based on clinical features and homozygosity for p.V377I mutation in MVK. Shorter episodes of fever and abdominal pain more consistent with FMF ensued since the age of 17 years. Genetic testing done for both patients and all other family members revealed simultaneous presence of mutations in MEFV and MVK but with a variable clinical spectrum ranging from asymptomatic to severe manifestations. Both of our patients are homozygous for p.V377I MVK mutation; the girl is a compound heterozygote for p.E148Q/p.P369S/p.R408G and p.E167D/p.F479L MEFV mutations whereas the brother is a compound heterozygote for p.E148Q/p.P369S/p.R408G and p.M680I MEFV mutations. The clinical implications of having more than one mutation in different genes of monogenic autoinflammatory diseases in the same individual are not clear but may explain atypical clinical manifestations such as the overlap features of both FMF and HIDS in this family.

Systematic review and meta-analysis of Japanese familial Alzheimer’s disease and FTDP-17

Mutations in APP, PSEN1 and PSEN2 as the genetic causes of familial Alzheimer's disease (FAD) have been found in various ethnic populations. A substantial number of FAD pedigrees with mutations have been reported in the Japanese population; however, it remains unclear whether the genetic and clinical features of FAD in the Japanese population differ from those in other populations. To address this issue, we conducted a systematic review and meta-analysis of Japanese FAD and frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) by literature search. Using this analysis, we identified 39 different PSEN1 mutations in 140 patients, 5 APP mutations in 35 patients and 16 MAPT mutations in 84 patients. There was no PSEN2 mutation among Japanese patients. The age at onset in Japanese FAD patients with PSEN1 mutations was significantly younger than that in patients with APP mutations. Kaplan–Meier analysis revealed that patients with MAPT mutations showed a shorter survival than patients with PSEN1 or APP mutations. Patients with mutations in different genes exhibit characteristic clinical presentations, suggesting that mutations in causative genes may modify the clinical presentations. By collecting and cataloging genetic and clinical information on Japanese FAD and FTDP-17, we developed an original database designated as Japanese Familial Alzheimer's Disease Database, which is accessible at http://alzdb.bri.niigata-u.ac.jp/.

Evidence of digenic inheritance in Alport syndrome

BackgroundAlport syndrome is a clinically heterogeneous, progressive nephropathy caused by mutations in collagen IV genes, namely COL4A3 and COL4A4 on chromosome 2 and COL4A5 on chromosome X. The wide phenotypic...

Whole Exome Sequencing Reveals DYSF, FKTN, and ISPD Mutations in Congenital Muscular Dystrophy Without Brain or Eye Involvement.

BackgroundCongenital muscular dystrophies (CMDs) are a genetically and clinically heterogeneous group of neuromuscular disorders. Several genes encoding extracellular matrix, nuclear envelope, sarcolemmal proteins and glycosylation enzymes have been implicated in CMDs. The large overlap of clinical presentations due to mutations in different genes poses a challenge for clinicians in determining disease etiology for each patient.ObjectiveWe investigated the use of whole exome sequencing (WES) in identifying the genetic cause of disease in 5 CMD patients from 3 families who presented with highly similar clinical features, including early-onset rapidly progressive weakness without brain or eye abnormalities.MethodsWhole exome sequencing was performed on DNA from affected individuals. Potential functional impacts of mutations were investigated by immunostaining on available muscle biopsies.ResultsPathogenic mutations in 3 different genes, DYSF, FKTN, and ISPD were identified in each family. Mutation in DYSF led to absence of dysferlin protein in patient muscle. Mutations in ISPD led to impaired ISDP function, as demonstrated by deficiency of α-dystroglycan glycosylation in patient muscle.ConclusionsThis study highlights the benefit of unbiased genomic approaches in molecular diagnosis of neuromuscular disorders with high clinical heterogeneity, such as the phenotypes observed in our patients. Our results suggest that dysferlin deficiency should be in the differential diagnosis of congenital and rapidly progressive muscular dystrophy, and therefore dysferlin antibody should be in the standard immunohistochemistry panel for muscle biopsies in cases with suspected CMD.

The successful use of sugammadex and uneventful recovery from general anaesthesia in a patient with myotonic dystrophy.

Sir, Myotonic dystrophy is an autosomal dominant multisystem disorder characterised by progressive weakness and wasting of muscles, cardiomyopathy and cardiac conduction defects, restrictive lung disease, sleep apnoea syndrome, slow gastric emptying, endocrine disorders and cataracts. This condition is often associated with prolonged muscle contractions with defective relaxation (myotonia). There are two major types of myotonic dystrophy: Type 1 and type 2. These are caused by mutations in different genes. Anaesthetic management of this condition can be challenging due to the potential for these patients to develop perioperative cardiorespiratory complications and because they may demonstrate abnormal responses to sedative and neuromuscular blocking agents. We present a report of the successful management of a 60-year-old gentleman with myotonic dystrophy type 1, weighing 70 kg, who presented for elective laparoscopic cholecystectomy. His symptoms had included progressive weakness of the upper limbs with dysarthria, soft voice and mild static swallowing impairment. He had dilated cardiomyopathy and had a pacemaker/defibrillator in situ. The patient also had moderate obstructive sleep apnoea and was saturating at 99% in room air. This patient had never undergone general anaesthesia, and all his previous investigations had been performed previously under local anaesthesia with sedation. Pre-operative examination findings included the characteristic facial features of myotonic dystrophy and a modified Mallampati class III. On the day of surgery, no oral pre-medication was given other than his usual medications. Intra-operative monitoring included five-lead electrocardiography, pulse oximetry, capnography, invasive blood pressure monitoring with a right radial artery catheter and bispectral index (BIS). Magnet and external defibrillator pads were placed over his chest. Following adequate pre-oxygenation, induction and maintenance of anaesthesia were carried out with 0.5 mg of midazolam followed by propofol and remifentanil target controlled infusion titrated to BIS between 40 and 60 and haemodynamic status. After the initial administration of 50 mg rocuronium for intubation anticipating approximately 2 h procedure, we had planned to give subsequent doses guided by a peripheral nerve stimulator. A grade II b laryngeal view was obtained with direct laryngoscopy and a size 8.5 endotracheal tube was inserted. The maintenance phase of anaesthesia was uneventful, and no other analgesia was given intra-operatively. Upper and lower body forced air warmers were used throughout. The surgery was uncomplicated and lasted 45 min. At the end of surgery as no twitches were observed with train-of-four (TOF) stimulus after the initial intubating dose of rocuronium, the decision was made to reverse neuromuscular blockade with an initial sugammadex dose of 200 mg and further administer additional doses if necessary. Interestingly with the initial dose, all the four twitches to TOF stimuli appeared with no fade, merely within 30 s. Furthermore, there was no obvious fade with double burst stimulation. The patient was extubated when fully awake after a smooth emergence. His oxygen saturation was 99% with 5 L/min of supplemental oxygen. The patient was admitted to the Intensive Care Unit for observation and had an uncomplicated recovery. Patients with myotonic dystrophy often have respiratory muscle weakness resulting in an ineffective cough. This can co-exist with a decreased ventilatory response to hypoxia and hypercapnia. Pharyngeal muscle weakness and prolonged gastric emptying may increase the risk of aspiration. As a consequence, there is a possibility of increased postoperative pulmonary complications in this patient group.[1] Suxamethonium can result in exaggerated contracture in patients with myotonic dystrophy.[1] It may be difficult to predict the reaction to neostigmine in these patients as it can result in aggravation of paralysis or myotonia.[2,3] Peripheral nerve stimulation itself can also result in a myotonic response that may be misinterpreted, as sustained tetanus.[1] Residual neuromuscular blockade may further increase the risk of postoperative pulmonary complications in these patients. Sugammadex, a modified gamma-cyclodextrin, selectively binds with rocuronium or vecuronium to reverse the neuromuscular blocking effect. It is devoid of the risks of neostigmine and with better efficacy at reversal, has been suggested as a superior alternative.[4,5,6] We had decided to use rocuronium and sugammadex combination, to avoid the possible need for neostigmine to reverse any other relaxant as well as because of lack of quantitative neuromuscular monitoring with which we could ensure complete reversal. In contrast to the previous reports, where sugammadex was either given to avoid the possibility of residual paralysis[4] or less than normal dose of rocuronium was given for intubation,[6] we observed that using sugammadex, we could easily reverse a complete paralysis following the usual intubating dose of rocuronium (approximately 0.6 mg/kg). We therefore suggest that sugammadex, if available, should be considered for reversal of neuromuscular blockade with steroidal non-depolarising muscle relaxants in patients with muscular disorders such as myotonic dystrophy, particularly in facilities where advanced neuromuscular monitoring is unavailable.

Locus heterogeneity disease genes encode proteins with high interconnectivity in the human protein interaction network

Mutations in genes potentially lead to a number of genetic diseases with differing severity. These disease genes have been the focus of research in recent years showing that the disease gene population as a whole is not homogeneous, and can be categorized according to their interactions. Locus heterogeneity describes a single disorder caused by mutations in different genes each acting individually to cause the same disease. Using datasets of experimentally derived human disease genes and protein interactions, we created a protein interaction network to investigate the relationships between the products of genes associated with a disease displaying locus heterogeneity, and use network parameters to suggest properties that distinguish these disease genes from the overall disease gene population. Through the manual curation of known causative genes of 100 diseases displaying locus heterogeneity and 397 single-gene Mendelian disorders, we use network parameters to show that our locus heterogeneity network displays distinct properties from the global disease network and a Mendelian network. Using the global human proteome, through random simulation of the network we show that heterogeneous genes display significant interconnectivity. Further topological analysis of this network revealed clustering of locus heterogeneity genes that cause identical disorders, indicating that these disease genes are involved in similar biological processes. We then use this information to suggest additional genes that may contribute to diseases with locus heterogeneity.

Somatic Mutations in Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are clonal stem cell neoplasms affecting patients usually over 60 years old that typically present into the clinic with common symptoms including cytopenias, recurrent infections, bleeding and bruising. Approximately 20-30% of MDS patients progress to acute myeloid leukemia (AML) and are associated with inferior survival1. Diagnosis of MDS relies on findings from peripheral blood counts, examination of bone marrow morphology and evaluation of cytogenetic profiles for chromosomal aberrations. Using the WHO 2008 criteria, the proportion of blasts in the bone marrow, the number of cell lineages affected and the presence of del(5q) are collectively evaluated to classify patients into one of the five MDS categories [refractory anemia, refractory anemia with ring sideroblasts, refractory cytopenia multilineage dysplasia, refractory anemia with excess blasts, MDS with del(5q)]. The International Prognostication Scoring System (IPSS & IPSS-R) is the most widely used prognostic system in MDS. IPSS utilizes morphological variables to assign patients into low, intermediate or high-risk groups2. Accurate classification into one of these prognostic categories is critical as it determines selection of therapy regimes. Recent systematic profiling screens of MDS genomes have unraveled a complex network of cellular pathways that are causally implicated in MDS pathogenesis. Mutations have now been characterized in a number of key components of the spliceosome machinery (SF3B1, SRSF2, U2AF1, U2AF2, ZRSR2), regulators of DNA methylation (DNMT3A, IDH1, IDH2, TET2), chromatin modification (ASXL1, EZH2), transcription (EVI1, RUNX1, GATA2), signal transduction (NRAS, JAK2, KRAS, CBL) and cell cycle control (TP53)3-9. Collectively, more than 40 genes are significantly mutated in MDS; these mutations account for nearly 90% of MDS patients. The majority of patients present with two or more oncogenic mutations at diagnosis, and significant patterns of gene-gene interactions and mutual exclusivity have been reported10,11. Systematic integration of mutation data with large and well-annotated clinical datasets offers an unprecedented opportunity to decipher both the diagnostic as well as prognostic potential of these mutations as clinical biomarkers. However, the underlying genetic heterogeneity imposes significant challenges and important considerations that need to be accounted for when interpreting observed correlations between genotype, morphology and patient outcome. To unravel the interlocking genetic heterogeneity in MDS, Bejar et al., Papaemmanuil et al., and Haferlach et al. have studied the prevalence of acquired gene mutations in MDS and closely related chronic myeloid neoplasms in ~ 2100 MDS patients with well-annotated diagnostic and clinical outcome variables10-12. Univariate analysis has identified more than 10 genes to be significantly correlated with clinical outcome, including SF3B1, SRSF2, ASXL1, RUNX1, TP53, BCOR, RUNX1, EZH2, IDH2, ZRSR2, U2AF1 and CUX1. The total number of oncogenic mutations identified in each patient is selected as one of the most significant genetic predictors of outcome. Mutations in gene components of the spliceosome machinery are observed in approximately 50% of MDS patients, identifying pre-mRNA splicing as the most frequently altered biological process in MDS. Additionally, clonal relationship analysis of these mutations identifies that mutations in splicing genes occur early, followed by mutations in preferred partner genes, and mutations in different genes of the spliceosome machinery are associated with distinct morphological classification groups. The present talk will provide an overview of our current understanding of the underlying molecular mechanisms that underpin MDS biology. It will also evaluate how the genetic architecture of MDS can be incorporated in developing reliable and informative patient classification as well as outcome prediction models that can support clinical decision making in the future.

Complexity of CEBPA Mutated Acute Myeloid Leukemia

Introduction:CEBPA mutations (mut) occur in 5-14% of patients with acute myeloid leukemia (AML). A difference in prognosis between CEBPA single- (sm) and CEBPA double-mutated (dm) cases has been reported with longer overall and event-free survival in dm cases. This may be due to differences in the amount and composition of concomitant mutations.Aims: 1) Elucidate the cause of the clinical difference between CEBPAsm and dm cases. 2) Evaluate the clonal architecture of CEBPAmut AML in a well characterized cohort (Fasan et al., Leukemia 2014).Patients: We investigated concomitant mutations in a cohort of 229 CEBPAmut AML cases (129 sm and 100 dm). The cohort was composed of 113 females and 116 males, median age was 67.1 y (range: 15.7-87.6 y). 218 cases showed intermediate risk (n=162 normal and n=56 aberrant karyotype), 11 cases adverse risk cytogenetics.Results:CEBPAdm (median age 56.8 y, range 15.7–87.6 y) were significantly younger compared to sm (median age 64.7 y, range 20.4–87.0 y, p=0.001). Furthermore, CEBPAdm occurred significantly more often in females than in males (61.0% vs 39.0%; p=0.002). With regard to cytomorphology and cytogenetics, no significant differences between CEBPAsm and dm cases were seen.Concomitant mutations were present in significantly more CEBPAsm cases compared to CEBPAdm cases (93.0% vs 79.0%; p=0.003). Furthermore, the amount of additional mutations was higher in CEBPAsm (mean: 2.1 mutations, range: 1-6) compared to CEBPAdm (1.3 mutations, range: 1-4). ASXL1 (p=0.009), DNMT3A (p<0.001), FLT3-ITD (p=0.007), IDH2 (p=0.042), NPM1 (p<0.001) and RUNX1 (p=0.004) mutations were significantly more frequent in CEBPAsm compared to dm. In contrast, GATA2 (p<0.001) and WT1 (p=0.039) were more frequently mutated in CEBPAdm. No respective differences were detected in the frequencies of MLL-PTD and of mutations in IDH1, KRAS, NRAS and TET2. We additionally analyzed concomitant mutations according to functional pathways. No differences between CEBPAsm and dm were detected in myeloid transcription factors (GATA2, RUNX1) and oncogenes (KRAS, NRAS). Significantly higher frequencies were found in CEBPAsm compared to dm for the following pathways: mutations affecting DNA-methylation (DNMT3A, IDH1/2, TET2; 64.3% vs 43.0%; p=0.001), nucleophosmin (30.2% vs 2.0%; p<0.001), signaling (FLT3-ITD, FLT3-TKD; 21.7% vs 8.0%; p=0.006) and chromatin modifiers (ASXL1, MLL-PTD; 27.1% vs 13.0%; p=0.009). In contrast, the tumor suppressor WT1 was significantly less frequently mutated in CEBPAsm (3.9% vs 12.0%; p=0.023). Regarding outcome, CEBPAdm cases with DNA methylation affecting mutations as compared to those without had significantly shorter EFS (median 11.3 vs 45.9 months in all others; p=0.052) and OS (median 29.3 months vs n.r.; p=0.009). Furthermore, mutations in chromatin modifiers negatively influenced EFS (median 5.4 vs 26.4 months; p=0.002) and OS (median 15.9 months vs n.r.; p=0.006) in CEBPAdm cases.We further compared clonal architecture between CEBPAsm and dm cases. As a first step we compared mean mutation loads of concomitant mutations in CEBPAsm and dm and observed no difference between the two entities. As a next step, cases with uniform mutation loads in all genes were considered monoclonal, while cases with heterogeneous mutation loads (range of loads for different mutations spanning >30%) were considered clonally diverse. CEBPAsm were significantly more often clonally diverse compared to dm (52.7% vs. 32.0%; p=0.002). In 63.3% of diverse CEBPAdm cases (n=19/30), the dominant clone comprised CEBPAmut compared to only 32.3% (n=22/68) of diverse CEBPAsm (p=0.009). In 20.0% (n=6/30) of CEBPAdm with subclonal CEBPAmut, the dominant clone was TET2mut and in 10.0% (n=3/30) WT1mut. In 2 cases (7.6%), the dominant clone had mutations in different genes. In 26.5% (n=18/68) of CEBPAsm with subclonal CEBPAmut, the dominant clone carried TET2mut, 8.8% (n=6/68) DNMT3Amut, 4.4% (n=3/68) ASXL1mut. In 27.9% (n=19/68) the dominant clone had mutations in different genes.Conclusions: 1. CEBPAsm occur preferentially in male patients and are correlated with older age. 2. Biological diversity of CEBPAmut cases is reflected by the amount and composition of additional mutations. 3. Prognosis in CEBPAdm cases is adversely influenced by mutations affecting DNA-methylation and chromatin modifiers. 4. Clonal architecture is more complex in CEBPAsm compared to dm cases. DisclosuresFasan:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Perglerová:MLL2 s.r.o.: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Assessment of curated phenotype mining in neuropsychiatric disorder literature

Clinical evaluation of patients and diagnosis of disorder is crucial to make decisions on appropriate therapies. In addition, in the case of genetic disorders resulting from gene abnormalities, phenotypic effects may guide basic research on the mechanisms of a disorder to find the mutated gene and therefore to propose novel targets for drug therapy. However, this approach is complicated by two facts. First, the relationship between genes and disorders is not simple: one gene may be related to multiple disorders and a disorder may be caused by mutations in different genes. Second, recognizing relevant phenotypes might be difficult for clinicians working with patients of closely related complex disorders. Neuropsychiatric disorders best illustrate these difficulties since phenotypes range from metabolic to behavioral aspects, the latter extremely complex.Based on our clinical expertise on five neurodegenerative disorders, and from the wealth of bibliographical data on neuropsychiatric disorders, we have built a resource to infer associations between genes, chemicals, phenotypes for a total of 31 disorders. An initial step of automated text mining of the literature related to 31 disorders returned thousands of enriched terms. Fewer relevant phenotypic terms were manually selected by clinicians as relevant to the five neural disorders of their expertise and used to analyze the complete set of disorders. Analysis of the data indicates general relationships between neuropsychiatric disorders, which can be used to classify and characterize them. Correlation analyses allowed us to propose novel associations of genes and drugs with disorders. More generally, the results led us to uncovering mechanisms of disease that span multiple neuropsychiatric disorders, for example that genes related to synaptic transmission and receptor functions tend to be involved in many disorders, whereas genes related to sensory perception and channel transport functions are associated with fewer disorders. Our study shows that starting from expertise covering a limited set of neurological disorders and using text and data mining methods, meaningful and novel associations regarding genes, chemicals and phenotypes can be derived for an expanded set of neuropsychiatric disorders. Our results are intended for clinicians to help them evaluate patients, and for basic scientists to propose new gene targets for drug therapies. This strategy can be extended to virtually all diseases and takes advantage of the ever increasing amount of biomedical literature.

Mutations in filamin C cause a new form of familial hypertrophic cardiomyopathy.

Mutations in different genes encoding sarcomeric proteins are responsible for 50-60% of familial cases of hypertrophic cardiomyopathy (HCM); however, the genetic alterations causing the disease in one-third of patients are currently unknown. Here we describe a case with familial HCM of unknown cause. Whole-exome sequencing reveals a variant in the gene encoding the sarcomeric protein filamin C (p.A1539T) that segregates with the disease in this family. Sequencing of 92 HCM cases identifies seven additional variants segregating with the disease in eight families. Patients with FLNC mutations show marked sarcomeric abnormalities in cardiac muscle, and functional analysis reveals that expression of these FLNC variants resulted in the formation of large filamin C aggregates. Clinical studies indicate that FLNC-mutated patients have higher incidence of sudden cardiac death. On the basis of these findings, we conclude that mutations in the gene encoding the sarcomeric protein filamin C cause a new form of familial HMC.

Molecular testing in lung cancer in the era of precision medicine.

The clinical expectations how pathologists should submit lung cancer diagnosis have changed dramatically. Until mid 90-ties a clear separation between small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) was mostly sufficient. With the invention of antiangiogenic treatment a differentiation between squamous and non-squamous NSCLC was requested. When epidermal growth factor receptor (EGFR) mutation was detected in patients with pulmonary adenocarcinomas and subsequent specific treatment with tyrosine kinase inhibitors (TKIs) was invented, sub-classification of NSCLC and molecular analysis of the tumor tissue for mutations was asked for. Pathologists no longer submit just a diagnosis, but instead are involved in a multidisciplinary team for lung cancer patient management. After EGFR several other driver genes such as echinoderm microtubule associated protein like 4-AL-Kinase 1 (EML4-ALK1), c-ros oncogene 1, receptor tyrosine kinase (ROS1), discoidin domain receptor tyrosine kinase 2 (DDR2), fibroblast growth factor receptor 1 (FGFR1) were discovered, and more to come. Due to new developments in bronchology (EUS, EBUS) the amount of tissue submitted for diagnosis and molecular analysis is decreasing, however, the genes to be analyzed are increasing. Many of these driver gene aberrations are inversions or translocations and thus require FISH analysis. Each of these analyses requires a certain amount of tumor cells or one to two tissue sections from an already limited amount of tissues or cells. In this respect new genetic test systems have been introduced such as next generation sequencing, which enables not only to detect multiple mutations in different genes, but also amplifications and fusion genes. As soon as these methods have been validated for routine molecular analysis this will enable the analysis of multiple genetic changes simultaneously. In this review we will focus on genetic aberrations in NSCLC, resistance to new target therapies, and also to methodological requirements for a meaningful evaluation of lung cancer tissue and cells.

Maturity onset diabetes of the young (MODY)—History, first case reports and recent advances

The world is seemingly facing a global increase in people suffering from diabetes especially in developing countries. The worldwide occurrence of diabetes for all age groups in year 2000 was estimated to be 2.8% and this number is most certainly expected to rise to 4.4% by 2030. Maturity-onset of diabetes of the young, or MODY, is a form of monogenic diabetes that is caused by mutations occurring in a number of different genes. Mutations in different genes tend to cause a slightly different variant of diabetes. MODY is typically diagnosed during late childhood, adolescence, or early adulthood and is usually observed to develop in adults during their late 50's. One of the main drawbacks in its diagnosis is that many people with MODY are misdiagnosed as having type 1 or type 2 diabetes. However, a molecular and genetic diagnosis can result in a better treatment and could also help in identifying other family members with MODY. This article explores the historical prospect and the genetic background of MODY, a brief summary of the first case reported and the significant factors that differentiate it from type 1 and type 2 diabetes.

Abstract 937: Molecular analysis of KRAS exon 2 wild type colorectal cancer patients enrolled in the CAPRI clinical trial reveals high degree of tumor heterogeneity

Abstract The CAPRI clinical trial (Optimal strategy in KRAS wild type metastatic colorectal cancer patients: cetuximab plus FOLFIRI followed by FOLFOX +/- cetuximab) enrolls metastatic colorectal carcinoma (mCRC) patients with no codon 12/13 (exon 2) KRAS mutations. Recent data showed that exon 3 and 4 KRAS mutations as well as NRAS exon 2, 3 and 4 mutations might confer resistance to EGFR monoclonal antibodies. In addition, somatic mutations in other genes such as BRAF, PIK3CA, PTEN, have been hypothesized to be prognostic and/or predictive in CRC. Therefore, molecular sub-classification of KRAS exon 2 wild type CRC might allow to identify subgroups of patients highly sensitive or resistant to anti-EGFR agents within the CAPRI trial. We analyzed 182 FFPE samples from mCRC patients enrolled in the CAPRI clinical trial by using a next generation sequencing (NGS) approach based on the Ion AmpliSeq™ technology. In particular, 10 ng of genomic DNA were analyzed with the Ion Ampliseq Colon and Lung cancer panel that targets over 500 hotspot mutations in 22 genes implicated in colon and lung cancers. Data analysis was performed using Ion Reporter™ Software. The analysis with the IonAmpliseq panel was successful in all the tested samples. Among the 182 analyzed cases, 58 (31.9%) showed no mutations in the investigated genes, whereas a total of 206 mutations in 14 genes were detected in 124 cases (68.1%). Surprisingly, 30 exon 2 KRAS mutations were identified in 29 patients (15.9%). Mutations at a frequency &amp;gt;4% were also found in: TP53 (39.5% of the cases mutant), KRAS exon 3 and 4 (7.5%), PIK3CA (13.2), BRAF (8.2%), NRAS (7.1%), FBXW7 (4.9%). A significant degree of tumor heterogeneity was observed: several tumors had multiple mutations in different genes, with some cases showing up to 5 mutations. In particular, 30/45 KRAS, 5/13 NRAS, 12/15 BRAF and 19/24 PIK3CA mutant cases showed variants in additional genes. The frequency of mutant alleles also differed significantly among tumors. For example, among the KRAS exon 2 mutant cases, the frequency of KRAS mutant alleles, after normalization for the neoplastic cell content, ranged between 6% and 130%, suggesting that some KRAS mutant cases were polyclonal and that in some other cases gain of mutant or loss of wild type KRAS alleles might have occurred. These data suggest that molecular classification of KRAS exon 2 wild type CRC with the Ion Ampliseq Colon and Lung cancer panel is feasible. This approach reveals mutations in several key genes involved in CRC tumor progression. The relatively high incidence of codon 12/13 KRAS mutations also suggests that an high rate of false negative results in KRAS testing occurs in clinical practice. Finally, the huge degree of tumor heterogeneity might have important implications for prognosis and response to therapy. Citation Format: Anna Maria Rachiglio, Matilde Lambiase, Francesca Fenizia, Claudia Esposito, Cristin Roma, Giuseppe Tonini, Saverio Cinieri, Giuseppe Colucci, Fortunato Ciardiello, Nicola Normanno. Molecular analysis of KRAS exon 2 wild type colorectal cancer patients enrolled in the CAPRI clinical trial reveals high degree of tumor heterogeneity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 937. doi:10.1158/1538-7445.AM2014-937

Update from the 2013 International Neurofibromatosis Conference.

The 2013 Neurofibromatosis (NF) Conference took place at the Portola Hotel and Spa, Monterey, CA, from June 8-11, 2013. This international meeting is sponsored annually by the Children's Tumor Foundation (CTF), with the goal of bringing together NF researchers and clinicians from disparate fields of expertise. The conference agenda included a range of preclinical topics with a focus on signaling pathways and mouse models and a number of clinical topics, including a symposium on the interaction of academics, government, and industry in NF clinical trials. Neurofibromatosis is a group of inherited genetic disorders – NF1, NF2, and schwannomatosis – that together affect about 100,000 persons in the US. NF1 is the most common, with an estimated birth prevalence of 1:3,000 [Friedman., 1999; Lammert et al., 2005]; for NF2 and schwannomatosis, the estimated birth prevalence is 1:25,000 and 1:40,000, respectively [Antinheimo et al., 2000; Evans et al., 2005]. NF1, NF2 and schwannomatosis have in common that they predispose affected individuals to develop Schwann cell tumors such as neurofibromas and schwannomas. At a lower frequency, they also predispose to the development of a number of other benign and malignant tumor types, as well as some developmental abnormalities including learning disabilities (in NF1). The disorders arise from mutations in different genes, each of which plays a key role in regulating cellular function. The NF1 gene on human chromosome 17 encodes an intracellular signaling molecule that functions as a GTPase activating protein for Ras proteins, whereas the NF2 gene on human chromosome 22 encodes a cytoskeletal-membrane linking protein with reported roles in the suppression of several different growth-associated signaling pathways. The biology of schwannomatosis remains understudied despite the identification of germline SMARCB1 mutations in schwannomatosis patients in 2007.

Alternating antibiotic treatments constrain evolutionary paths to multidrug resistance.

Alternating antibiotic therapy, in which pairs of drugs are cycled during treatment, has been suggested as a means to inhibit the evolution of de novo resistance while avoiding the toxicity associated with more traditional combination therapy. However, it remains unclear under which conditions and by what means such alternating treatments impede the evolution of resistance. Here, we tracked multistep evolution of resistance in replicate populations of Staphylococcus aureus during 22 d of continuously increasing single-, mixed-, and alternating-drug treatment. In all three tested drug pairs, the alternating treatment reduced the overall rate of resistance by slowing the acquisition of resistance to one of the two component drugs, sometimes as effectively as mixed treatment. This slower rate of evolution is reflected in the genome-wide mutational profiles; under alternating treatments, bacteria acquire mutations in different genes than under corresponding single-drug treatments. To test whether this observed constraint on adaptive paths reflects trade-offs in which resistance to one drug is accompanied by sensitivity to a second drug, we profiled many single-step mutants for cross-resistance. Indeed, the average cross-resistance of single-step mutants can help predict whether or not evolution was slower in alternating drugs. Together, these results show that despite the complex evolutionary landscape of multidrug resistance, alternating-drug therapy can slow evolution by constraining the mutational paths toward resistance.

Association of brain-derived neurotrophic factor (BDNF) and elongator protein complex 4 (ELP4) polymorphisms with benign epilepsy with centrotemporal spikes in a Greek population

Benign epilepsy with centrotemporal spikes (BECTS) is considered to be the most common childhood epileptic syndrome. Different mutations in genes that control the excitability of neurons have been described. Recent reports on the involvement of the BDNF and ELP4 genes in cell motility, migration, and adhesion raise the possibility that these genes are involved in pathogenesis of BECTS. We conducted a case-control association study on 60 patients with BECTS and 60 control participants to assess the influence of the BDNF and ELP4 polymorphisms on BECTS. The polymorphisms were detected with a PCR-RFLP method. Moreover, we explored the possible association of these polymorphisms with clinical and electroencephalographic parameters of patients with BECTS. Our results show no difference in BDNF and ELP4 genotype frequencies between patients and controls. Haplotype analysis also revealed no statistical difference. The role of BDNF and ELP4 polymorphisms remains controversial.

G.P.23: Diagnostic application of targeted NGS in early onset myopathies

Congenital myopathies and congenital muscular dystrophies are groups of clinically, pathologically, and genetically heterogeneous disorders. Even for the experienced clinicians, an accurate genetic diagnosis has often been challenging due to the heterogeneity and complexity of these groups of disorders. One gene can cause a wide variety of clinical and/or pathological features, while similar clinical features can be caused by mutations in different genes. Since next generation sequencing (NGS) is an effective diagnostic tool for the parallel investigation of a large number of genes, it has been increasingly used in recent clinical practices to diagnose these genetically and phenotypically heterogeneous diseases. Here, we present the result of a targeted NGS panel analysis in early onset myopathies. We selected 703 known pathogenic genes causing congenital myopathies, congenital muscular dystrophies, metabolic and mitochondrial myopathies, distal myopathies, channelopathies, neuromuscular junction disorders, and diseases of peripheral nerve. Total 42 infants or children with early onset ( COL6A1 (5), COL6A3 (1), LMNA (3), ACTA1 (2), MTM1 (1), DOK7 (1), and GARS (1). Our results suggest that targeted NGS has a significant potential to synergy with clinical and pathological analysis for an effective diagnosis of primary myopathies.

G.P.80: Unusual association of juvenile polyarthritis and granulomatous myositis in three siblings from a moroccan consanguineous family

We described an unusual clinical phenotype of three siblings, one male and two females aged 25, 24 and 16 years respectively, of first-degree cousins Moroccan parents; another brother and sister were not affected. At the age of three years for the young sister and six years for the two older, they presented with acute appearance of an inflammatory clinical phenotype characterized by joint stiffness, redness and pain, associated with rapidly evolution of proximal muscle weakness leading to difficulties in walking, arising from the floor and running. CK blood levels were normal. The diagnosis of juvenile polyarthritis was proposed and the joints inflammatory phenotype successfully treated with TNF α inhibitor. A muscle biopsy was performed in the oldest and the youngest at 3 and 7 years respectively from the onset of symptoms. The biopsy revealed a granulomatous myositis with CD68 and CD3 positive inflammatory infiltrate and muscular necrosis. Mycobacterium tuberculosis and non tuberculosis were excluded by PCR as were other sporadic causes of granulomatous myositis such as sarcoidosis, infection and neoplastic diseases. Thirteen to nineteen years after the symptoms, all three siblings show joint contractures especially in proximal limb segment as well as in the shoulders, hips and knees. Atrophy of the distal muscles of the lower limbs is clearly noticeable, without muscle weakness. Recently, the younger sister have been investigated by muscular MRI that showed an intramuscular edema of the thigh and twins muscles. Recently, mutations in different genes associated with Mendelian transmission of dysimmunity disease have been demonstrated, and it is likely that this until now unreported association belongs to this emerging group of diseases.