Mutation prevalence of cerebral cavernous malformation genes in Spanish patients

[Objective] To study the molecular genetic and clinical features of cerebral cavernous malformations (CCM) in a cohort of Spanish patients.[Methods] We analyzed the CCM1, CCM2, and CCM3 genes by MLPA and direct sequencing of exons and intronic boundaries in 94 familial forms and 41 sporadic cases of CCM patients of Spanish extraction. When available, RNA studies were performed seeking for alternative or cryptic splicing.[Results] A total of 26 pathogenic mutations, 22 of which predict truncated proteins, were identified in 29 familial forms and in three sporadic cases. The repertoire includes six novel non-sense and frameshift mutations in CCM1 and CCM3. We also found four missense mutations, one of them located at the third NPXY motif of CCM1 and another one that leads to cryptic splicing of CCM1 exon 6. We found four genomic deletions with the loss of the whole CCM2 gene in one patient and a partial loss of CCM1and CCM2 genes in three other patients. Four families had mutations in CCM3. The results include a high frequency of intronic variants, although most of them localize out of consensus splicing sequences. The main symptoms associated to clinical debut consisted of cerebral haemorrhage, migraines and epileptic seizures. The rare co-occurrence of CCM with Noonan and Chiari syndromes and delayed menarche is reported.[Conclusions] Analysis of CCM genes by sequencing and MLPA has detected mutations in almost 35% of a Spanish cohort (36% of familial cases and 10% of sporadic patients). The results include 13 new mutations of CCM genes and the main clinical symptoms that deserves consideration in molecular diagnosis and genetic counselling of cerebral cavernous malformations.This work has been supported by grants CP10/00526 (Instituto de Salud Carlos III, Spain) and P07-CVI-02790 (Junta de Andalucía, Spain). RM received a fellowship of José Luis Castaño Foundation.Peer Reviewe

Clinical and molecular study of the extracellular matrix protein 1 gene in a spanish family with lipoid proteinosis

[Background] Lipoid proteinosis (LP) is a rare autosomal recessive disorder characterized by a hoarse voice, variable scarring, and infiltration of the skin and mucosa. This disease is associated with mutations of the gene encoding extracellular matrix protein 1 (ECM1). [Case Report]This was a clinical and molecular study of a new case of LP with a severe phenotype. A 35-year-old female born to nonconsanguineous parents developed dermatological and extracutaneous symptoms in her 9th month of life. The neurological abnormalities of the disease began to appear at the age of 19 years. Computed tomography revealed cranial calcifications. [Conclusions]The diagnosis of LP was confirmed by histopathological findings and direct sequencing of ECM1. A new homozygous nonsense mutation was identified in exon 7 of ECM1, c.1076G>A (p.Trp359*). This mutation was not detected in 106 chromosomes of healthy individuals with a similar demographic origin. Microsatellite markers around ECM1 were used to construct the haplotype in both the parents and the patient. Reports on genotype-phenotype correlations in LP point to a milder phenotype in carriers of missense mutations in the Ecm1a isoform, whereas mutations in the Ecm1b isoform are thought to be associated with more severe phenotypes. The present findings in a Spanish patient carrying a truncating mutation in exon 7 revealed complete dermatological and neurological manifestations. © 2014 Korean Neurological Association.The authors thank the patient and her family for their participation, and the financial support of grants from MICINN (no. SAF2007-60508) and Consejería de Ciencia Junta de Andalucía (no. CVI02790).Peer Reviewe

Mutation Prevalence of Cerebral Cavernous Malformation Genes in Spanish Patients

Objective: To study the molecular genetic and clinical features of cerebral cavernous malformations (CCM) in a cohort of Spanish patients. Methods: We analyzed the CCM1, CCM2, and CCM3 genes by MLPA and direct sequencing of exons and intronic boundaries in 94 familial forms and 41 sporadic cases of CCM patients of Spanish extraction. When available, RNA studies were performed seeking for alternative or cryptic splicing. Results: A total of 26 pathogenic mutations, 22 of which predict truncated proteins, were identified in 29 familial forms and in three sporadic cases. The repertoire includes six novel non-sense and frameshift mutations in CCM1 and CCM3. We also found four missense mutations, one of them located at the third NPXY motif of CCM1 and another one that leads to cryptic splicing of CCM1 exon 6. We found four genomic deletions with the loss of the whole CCM2 gene in one patient and a partial loss of CCM1and CCM2 genes in three other patients. Four families had mutations in CCM3. The results include a high frequency of intronic variants, although most of them localize out of consensus splicing sequences. The main symptoms associated to clinical debut consisted of cerebral haemorrhage, migraines and epileptic seizures. The rare co-occurrence of CCM with Noonan and Chiari syndromes and delayed menarche is reported. Conclusions: Analysis of CCM genes by sequencing and MLPA has detected mutations in almost 35% of a Spanish cohort (36% of familial cases and 10% of sporadic patients). The results include 13 new mutations of CCM genes and the main clinical symptoms that deserves consideration in molecular diagnosis and genetic counselling of cerebral cavernous malformations.Instituto de Salud Carlos III CP10/00526Junta de Andalucía P07-CVI-02790Fundación José Luis Castañ

Mutation prevalence of cerebral cavernous malformation genes in Spanish patients.

Journal Article; Research Support, Non-U.S. Gov't;OBJECTIVE To study the molecular genetic and clinical features of cerebral cavernous malformations (CCM) in a cohort of Spanish patients. METHODS We analyzed the CCM1, CCM2, and CCM3 genes by MLPA and direct sequencing of exons and intronic boundaries in 94 familial forms and 41 sporadic cases of CCM patients of Spanish extraction. When available, RNA studies were performed seeking for alternative or cryptic splicing. RESULTS A total of 26 pathogenic mutations, 22 of which predict truncated proteins, were identified in 29 familial forms and in three sporadic cases. The repertoire includes six novel non-sense and frameshift mutations in CCM1 and CCM3. We also found four missense mutations, one of them located at the third NPXY motif of CCM1 and another one that leads to cryptic splicing of CCM1 exon 6. We found four genomic deletions with the loss of the whole CCM2 gene in one patient and a partial loss of CCM1and CCM2 genes in three other patients. Four families had mutations in CCM3. The results include a high frequency of intronic variants, although most of them localize out of consensus splicing sequences. The main symptoms associated to clinical debut consisted of cerebral haemorrhage, migraines and epileptic seizures. The rare co-occurrence of CCM with Noonan and Chiari syndromes and delayed menarche is reported. CONCLUSIONS Analysis of CCM genes by sequencing and MLPA has detected mutations in almost 35% of a Spanish cohort (36% of familial cases and 10% of sporadic patients). The results include 13 new mutations of CCM genes and the main clinical symptoms that deserves consideration in molecular diagnosis and genetic counselling of cerebral cavernous malformations.This work has been supported by grants CP10/00526 (Instituto de Salud Carlos III, Spain) and P07-CVI-02790 (Junta de Andalucía, Spain) and José Luis Castaño Foundation.Ye

Identification of tipifarnib sensitivity biomarkers in T-cell acute lymphoblastic leukemia and T-cell lymphoma

Patients diagnosed with T-cell leukemias and T-cell lymphomas (TCLs) still have a poor prognosis and an inadequate response to current therapies, highlighting the need for targeted treatments. We have analyzed the potential therapeutic value of the farnesyltransferase inhibitor, tipifarnib, in 25 TCL cell lines through the identification of genomic and/or immunohistochemical markers of tipifarnib sensitivity. More than half of the cell lines (60%) were considered to be sensitive. Tipifarnib reduced cell viability in these T-cell leukemia and TCL cell lines, induced apoptosis and modified the cell cycle. A mutational study showed TP53, NOTCH1 and DNMT3 to be mutated in 84.6%, 69.2% and 30.0% of sensitive cell lines, and in 62.5%, 0% and 0% of resistant cell lines, respectively. An immunohistochemistry study showed that p-ERK and RelB were associated as potential biomarkers of tipifarnib sensitivity and resistance, respectively. Data from RNA-seq show that tipifarnib at IC50 after 72 h downregulated a great variety of pathways, including those controlling cell cycle, metabolism, and ribosomal and mitochondrial activity. This study establishes tipifarnib as a potential therapeutic option in T-cell leukemia and TCL. The mutational state of NOTCH1, p-ERK and RelB could serve as potential biomarkers of tipifarnib sensitivity and resistance.The research was supported by grants from the Instituto de Salud Carlos III, from the Ministerio de Economía, Industria y Competitividad [SAF2013-47416-R, CIBERONC-ISCIII, ISCIII-MINECO-AES-FEDER (Plan Estatal I + D + I 2013–2016)}

Clinical description of patients with mutations detected in CCM genes.

a<p>Size of the larger lesion.</p>b<p>Sporadic patient.</p

Quantitative MLPA analysis of (a) P130 and (b) P131 probemix (MRC Holland).

<p>Black columns represent CV146 index patient and grey columns represent a healthy control. The deletion of exons 12 to 16 of <i>CCM1</i> is shown.</p

Mutation analysis in the CV150 patient.

<p>(a) Sequencing of exon 6 (<i>CCM1</i> gene), which shows the A>G transition at position 842. (b) cDNA sequencing of exons 5 to 8 of a healthy subject and (c) the CV150 patient. A new 5′ splice site is created at the mutation site. The new splicing alters the open reading frame of exon 7 and generates a premature stop codon (p.Asp281GlyfsX5). (d) Diagram showing the cryptic splicing of exon 6 to exon 7 in the patient. Codons are shown in consecutive blue and black colour and premature stop codon in red colour. The arrow shows the novel donor splice site.</p

Mutations identified within the <i>CCM1</i> gene (NM_194455.1).

a<p>New mutation non-previously reported.</p>b<p>Sporadic patient.</p>c<p>Exon count begins with exon 1 as the first coding exon for <i>CCM1</i>.</p><p>NA: not avialable. NC: 5′ untranslated exons.</p

An integrated prognostic model for diffuse large B-cell lymphoma treated with immunochemotherapy

Diffuse large B-cell lymphoma (DLBCL), the most frequent non-Hodgkin's lymphoma subtype, is characterized by strong biological, morphological, and clinical heterogeneity, but patients are treated with immunochemotherapy in a relatively homogeneous way. Here, we have used a customized NanoString platform to analyze a series of 197 homogeneously treated DLBCL cases. The platform includes the most relevant genes or signatures known to be useful for predicting response to R-CHOP (Rituximab, Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone) in DLBCL cases. We generated a risk score that combines the International Prognostic Index with cell of origin and double expression of MYC/BCL2, and stratified the series into three groups, yielding hazard ratios from 0.15 to 5.49 for overall survival, and from 0.17 to 5.04 for progression-free survival. Group differences were highly significant (p < 0.0001), and the scoring system was applicable to younger patients (<60 years of age) and patients with advanced or localized stages of the disease. Results were validated in an independent dataset from 166 DLBCL patients treated in two distinct clinical trials. This risk score combines clinical and biological data in a model that can be used to integrate biological variables into the prognostic models for DLBCL cases.Funding information: GILEAD, Grant/Award Numbers: PIE15/0081, PI16/01294, PI17/2172, PI17/00272, PI19/00715, GL18/00019; Asociación Española Contra el Cáncer, Grant/Award Number: PROYE18054PIRI; Instituto de Salud Carlos III, Grant/Award Number: PT17/0015/0024; Xarxa de Bancs de Tumors de Catalunya; Biobank do Complexo Hospitalario Universitario de Santiago de Compostela, Grant/Award Number: PT17/0015/0002; Hospital Universitario Virgen del Rocío-Instituto de Biomedicina de Sevilla Biobank, Grant/Award Number: PT17/0015/0041; Valdecilla Biobank, Grant/Award Number: PT17/0015/0019;MD Anderson Biobank, Grant/Award Number: PT17/0015/0008; AIRC (Italian Association for Cancer Research, Milan, Italy), Grant/Award Number: 5×1000 n. 21198; Marie Skłodowska-Curie Individual Fellowship, Grant/Award Number: 882597; CIBERONC, Grant/Award Number: CB16/12/00291; Comunidad Autonoma de MadridDLBCLGene expressionImmunochemotherapyDiffuse large B-cell lymphomaPrognosi