Original Mutation Research Articles

Induced pluripotent stem cells derived from Bernard-Soulier Syndrome patient's peripheral blood cells with a p.Phe55Ser mutation in the GPIX gene

Bernard Soulier Syndrome (BSS) is a rare autosomal platelet disorder characterized by mutations in the von Willebrand factor platelet receptor complex GPIb-V-IX. In this work we have generated an induced pluripotent stem cell (BSS3-PBMC-iPS4F8) from peripheral blood mononuclear cells of a BSS patient with a p.Phe55Ser mutation in the GPIX gene. Characterization of BSS3-PBMC-iPS4F8 showed that these cells maintained the original mutation present in the BSS patient, expressed pluripotent stem cell markers and were able to differentiate into the three germline layers. This new iPSC line will contribute to better understand the biology of BSS disease.

Brain metastasis in non-small cell lung cancer (NSCLC) patients with uncommon EGFR mutations: a report of seven cases and literature review.

Brain metastasis (BM) arising from non-small cell lung cancer (NSCLC) with rare epidermal growth factor receptor (EGFR) mutations is quite rare. The prognosis and therapeutic effects of BM remain enigmatic. To the best of our knowledge, this is the first report to make a separate analysis of BM from NSCLC patients with original uncommon EGFR mutations. We retrospectively reviewed 7 cases of BM arising from 42 cases of uncommon EGFR mutated lung cancer in Tianjin Medical University Cancer Institute and Hospital. We also performed a literature review to assess therapeutic features and outcomes.

P3.02b-109 Molecular Profiling of EGFR-Positive NSCLC with Secondary T790M Resistance Mutation and Tertiary Transformation into Small-Cell Lung Cancer: Topic: EGFR RES

In advanced stage NSCLC, activating EGFR-mutations are prognostic and predictive factors for treatment with an EGFR-tyrosine kinase inhibitor (TKI). However, invariably, resistance to EGFR-TKI develops. Resistance is primarily driven by T790M mutations present in approximately 50% of EGFR-mutation positive NSCLC at progression. A different mechanism of EGFR-TKI-resistance is transformation into SCLC which has been reported in very rare cases. Employing Sanger sequencing and targeted next generation sequencing, we performed full histopathological workup and molecular profiling of all tumor biopsies obtained during the course of disease. Both molecular and histopathological data were correlated with the clinical course. We report the case of a 70 year old patient, ECOG 1, presenting with dyspnea and fatigue due to a predominantly acinar adenocarcinoma with extensive metastatic disease. Using Sanger sequencing, we detected a del Exon19-EGFR mutation. Based on fulfilled response criteria for EGFR-TKI therapy, erlotinib 150mg was initiated with rapid improvement of dyspnoe and fatigue. After one month, erlotinib was reduced to 100mg due to dermatotoxicity. After 5 weeks, CT showed partial remission. After 4½ months, CT revealed mixed response with resolved pleural effusion but slowly progressive pulmonary metastases. The brain metastases present at diagnosis had regressed but a new lesion was detected. Due to ongoing clinical benefit, erlotinib was continued beyond progression. After 7 months, the patient deteriorated clinically (ECOG 2) with CT showing progression of all tumor sites including. FNAC of a progressive mediastinal lymph node was submitted for histopathological and molecular work-up. In parallel one cycle of systemic chemotherapy with carboplatin/gemcitabine was given, and cerebral radiation was delivered. Following detection of a T790M mutation, chemotherapy was stopped and therapy with osimertinib 80mg (CUP) was started with prompt improvement of the clinical state. CT after 6 weeks confirmed partial remission. However, 10 weeks later, the patient’s condition rapidly deteriorated. CT detected complete remission of brain metastasis but rapid progression of the primary tumor, mediastinal lymphadenopathy, and hepatic metastases. An endobronchial kryobiopsy revealed small cell lung cancer as the underlying cause. EGFR analysis revealed the presence of the original exon19 mutation which had been present in the previous biopsies showing NSCLC histology. Complementing these preliminary results, a full molecular workup using next generation sequencing is currently being performed across all biopsies and will be presented. Integrated analysis of clinical, histopathological and molecular characteristics reveals tumor evolution over time and leads to highly individual therapeutic management benefiting the patient.

The Slavic NBN Founder Mutation: A Role for Reproductive Fitness?

The vast majority of patients with Nijmegen Breakage Syndrome (NBS) are of Slavic origin and carry a deleterious deletion (c.657del5; rs587776650) in the NBN gene on chromosome 8q21. This mutation is essentially confined to Slavic populations and may thus be considered a Slavic founder mutation. Notably, not a single parenthood of a homozygous c.657del5 carrier has been reported to date, while heterozygous carriers do reproduce but have an increased cancer risk. These observations seem to conflict with the considerable carrier frequency of c.657del5 of 0.5% to 1% as observed in different Slavic populations because deleterious mutations would be eliminated quite rapidly by purifying selection. Therefore, we propose that heterozygous c.657del5 carriers have increased reproductive success, i.e., that the mutation confers heterozygote advantage. In fact, in our cohort study of the reproductive history of 24 NBS pedigrees from the Czech Republic, we observed that female carriers gave birth to more children on average than female non-carriers, while no such reproductive differences were observed for males. We also estimate that c.657del5 likely occurred less than 300 generations ago, thus supporting the view that the original mutation predated the historic split and subsequent spread of the ‘Slavic people’. We surmise that the higher fertility of female c.657del5 carriers reflects a lower miscarriage rate in these women, thereby reflecting the role of the NBN gene product, nibrin, in the repair of DNA double strand breaks and their processing in immune gene rearrangements, telomere maintenance, and meiotic recombination, akin to the previously described role of the DNA repair genes BRCA1 and BRCA2.

Mutational Shift in FLT3 and NPM1-Positive Acute Myeloid Leukemia (AML) Relative to Therapy and Disease Progression

Introduction:Clonal myeloid malignancies such as AML are often characterized and treated based on specific mutation profiles identified at diagnosis. Routine use of clinical cancer next-generation sequencing (NGS) in diagnosis and disease monitoring has resulted in sequential mutation profiles of individual patients. On NGS profiling at interval follow-ups, mutations identified at diagnosis may change, or shift, through disease course. Here we track FLT3 and NPM1 mutations in the context of therapy, disease progression, clinical course, and pathology.Methods:Patients with AML or MDS whose blood or marrow specimens were sequenced using the Penn hematological-NGS panel (68 genes with full exon coverage) on at least 2 occasions and found at least once to have FLT3 or NPM1 mutations were included. An R script was written to track mutations and allele frequencies over time. These data were integrated with corresponding pathology and clinical data to evaluate mutation profiles in relation to disease status, therapy and progression. This study was approved by the institutional review board.Results:Review of clinical NGS data identified 37 patients with pathogenic mutations in FLT3 (n=15), NPM1 (n=5), or FLT3 and NPM1 (n=17) in samples characterized as de novo AML (AML), recurrent AML (rAML), myelodysplastic syndrome (MDS), recurrent MDS (rMDS), AML transformed from MDS (tAML), or AML in remission (R). Patients were divided into 3 mutational groups: FLT3 only, FLT3 and NPM1, and NPM1 only. The average number of pathogenic mutations (including mutations in genes other than FLT3 or NPM1) at initial and second testing was 1.9/3.1; 3.5/3.4 and 3.2/1.8, respectively.Tracking of FLT3 and NPM1 allele frequencies (>10% change) revealed changes in populations of multiple subclones at different time points. In the FLT3 and FLT3 and NPM1 groups, the original FLT3 and/or NPM1 mutation often became undetectable at subsequent testing. This frequently coincided with the emergence of new clones with different mutations. Nineteen patients showed loss of an original FLT3 mutation at subsequent testing, with 11 of these patients showing emergence of a new FLT3 clone with a different mutation. All 11 patients with loss of an original NPM1 clone had a new NPM1 clone with a different mutation appear. The NPM1 only group did not show loss of the original NPM1 clone unless it was at remission. All five patients in the NPM1 only group (3 AML, 2 rAML at initial CPD testing) achieved clinical and histologic remission with concordant loss of NPM1 mutations. In four patients, the detectable mutations remaining at remission were exclusively from the original clone (DNMT3A, DNMT3A/TET2, DNMT3A/IDH2/TET2, IDH2). The fifth patient had a newly detected SRSF2 mutation in addition to the original IDH2 mutation. All five patients are alive, and do not have overt morphologic evidence of recurrent AML on bone marrow biopsies that were performed within the last year.In four AML patients with FLT3 mutations, of which three were on FLT3 inhibitor therapy, the loss of a FLT3 mutation was followed by the emergence of a new NRAS mutation. Three patients achieved remission, but two died soon after of complications from graft versus host disease. The fourth patient achieved remission and had a detectable mutation only in IDH2, but on subsequent NGS testing showed reemergence of the previous NRAS mutation and relapsed three months later.Conclusions:Monitoring of FLT3 and NPM1 mutations in AML patients by NGS rather than single gene assays is crucial due to the frequent findings of other pathogenic co-mutations, mutational shift in response to disease progression and to therapy, and emergence of new disease associated clones at recurrence. DisclosuresNo relevant conflicts of interest to declare.

High Efficiency Gene Correction in Hematopoietic Cells By Template-Free Crispr/Cas9 Genome Editing

IntroductionA significant fraction of inherited monogenic disorders is caused by patient-specific mutations dispersed over the entire locus of the affected gene. Correcting these mutations by introducing healthy gene copies into the genome of the diseased cells proved successful in several clinical gene therapy trials. Most of these trials employed retroviral vectors, which by inserting randomly throughout the genome deprived the transduced genes of their endogenous control and caused insertional mutations leading to secondary disease. The development of genome editing tools capable of modifying any prespecified genomic sequence with unprecedented accuracy opened up a wide range of new possibilities in gene manipulation including targeted gene repair. In particular, CRISPR/Cas9, a prokaryotic adaptive immune system, and its swift repurposing for genome editing was widely adopted as the hitherto simplest genome editing tool. CRISPR/Cas9 is an RNA guided endonuclease that uses RNA-DNA base pairing to target genomic DNA. Bound to its target via the guide (g)RNA, Cas9 induces DNA double strand breaks (DBS) at prespecified genomic sites that promptly activate the endogenous DNA repair machinery. DSB repair is accomplished by either non-homologous end joining (NHEJ) or homology directed repair (HDR). Thus far, correction of human mutations in hematopoietic cells relied entirely on HDR requiring gene specific donor templates in addition to the RNA guided endonucleases (RGNs). However, although the HDR offers precision, its efficiency is low and requires positive selection to enrich for gene corrected cell. Because in mammalian cells DSB repair by NHEJ significantly exceeds HDR and even more importantly, is the dominant DSB repair pathway in hematopoietic stem- and progenitor cells (HSPC), we thought to exploit NHEJ for gene therapy because in theory, approximately one third of the indels associated with NHEJ should restore the open reading frame (ORF) disrupted by a disease mutation. This would lead to a significant number of ORF reconstitutions of which some, depending on the position and type of the original mutation, should either completely or partially recover protein function.ResultsTo test gene repair efficiency by NHEJ in human hematopoietic cells, we generated PLB-985 (PLB) reporter cells expressing mutationally inactivated EGFP (mEGFP). Transduction of mEGFP expressing PLBs (mEGFP-PLB) with integrase-deficient lentiviral (IDLV) particles encoding for RGNs targeting the EGFP mutation reconstituted EGFP expression in up to 27% of the mEGFP PLB cells. Indel analysis revealed that 13 out of 28 (46%) restored the EGFP-open reading frame of which 7 (25%) reconstituted EGFP activity. To test whether the donor-template free IDLV strategy would also effectively correct bone fide disease mutations, we performed similar experiments with X-CGD PLB cells expressing transgenes encoding patient-specific frameshift, missense and nonsense CYBB mutations causing X-linked chronic granulomatous disease (X-CGD) which is an inherited, life threatening immunodeficiency disorder. Transduction of the cells with IDLVs carrying RGNs directed against each of these mutations restored CYBB function in up to 10% of cells harboring frameshift mutations which is sufficient to protect X-CGD patients from microbial infections. However, RGNs directed against the nonsense or missense mutations restored CYBB function in only 1-2% of the cells, suggesting that these mutations are less amenable to CRISPR/Cas9 mediated repair. As Cas9 frequently tolerates single nucleotide mismatches, selection against solitary nucleotide substitutions may explain this low efficiency of gene repair.ConclusionsFrameshift mutations can be effectively repaired by NHEJ in hematopoietic cells by CRISPR/Cas9 transducing IDLVs. As about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that about a quarter of patients suffering from monogenic blood disorders could benefit from personalized, template free CRISPR/Cas9 gene therapy. DisclosuresNo relevant conflicts of interest to declare.

Analysis of mutations in DNA gyrase and topoisomerase IV of Ureaplasma urealyticum and Ureaplasma parvum serovars resistant to fluoroquinolones

This study aims to determine the prevalence of fluoroquinolone resistance of Ureaplasma biovars and serovars isolated from urogenital clinical samples and determine the underlying molecular mechanism for quinolone resistance for all resistant isolates.Of 105 samples confirmed as positive for U. urealyticum/U. parvum, 85 were resistant to quinolones by the Mycoplasma–IST2 kit. However, only 43 out of 85 quinolone resistant isolates had amino acid substitutions in GyrA, GyrB, ParC and ParE proteins underlining that this assay have mis-identified as fluoroquinolone resistant 42 isolates. The known ParC E87K and ParC S83L mutations were found in 1 and 10 isolates, respectively. An original mutation of ureaplasmal ParC (E87Q, 1 isolate) was found. Furthermore, we found a ParE R448K mutation in one isolate, already described. Among the additional alterations detected, the most prevalent mutation found was L176F in GyrA protein in 18 isolates with single infection and in 3 isolates with mixed ureaplasma infections. Mutations in GyrB (E502Q, 4 isolates), ParE (Q412K, Q412P, Q412T, 3 independent isolates), whose role is unknown, were also found. Other sporadic mutations in the four genes were identified.This investigation is the result of monitoring the data for molecular fluoroquinone resistance in Ureaplasma spp. in Italy. Resulting that this acquired resistance is high and that continued local epidemiological studies are essential to monitor and document their antimicrobial resistance trends.

Small cell lung cancer transformation and the T790M mutation: A case report of two acquired mechanisms of TKI resistance detected in a tumor rebiopsy and plasma sample of EGFR-mutant lung adenocarcinoma.

The present study describes the case of a 45-year-old man diagnosed with metastatic lung adenocarcinoma, which harbored a deletion within exon 19 of the epidermal growth factor receptor (EGFR) gene. The patient was subsequently treated with gefitinib (250 mg/day orally from May 2013 to March 2014), but developed acquired resistance to the drug following 11 months of treatment. Tumor burden molecular analysis was performed on a tumor rebiopsy and plasma sample, and histological analysis was also performed on the tumor rebiopsy. A small cell transformation retaining the original EGFR mutation was detected in the tumor rebiopsy, while the T790M mutation together with the activating ex19del mutation were identified only in the plasma sample. The patient was treated with cytotoxic chemotherapy (off-label schedule with epirubicin 80 mg/mq and paclitaxel 160 mg/mq every 21 days for 6 cycles) and radiation (50.4 Gy administered in 28 fractions of 1.8 Gy once daily for 5.5 weeks) specific for small cell lung cancer, and may also have benefitted from treatment with a third generation T790M-specific EGFR-TKI. To better describe the mechanisms of resistance to TKI inhibitors and to optimize therapeutic regimens, the simultaneous analysis of tumor biopsies and circulating tumor DNA should be considered.

Genomics of Natural Populations: How Differentially Expressed Genes Shape the Evolution of Chromosomal Inversions in Drosophila pseudoobscura.

Chromosomal rearrangements can shape the structure of genetic variation in the genome directly through alteration of genes at breakpoints or indirectly by holding combinations of genetic variants together due to reduced recombination. The third chromosome of Drosophila pseudoobscura is a model system to test hypotheses about how rearrangements are established in populations because its third chromosome is polymorphic for >30 gene arrangements that were generated by a series of overlapping inversion mutations. Circumstantial evidence has suggested that these gene arrangements are selected. Despite the expected homogenizing effects of extensive gene flow, the frequencies of arrangements form gradients or clines in nature, which have been stable since the system was first described >80 years ago. Furthermore, multiple arrangements exist at appreciable frequencies across several ecological niches providing the opportunity for heterokaryotypes to form. In this study, we tested whether genes are differentially expressed among chromosome arrangements in first instar larvae, adult females and males. In addition, we asked whether transcriptional patterns in heterokaryotypes are dominant, semidominant, overdominant, or underdominant. We find evidence for a significant abundance of differentially expressed genes across the inverted regions of the third chromosome, including an enrichment of genes involved in sensory perception for males. We find the majority of loci show additivity in heterokaryotypes. Our results suggest that multiple genes have expression differences among arrangements that were either captured by the original inversion mutation or accumulated after it reached polymorphic frequencies, providing a potential source of genetic variation for selection to act upon. These data suggest that the inversions are favored because of their indirect effect of recombination suppression that has held different combinations of differentially expressed genes together in the various gene arrangement backgrounds.

Diageotropica and Lateral Rooting, the Rest of the Story

Diageotropica and Lateral Rooting, the Rest of the Story

Playing Hide-and-Seek with Yeast

I like to think of the late 80s and early 90s as extremely exciting times in my career. I was working as a visiting graduate student in Rick Gaber’s laboratory at Northwestern University, and some of the work I did during those years turned out to be seminal for the then-fledgling field of epigenetics. Together with other scientists, I provided long-awaited in vivo functional evidence for a model proposed by Vince Allfrey in 1964, postulating that post-translational modifications of histones, particularly acetylation and deacetylation, are crucial for transcriptional regulation.

Gaussian Barebones Differential Evolution with Random-type Gaussian Mutation Strategy

This study attempts to propose a random-type Gaussian mutation strategy to improve the solution accuracy of Gaussian barebones differential evolution (GBDE). The proposed Gaussian mutation strategy is not only parameter free, but also employed to enhance the population diversity and global searching ability of the original mutation strategy. The search performance of GBDE with the proposed mutation strategy is compared with two standard DEs (DE/rand/1 and DE/best/1), the original GBDE and its modified version in terms of solution accuracy. Simulation results on two real-world optimal control problems given in IEEE - CEC 2011 evolutionary algorithm competition demonstrate the effectiveness of the proposed GBDE algorithm.

Abstract 4043: Establishment of a mouse skin squamous cell carcinoma allograft model for in vivo pharmacological analysis of immunotherapy

Abstract The recent clinical success of novel therapeutics blocking immune checkpoints cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) and programmed cell death 1 (PD-1) has fueled an intense interest around immuno-oncology. However, the lack of relevant animal models is a major bottleneck for understanding the mechanism of action and evaluating the efficacy of such therapeutics. Syngeneic mouse tumor models, despite being widely used as experimental models for efficacy studies, are limited by the restricted number of models available and responsive to current checkpoint inhibitors. Genetically engineered mouse models (GEMMs) of human cancer are effective tools for mechanistic analysis, but they are often not suitable for efficacy studies due to usually un-synchronized tumor progression. Syngeneic allografts of spontaneous mouse tumors derived from GEMMs (MuPrime®) may be used as a new type of immuno-oncology models with the following advantages: 1) their primary nature of “stem cell diseases” and relevant tumor microenvironment mirror the patient derived xenograft (PDX) models; 2) the availability of diverse cancer types and oncogenic drivers deriving from a wide range of GEMMs. We set out to build a library of allografts of spontaneous mouse tumors, including those derived from GEMM, in an immunocompetent host to support pharmacological investigation, particularly of immuno-oncology agents. Recently, we observed the growth of spontaneous cutaneous tumors on the neck of the C57BL/6J-ApcMin/J GEMM, which is heterozygous for the Apc (APCMin/+). Histopathology suggests the tumors are well-differentiated skin squamous cell carcinoma. We subcutaneously transplanted these skin tumors into the syngeneic C57BL/6J mice. The allografts grow well, are serially transplantable and maintain the histopathology of the primary tumor. Transcriptome sequencing revealed that the allografts maintain the original ApcMin mutation, express high levels of HER2 (also confirmed by immunochemistry, or IHC), and present frameshift mutations in both c-met and EGFR. In vivo pharmacological assessment indicate that tumors respond to 5-FU, paclitaxel, gemcitabine, docetaxel, and cisplatin chemotherapy. They is also sensitive to anti-mouse CTLA-4 antibody, and the response is associated with an increased number of tumor infiltrating immune cells, including TILs. Alltogether, here we show that we have established an allograft model suitable for in vivo efficacy analysis of immunotherapy using surrogate anti-mouse antibodies. Citation Format: Gavin Jiagui Qu, Annie Xiaoyu An, Jinping Liu, Davy Xuesong Ouyang, Likun Zhang, Jie Cai, Jean Pierre Wery, Henry Q. X. Li. Establishment of a mouse skin squamous cell carcinoma allograft model for in vivo pharmacological analysis of immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4043.

A Spontaneous Eggplant (Solanum melongena L.) Color Mutant Conditions Anthocyanin-free Fruit Pigmentation

We identified a single plant in a grow out of the eggplant (Solanum melongena L.) variety ‘Black Beauty’ bearing green fruit. ‘Black Beauty’ normally produces violet/black pigmented fruit attributed to anthocyanin accumulation. We selected the green-fruited true-breeding genotype E13GB42 from the S2 generation obtained from selfing of the S0 green-fruited color mutant. Characterization of 12 simple sequence repeat (SSR) markers, eight fruit morphological attributes and fruit yield support E13GB42 arising as a spontaneous mutant of ‘Black Beauty’. With the exception of fruit calyx prickliness, E13GB42 was not significantly different from ‘Black Beauty’ for fruit morphological attributes and yield. E13GB42 exhibited an SSR marker profile identical to that of ‘Black Beauty’ but polymorphic with that of eight violet/black-fruited modern eggplant hybrids, older open-pollinated varieties and landraces. Transcript levels of key anthocyanin biosynthetic genes (Chs, Dfr, and Ans) and regulatory genes (MybC, Myc, and Wd) were significantly lower in the green-fruited E13GB42 mutant in comparison with the black-fruited variety ‘Black Beauty’ at various stages of fruit development ranging from small post-anthesis fruit to full-size marketable fruit. Progeny obtained from selfing of the original mutant and reciprocal crosses with ‘Black Beauty’ produced violet, green, and green with violet striped color classes that together were not compatible with one or two gene inheritance models, suggesting that the mutation responsible for the E13GB42 phenotype influences multiple genetic factors that control fruit pigmentation.

Testing access control policy through Change Rule and Swap Rule Algorithm (CRSR)

We propose an algorithm for testing policies based on XACML Context Schema, known as Change Rule and Swap Rule Algorithm (CRSR). Compared to other testing techniques and tools for testing access control policies, where policy set or policy is evaluated first, this algorithm focuses on the rule and target of a policy set or policy. Our approach represents policy as a vector of bits. A Boolean variable 1 is used to represent the applicability of a policy to a request and a Boolean variable 0 is used to represent the non-applicability of a policy to a request. We assume that a correct policy evaluates to 1, indicating that all the elements, attributes ID and their values are correctly enforced. First of all we identify and extract the rule and target from the policy and then generate mutant policies and requests by applying the proposed algorithm. The rule and target are then evaluated using the original policies, mutant policies and requests generated. We compare our results against mutants generated using mutant operators, the Targen tool and the simple combinatorial approach to see the fault detection capability. The proposed algorithm is found to be effective, efficient and of high fault detection capability in testing access control policies, as it ensures a total coverage of the various elements of a policy based on the XACML Context Schema.

Heterogeneity in clinical features and disease severity in ataxia-associated SYNE1 mutations.

The autosomal recessive spinocerebellar ataxias are an exciting field of study, with a growing number of causal genes and an expanding phenotypic spectrum. SYNE1 was originally discovered in 2007 as the causal gene underlying autosomal recessive spinocerebellar ataxia 1, a disease clinically thought to manifest with mainly pure cerebellar ataxia. Since the original report SYNE1 mutations have also been identified in families with motor neuronopathy and arthrogryposis but few families have been screened as the gene is very large at 146 exons in length. We screened 196 recessive and sporadic ataxia patients for mutations in SYNE1 using next generation sequencing in order to assess its frequency and extend the clinicogenetic spectrum. We identified four novel truncating mutations spread throughout the SYNE1 gene from three families living in London that originated from England, Turkey and Sri Lanka. The phenotype was mainly pure cerebellar ataxia in two families, cognitive decline was present in all three families, axonal neuropathy in one family and marked spasticity in the Turkish family, with a range of disease severities. Searching for genotype–phenotype correlations in the SYNE1 gene, defects located near the 3′ prime end of the gene are more frequently associated with motor neuron or neuromuscular involvement so far. Our data indicate SYNE1 mutations are not an uncommon cause of recessive ataxia with or without additional clinical features in patients from various ethnicities. The use of next generation sequencing allows the rapid analysis of large genes and will likely reveal more SYNE1 associated cases and further expand genotype–phenotype correlations.Electronic supplementary materialThe online version of this article (doi:10.1007/s00415-016-8148-6) contains supplementary material, which is available to authorized users.

Temporal changes of EGFR mutations and T790M levels in tumour and plasma DNA following AZD9291 treatment

AZD9291, a T790M specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), has demonstrated impressive response rates in tumours harbouring the EGFR T790M resistance mutation. Emergence of resistance to AZD9291 has been shown to occur through several different mechanisms including the development of new mutations (e.g. C797S) in the EGFR tyrosine kinase domain. We studied two patients with paired tumour biopsies and blood samples pre- and post-progression on AZD9291 to explore possible resistance mechanisms.Pre- and Post-AZD9291 tumour biopsies as well as serial plasma samples were collected from two patients on the AURA clinical study (AZD9291 First Time in Patients Ascending Dose study). Droplet digital PCR (ddPCR) assays were used to quantify T790M, the driver EGFR mutation, and the C797S mutation in genomic DNA from paired tumour biopsies and plasma cell-free DNA.In the first patient, both EGFR T790M and L858R became undetectable in the plasma within 1 month after treatment with AZD9291. However, the T790M and the original L858R mutation re-emerged with radiologically confirmed resistance to AZD9291. In patient two, the levels of T790M were undetectable at the time of radiological resistance to AZD9291 but increasing levels of the original EGFR exon 19 deletion was detected. MET amplification was detected in a biopsy performed on progression. The EGFR C797S mutation was not detected in either patient at the time of relapse.ddPCR of cell free DNA enables real time monitoring of patients on 3rd generation TKIs. As resistance mechanisms are variable, monitoring levels of the initial activating EGFR mutation may facilitate more reliable detection of progression.

HIV Immunology Goes Out On a Limb

Vaccines against HIV most likely need to elicit broadly neutralizing antibodies. In this issue of Immunity, Poignard and colleagues describe the co-evolution of a broadly neutralizing antibody and the virus that triggered it, providing a template for HIV vaccine design.

Stationary mutation models

Probability calculations for relationship inference based on DNA tests are often performed with computer packages such as Familias. When mutations are assumed to be a possibility, one may notice a curious and problematic effect of including untested parents: results tend to change slightly. In this paper, we trace this effect back to fundamental model-formulating issues which can only be resolved by using stationary mutation models. We present several methods for obtaining such stationary mutation matrices from original mutation matrices, and evaluate essential properties of these methods. Our conclusion is that typically, stationary mutation models can be obtained, but for many types of markers, it may be impossible to combine specific biologically reasonable requirements for a mutation matrix with the requirement of stationarity.

MiRNA-146b-5p upregulates migration and invasion of different Papillary Thyroid Carcinoma cells.

BackgroundTumor invasiveness is directly related to the ability of tumor cells to migrate and invade surrounding tissues, usually degrading extracellular matrix. Despite significant progress in the knowledge about migration and invasion, there is much more to elucidate about their regulatory mechanisms, especially in cancer cells. MicroRNAs (miRs) were recently described as important regulators of migration. Differential expression of miRs in cancer is frequently associated with progression, invasion and metastasis. In papillary thyroid carcinoma (PTC), miR-146b-5p is highly expressed and positively correlated to the degree of malignancy.MethodsThis study aimed to investigate the role of miR-146b-5p on the migratory and invasive behaviors of thyroid cells, using a non tumor rat thyroid follicular cell line (PCCl3) transfected with the miR-146b-5p genomic region, and two PTC cell lines (TPC-1 and BCPAP, bearing distinct oncogenic backgrounds), which express high levels of miR-146b-5p, after miR-146b inhibition by antagomiR and miR-146b overexpression by mimics-miR. Migration and invasion were studied by time-lapse and transwell assays (with and without Matrigel®). Gelatin degradation assays were also employed, as well as F-actin staining.ResultsMigration and invasion of PCCl3 were increased 2-3x after miR-146b-5p overexpression (10X) and large lamellipodia were evident in those cells. After miR-146b-5p inhibition, TPC-1 and BCPAP migration and invasion were significantly reduced, with cells showing several simultaneous processes and low polarity. Gelatin degradation was inhibited in TPC-1 cells after inhibition of miR-146b-5p, but was unaffected in BCPAP cells, which did not degrade gelatin. The inhibition of miR-146b-5p in PCCl3 also inhibited migration and invasion, and additional (exogenous) overexpression of this miR in TPC-1 and BCPAP cells increased migration and invasion, without effects on cell morphology or gelatin degradation. The overexpression of SMAD4 in BCPAP cells, a validated target of miR-146b-5p and key protein in the TGF-β signaling pathway, inhibited migration similarly to the effects observed with the antagomiR 146b-5p.ConclusionsmiR-146b-5p positively regulates migration and invasion of thyroid normal and tumor follicular cells (independently from their original mutation, either BRAF or RET/PTC), through a mechanism that involves the actin cytoskeleton but not an increased capacity of matrix degradation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2146-z) contains supplementary material, which is available to authorized users.