In-frame Mutations Research Articles

An In-Frame Exon-Skipping MUTYH Mutation Is Associated With Early-Onset Colorectal Cancer

MUTYH-associated polyposis syndrome is inherited as a recessive trait and is characterized by the presence of colorectal adenomas and cancer predisposition. Carriers of biallelic mutations in the base excision repair gene MUTYH are exposed to an increased risk of colorectal cancer. The aim of this study was to investigate the pathogenicity of the 13-base-pair deletion, c.504 + 19_504 + 31del13, located on intron 6 of the MUTYH gene, which was identified in 2 unrelated Greek patients with MUTYH-associated polyposis. Genomic DNA and total RNA were extracted from peripheral blood lymphocytes of 2 unrelated families with polyposis. Screening of the entire coding region of the APC gene and MUTYH gene was performed by direct sequencing. cDNA analysis of a patient homozygous for the c.504 + 19_504 + 31del13 mutation, and a patient heterozygous for the c.504 + 19_504 + 31del13/c.1437_1439delGGA mutations, revealed complete skipping of MUTYH exon 6. A part of the glycosylase catalytic domain, the pseudo-helix-hairpin-helix motif, is located within exon 6, which enhances the pathogenic effect of the mutation. This study demonstrates a rare pathogenic MUTYH mutation that is possibly associated with a relatively severe MUTYH-associated polyposis phenotype, highlighted by early-onset colorectal cancer. These data can become useful in clinical practice for the appropriate surveillance and management of families with MUTYH-associated polyposis. It is evident that each MUTYH-associated polyposis case should be handled according to its distinct genotype, following a specific prophylaxis protocol to ensure cancer prevention.

Congenital insensitivity to pain: novel SCN9A missense and in-frame deletion mutations.

SCN9A encodes the voltage-gated sodium channel Nav1.7, a protein highly expressed in pain-sensing neurons. Mutations in SCN9A cause three human pain disorders: bi-allelic loss of function mutations result in Channelopathy-associated Insensitivity to Pain (CIP), whereas activating mutations cause severe episodic pain in Paroxysmal Extreme Pain Disorder (PEPD) and Primary Erythermalgia (PE). To date, all mutations in SCN9A that cause a complete inability to experience pain are protein truncating and presumably lead to no protein being produced. Here, we describe the identification and functional characterization of two novel non-truncating mutations in families with CIP: a homozygously-inherited missense mutation found in a consanguineous Israeli Bedouin family (Nav1.7-R896Q) and a five amino acid in-frame deletion found in a sporadic compound heterozygote (Nav1.7-ΔR1370-L1374). Both of these mutations map to the pore region of the Nav1.7 sodium channel. Using transient transfection of PC12 cells we found a significant reduction in membrane localization of the mutant protein compared to the wild type. Furthermore, voltage clamp experiments of mutant-transfected HEK293 cells show a complete loss of function of the sodium channel, consistent with the absence of pain phenotype. In summary, this study has identified critical amino acids needed for the normal subcellular localization and function of Nav1.7. © 2010 Wiley-Liss, Inc.

Characterization of a lipopolysaccharide mutant of Leptospira derived by growth in the presence of an anti-lipopolysaccharide monoclonal antibody

A lipopolysaccharide mutant of Leptospira interrogans (LaiMut) was obtained by growth in the presence of an agglutinating monoclonal antibody (mAb) against lipopolysaccharide. Agglutination reactions with anti-lipopolysaccharide mAbs and polyclonal antibodies showed that LaiMut had lost some serogroup Icterohaemorrhagiae agglutinating epitopes. However, LaiMut displayed an increased reactivity to antisera against related serogroups, suggesting that the disruption of some lipopolysaccharide epitopes resulted in greater exposure to cross-reactive epitopes, not accessible to antibodies in the wild type (LaiWT). Comparison of the nucleotide sequences of the lipopolysaccharide loci of LaiMut and Lai wild type (LaiWT) strains showed an inframe stop mutation in the gene encoding undecaprenyl-galactosyltransferase, a protein that provides a fundamental and nonredundant function essential for lipopolysaccharide biosynthesis. Despite this, the biosynthesis of lipopolysaccharide agglutinating antigens was not abolished by the mutation. Based on the phenotype of LaiMut and analysis of the domain structure of the undecaprenyl-galactosyltransferase in relation to the mutation, we propose that the mutation results in the expression of two functional proteins in place of the undecaprenyl-galactosyltransferase. We hypothesize that the loss of coordination of the coupled function afforded by the intact dual function protein present in the parent strain results in an inefficient production of lipopolysaccharide in LaiMut.

Dominant-Negative Mutations in α-II Spectrin Cause West Syndrome with Severe Cerebral Hypomyelination, Spastic Quadriplegia, and Developmental Delay

A de novo 9q33.3-q34.11 microdeletion involving STXBP1 has been found in one of four individuals (group A) with early-onset West syndrome, severe hypomyelination, poor visual attention, and developmental delay. Although haploinsufficiency of STXBP1 was involved in early infantile epileptic encephalopathy in a previous different cohort study (group B), no mutations of STXBP1 were found in two of the remaining three subjects of group A (one was unavailable). We assumed that another gene within the deletion might contribute to the phenotype of group A. SPTAN1 encoding alpha-II spectrin, which is essential for proper myelination in zebrafish, turned out to be deleted. In two subjects, an in-frame 3 bp deletion and a 6 bp duplication in SPTAN1 were found at the initial nucleation site of the alpha/beta spectrin heterodimer. SPTAN1 was further screened in six unrelated individuals with WS and hypomyelination, but no mutations were found. Recombinant mutant (mut) and wild-type (WT) alpha-II spectrin could assemble heterodimers with beta-II spectrin, but alpha-II (mut)/beta-II spectrin heterodimers were thermolabile compared with the alpha-II (WT)/beta-II heterodimers. Transient expression in mouse cortical neurons revealed aggregation of alpha-II (mut)/beta-II and alpha-II (mut)/beta-III spectrin heterodimers, which was also observed in lymphoblastoid cells from two subjects with in-frame mutations. Clustering of ankyrinG and voltage-gated sodium channels at axon initial segment (AIS) was disturbed in relation to the aggregates, together with an elevated action potential threshold. These findings suggest that pathological aggregation of alpha/beta spectrin heterodimers and abnormal AIS integrity resulting from SPTAN1 mutations were involved in pathogenesis of infantile epilepsy.

A Splice-Site Mutation in a Retina-Specific Exon of BBS8 Causes Nonsyndromic Retinitis Pigmentosa

Tissue-specific alternative splicing is an important mechanism for providing spatiotemporal protein diversity. Here we show that an in-frame splice mutation in BBS8, one of the genes involved in pleiotropic Bardet-Biedl syndrome (BBS), is sufficient to cause nonsyndromic retinitis pigmentosa (RP). A genome-wide scan of a consanguineous RP pedigree mapped the trait to a 5.6 Mb region; subsequent systematic sequencing of candidate transcripts identified a homozygous splice-site mutation in a previously unknown BBS8 exon. The allele segregated with the disorder, was absent from controls, was completely invariant across evolution, and was predicted to lead to the elimination of a 10 amino acid sequence from the protein. Subsequent studies showed the exon to be expressed exclusively in the retina and enriched significantly in the photoreceptor layer. Importantly, we found this exon to represent the major BBS8 mRNA species in the mammalian photoreceptor, suggesting that the encoded 10 amino acids play a pivotal role in the function of BBS8 in this organ. Understanding the role of this additional sequence might therefore inform the mechanism of retinal degeneration in patients with syndromic BBS or other related ciliopathies.

Keratin K6c Mutations Cause Focal Palmoplantar Keratoderma

The palmoplantar keratodermas (PPKs) are a large group of clinically and genetically heterogeneous genodermatoses. The gene defects underlying many PPKs still need to be resolved to facilitate definitive molecular diagnosis and genetic counseling. Dominant-negative mutations in any of the four identified keratin genes, KRT6A, KRT6B, KRT16, or KRT17, cause pachyonychia congenita (PC), characterized by hypertrophic nail dystrophy and other ectodermal features. In PC, focal PPK (FPPK) is the most painful and debilitating phenotypic feature. Some families presenting with FPPK alone, or with minimal nail changes, carry mutations in KRT16; however, most FPPK families do not harbor mutations in any of these keratin genes. Here, we report three unrelated families who presented with familial FPPK with minor or absent nail changes. The four PC/FPPK-related keratin genes were excluded; however, mutational analysis of the recently identified KRT6C gene, encoding keratin K6c, showed heterozygous in-frame deletion mutations in all three kindreds. Affected members of Families 1 and 2 carried the same mutation, p.Asn172del. In Family 3, the mutation p.Ile462-Glu470del co-segregated with the disease. KRT6C was shown to be expressed in the plantar epidermis using reverse transcription-PCR, consistent with the phenotype observed in this tissue. These data expand the genetic testing repertoire for the PPKs.

Identification of a Heterozygous Sulfonylurea Receptor 1 Mutation that Exerts a Strong Dominant-Negative Effect on KATP Channel Response to MgADP

ATP-sensitive potassium (KATP) channels couple cell metabolism to cell excitability thus mediating a range of physiological responses to metabolic stress. In pancreatic β-cells, KATP channels regulate insulin secretion according to plasma glucose concentrations. Mutations in the channel genes ABCC8 encoding the regulatory sulfonylurea receptor 1 or KCNJ11 encoding the pore-forming inwardly rectifying potassium channel Kir6.2 that lead to loss of channel function are causes of congenital hyperinsulinism, characterized by inappropriate insulin secretion despite severe hypoglycemia. The disease-causing mutations can be recessively inherited, which are usually associated with severe disease phenotype, or dominantly inherited, which are commonly associated with less severe disease phenotype and are clinically responsive to the KATP channel opener diazoxide. The most prominent channel gating defects caused by mutations identified in congenital hyperinsulinism is loss of channel response to the stimulatory effect of MgADP and diazoxide. Here, we have identified a heterozygous in-frame insertion mutation in exon 37 of the ABCC8 gene that results in duplication of two amino acids ala-ser at position 1508 in the second nucleotide binding fold 2 (NBF2) from a patient with severe congenital hyperinsulinism unresponsive to diazoxide. Functional characterization of mutant channels reconstituted in COS cells show that the mutation does not disrupt surface expression of the channel but abolishes channel response to MgADP and diazoxide. Strikingly, in simulated heterozygous expression condition, the mutant SUR1 subunit exhibited a strong dominant negative effect on WT SUR1 subunit such that the MgADP and diazoxide response are nearly identical to homomeric mutant channels. This clinical and in-vitro strong dominant negative effect is distinct from other heterozygous mutations reported previously present an interesting case for understanding the structural mechanisms underlying channel response to MgADP and diazoxide.

Contributions of Four Cortex Lytic Enzymes to Germination of Bacillus anthracis Spores

Bacterial spores remain dormant and highly resistant to environmental stress until they germinate. Completion of germination requires the degradation of spore cortex peptidoglycan by germination-specific lytic enzymes (GSLEs). Bacillus anthracis has four GSLEs: CwlJ1, CwlJ2, SleB, and SleL. In this study, the cooperative action of all four GSLEs in vivo was investigated by combining in-frame deletion mutations to generate all possible double, triple, and quadruple GSLE mutant strains. Analyses of mutant strains during spore germination and outgrowth combined observations of optical density loss, colony-producing ability, and quantitative identification of spore cortex fragments. The lytic transglycosylase SleB alone can facilitate enough digestion to allow full spore viability and generates a variety of small and large cortex fragments. CwlJ1 is also sufficient to allow completion of nutrient-triggered germination independently and is a major factor in Ca(2+)-dipicolinic acid (DPA)-triggered germination, but its enzymatic activity remains unidentified because its products are large and not readily released from the spore's integuments. CwlJ2 contributes the least to overall cortex digestion but plays a subsidiary role in Ca(2+)-DPA-induced germination. SleL is an N-acetylglucosaminidase that plays the major role in hydrolyzing the large products of other GSLEs into small, rapidly released muropeptides. As the roles of these enzymes in cortex degradation become clearer, they will be targets for methods to stimulate premature germination of B. anthracis spores, greatly simplifying decontamination measures.

A family harboring a germ‐line N‐terminal C/EBPα mutation and development of acute myeloid leukemia with an additional somatic C‐terminal C/EBPα mutation

C/EBPalpha plays an essential role as a transcription factor in myeloid cell differentiation. Here, we describe a Japanese family in which two individuals with acute myeloid leukemia (AML) and one healthy individual had an identical 4-base pair insertion in the N-terminal region of CEBPA (350_351insCTAC), resulting in the termination at codon 107 (I68fsX107). The father and a son at diagnosis of AML had different in-frame insertion mutations in the C-terminal region of C/EBPalpha. These C-terminal mutations disappeared upon remission in both patients. Interestingly, the father showed different in-frame insertion mutations in the C-terminal CEBPA at the time of diagnosis and relapse. These data strongly suggest that the N-terminal C/EBPalpha mutation predisposes to the occurrence of a C-terminal C/EBPalpha mutation as a secondary genetic hit, causing AML.

In Vitro Analysis of LIPH Mutations Causing Hypotrichosis Simplex: Evidence Confirming the Role of Lipase H and Lysophosphatidic Acid in Hair Growth

Hypotrichosis simplex (HS) is a group of isolated alopecias that can be inherited as an autosomal-dominant or an autosomal-recessive trait. Hair loss usually begins in early childhood, and is diffuse and progressive. Mutations in LIPH, which encodes lipase member H, have recently been shown to cause an autosomal-recessive form of HS. Here we describe an Austrian HS patient who was found to be carrying compound heterozygous mutations in the LIPH gene: a 7-bp frameshift duplication (c.403_409dup; p.Gln137HisfsX1) and a recently reported 30-amino acid in-frame duplication (c.280_369dup; p.Gly94_Lys123dup). To examine the impact of LIPH mutations on lipid metabolism, we established an in vitro assay to measure the action of this phospholipase in a cell-based system. Both the 7-bp duplication frameshift mutation and all known in-frame mutations were observed to reduce the in vitro activity of the lipase in response to the addition of phosphatidic acid, the substrate of lipase H. The reduced production of lysophosphatidic acid (LPA) led to a reduced response of cells expressing the human G-protein-coupled receptor p2y5 (p2y5) receptor. Our study increases the spectrum of known LIPH mutations and provides biochemical evidence for the important role of lipase H and its product LPA in human hair growth.

Characterization of a spontaneous avirulent mutant of Legionella pneumophila Serogroup 6: Evidence of DotA and flagellin involvement in the loss of virulence

The pathogenesis of Legionella pneumophila mainly resides in its ability to inhibit the phagosome-lysosome fusion, which normally prevents the killing of the host cells. In order to characterize the molecular alterations that occurred in a spontaneous avirulent mutant of Legionella pneumophila serogroup 6, named Vir-, we investigated the ability of the mutant to adhere to and multiply in the WI26VA4 alveolar epithelial cell line and in human macrophages, when compared to its parental strain, Vir+. We also determined the colocalization of bacteria with LAMP-1 to gain an insight into the phagosome-lysosome fusion process. Additionally, we determined the flagellin expression and dotA nucleotide sequencing. We observed a lack of expression of flagellin and an in-frame mutation in the dotA. gene. The data obtained strongly suggest the loss of virulence of the mutant could probably be due to the absence of flagellin and the dysfunctional type IV secretion System, resulting from the DotA protein being severely compromised.

Characterization of CEBPA Mutations and Promoter Hypermethylation in Pediatric Acute Myeloid Leukemia.

Abstract 1605▪▪This icon denotes an abstract that is clinically relevant.Poster Board I-631CCAAT/enhancer binding protein alpha (C/EBPá) function is frequently disrupted in acute myeloid leukemia (AML). This can be caused by different mechanisms, including mutations in CEBPA, the gene encoding for C/EBPá. Recently, promoter hypermethylation resulting in CEBPA silencing has been described. CEBPA mutations are associated with a favorable outcome in adult AML. Recent studies however suggested that the favorable outcome is uniquely associated with CEBPA double-mutated AML (CEBPAdoublemut) (presence of >1 CEBPA mutation), and not with CEBPA single-mutated AML (CEBPAsinglemut). In pediatric AML, data on outcome of CEBPA-mutated AML is limited to one study, showing favorable outcome for CEBPAdoublemut as well as CEBPAsinglemut, which is in contrast with the adult data. So far, data on CEBPA hypermethylation in pediatric AML is lacking.We therefore studied a large pediatric AML cohort (n=252) to characterize CEBPA mutations by sequencing the entire coding region, and CEBPA promoter hypermethylation by methylation-specific PCR (MSP). Survival analyses were performed in 185 patients with de novo AML (excluding t(15;17) and secondary AML) treated on uniform DCOG and BFM protocols. Furthermore, we generated gene expression profiles using the Affymetrix HGU133 plus 2.0 microarrays to compare subgroups with CEBPA aberrations.Thirty four CEBPA mutations were identified in 20/252 diagnostic samples (7.9%). In 14 cases double mutations were present, which combined an N-terminal frame shift mutation with an in-frame mutation in the bZIP region (n=13) or with a frame shift-causing insertion before bZIP (n=1). In 6 cases a single mutation was present; i.e. in-frame bZIP mutation (n=4), or frame shift mutation respectively in the TAD2 domain (n=1) or before the bZIP domain (n=1). CEBPAdoublemut were only present in children above 3 years of age and in FAB M1/2 subtypes, in contrast to CEBPAsinglemut, which presented also in children <3 years of age (1/6) and in other FAB subtypes (3/6). CEBPAdoublemut and CEBPAsinglemut were both exclusively found in cases with a normal karyotype (57% and 33%, respectively) and in cases with ‘other' karyotypes (defined as 'other than t(8;21), inv(16), t(15;17) and MLL-rearrangements'), in 36% and 50%, respectively. However, in both subgroups additional molecular aberrations, i.e. in RAS, FLT3/ITD and WT1, were equally distributed. CEBPAdoublemut patients (n=10) had a significantly better overall survival compared with CEBPAsinglemut (n=5) (5-years pOS 79±13% vs. 25±22%, p=0.04; pEFs 58±16% vs. 30±24%, p=0.16). Furthermore, they showed a trend for favorable outcome compared with CEBPA wild-type AML patients, after excluding CBF-AML cases (n=120; pOS 79±13% vs. 47±5%, p=0.07; pEFS 58±16% vs. 34±4%; p=0.06). Their survival was comparable to the CBF-AML subgroup (n=50) (pOS 91±4%, pEFS 61±8%). Multivariate analysis, including age, WBC, CBF-AML, NPM1 mutations and FLT3/ITD, showed that CEBPAdoublemut were an independent favorable prognostic factor for pOS (HR 0.23; p=0.04) and pEFS (HR 0.32; p=0.03). CEBPA promoter hypermethylation was detected in 3/237 cases, which resulted in CEBPA silencing. Using an unsupervised clustering analysis as previously published by Valk et al. (NEJM 2004) of our de novo AML cases (n=237), CEBPA-mutated cases predominantly aggregated in 1 cluster with the CEBPAsil cases, revealing a common underlying gene expression profile. Two additional cases with silenced CEBPA (CEBPAsil) were identified in this cluster, resulting in 5/237 (2.1%) CEBPAsil cases. All CEBPAsil showed T-lymphoid characteristics, (e.g. high expression of CD7 and high LCK expression). However, NOTCH1 mutations were not found. Three of 5 patients relapsed within 1 year of diagnosis, but the other 2 are in continuous complete remission for 4.8 and 8.5 yrs.In conclusion, CEBPAdoublemut were identified as an independent predictor of good clinical outcome. Hence, if these results could be confirmed in a prospective serie, CEBPAdoublemut may be used for further refinement of risk-group stratification. Of interest, the other cases with CEBPA aberrations, i.e. CEBPAsinglemut and CEBPAsil, seem to predict for poor outcome, in line with data presented in adults. The subgroup with CEBPAsil due to hypermethylation may potentially benefit from the use of demethylating agents. DisclosuresNo relevant conflicts of interest to declare.

Prevalence and Prognostic Impact of CEBPA mutations in Children with AML Treated with the AIEOP-LAM 2002/01 Protocol.

Abstract 2643Poster Board II-619C/EBP is a transcription factor that regulates terminal granulocytic differentiation. CEBPA mutations have been associated with improved outcome in both adult and pediatric patients with acute myeloid leukemia (AML). However, the impact in different treatment protocols, as well as the different outcome between single and double mutants, is still to be definitively established. We evaluated the prevalence and prognostic significance of CEBPA mutations in children with de novo non promyelocitic AML treated in Italy with the AIEOP-LAM 2002/01 pediatric protocol. Among 205 patients enrolled in the protocol between December 2002 and December 2007, 103 were successfully analyzed for CEBPA mutations by PCR amplification of TAD and bZIP domains of the gene, and through sequencing of positive cases after DHPLC analysis. Characteristics of analyzed and non analyzed patients were not statistically different. Two types of CEBPA mutations, N-terminal (TAD) truncating mutations and in-frame bZip domain mutations, were detected in 13/103 (12.6%) patients tested; 8 of them (61.5%) harboured both mutation types.Laboratory, clinical characteristics and outcomes for patients with CEBPA mutations were compared to those of patients with wild-type CEBPA. CEBPA mutations were significantly more common in older patients (8/13 vs 30/90 children were older than 10 years), in patients with FAB M1 (7/13 vs 4/90), and in patients with normal cytogenetics (13/13). None of the CEBPA mutated cases carried either FLT3 or NPM1 mutations. Only 1 mutated case was found in Standard Risk patients (defined as children carrying isolated CBF abnormality and achieving complete remission after 1 cycle of induction therapy), while the other 12 patients belonged to the High Risk group.Although the values did not reach statistical significance because of the low prevalence of CEBPA mutations, with a median follow up of 39 months (range 4–86) the event-free survival probability at 5 years was 76.9% vs. 59.6% for children with or without CEBPA mutations, respectively. The values for Disease-Free Survival were 83.3% vs. 65.4% and those for Overall Survival were 90.0%. 66.4%, respectively. No difference in terms of outcome was found between patient with a single and those with double mutants, neither between those with TAD- and bZIP- mutations.Therefore, patients in the AIEOP-LAM 2002/01 pediatric trial carrying CEBPA mutations seem to have a lower relapse rate and improved outcome, their overall survival approaching that of children belonging to the Standard Risk group (90% vs. 97%). If confirmed in a larger cohort of patients and with longer follow-up, these data suggest that CEBPA mutation analysis could be usefully employed to identify patients at lower risk of treatment failure and for allocating them into different classes of risk. Disclosures:No relevant conflicts of interest to declare.

Mutations in NR5A1 Associated With Ovarian Insufficiency

Primary ovarian insufficiency, also known as premature ovarian failure, is characterized by loss of function of the ovaries before the age 40. Observational evidence of familial associations suggests a genetic basis for ovarian insufficiency. Despite extensive research, specific genetic causes have not been identified. A nuclear receptor, NR5A1, also called Ad4 binding protein or steroidogenic factor 1, is a protein that regulates the transcription of genes involved in the hypothalamic―pituitary―steroidogenic axis that play a key role in sexual development and reproduction. NR5A1 I is expressed in multiple ovarian cell types during fetal development, postnatal and prepubertal growth, and at maturity. In this study, the investigators tested the hypothesis that mutations in NR5A1 are associated with disorders of ovarian development and function. NR5A1 was sequenced in affected study subjects who were members of 4 families with a history of 46,XY disorders of sex development and 46,XX ovarian insufficiency and 25 women with 46,XX sporadic ovarian insufficiency. A panel of 1465 subjects of various ancestral origins who did not carry NRSA1 mutations served as controls. There was no clinical evidence of adrenal insufficiency among any of the affected patients. Mutations in the NR5A1 gene were found among members of each of the 4 families and 2 of the 25 subjects with isolated ovarian insufficiency. Analysis of the NRSA1 gene revealed a series of in-frame, frame-shift and missense mutations. Testing the effect of each of the NRSA1 mutations on protein function revealed a severe quantitative impairment of NRSA1 transactivational activity. A range of ovarian anomalies, including 46,XY gonadal dysgenesis and 46,XX primary ovarian insufficiency, were associated with the mutations. None of these mutations were found in any of the unaffected control subjects. These findings indicate that mutations in NRSA1 are associated with a number of ovarian anomalies characterized by loss of ovarian reproductive capacity.

Exon‐skipping therapy for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal muscle disorder caused by mutations in the DMD gene for which no mutation-targeted therapy has been available thus far. However, exon-skipping mediated by antisense oligonucleotides (AOs), which are short single-strand DNAs, has considerable potential for DMD therapy, and clinical trials in DMD patients are currently underway. This exon-skipping therapy changes an out-of-frame mutation into an in-frame mutation, aiming at conversion of a severe DMD phenotype into a mild phenotype by restoration of truncated dystrophin expression. Recently, stable and less-toxic AOs have been developed, and their higher efficacy was confirmed in mice and dog models of DMD. In this review, we briefly summarize the genetic basis of DMD and the potential and perspectives of exon skipping as a promising therapy for this disease.

The PEPvIII-KLH (CDX-110) vaccine in glioblastoma multiforme patients

Conventional therapies for glioblastoma multiforme (GBM) fail to target tumor cells exclusively, resulting in non-specific toxicity. Immune targeting of tumor-specific mutations may allow for more precise eradication of neoplastic cells. EGFR variant III (EGFRvIII) is a tumor-specific mutation that is widely expressed in GBM and other neoplasms and its expression enhances tumorigenicity. This in-frame deletion mutation splits a codon, resulting in a novel glycine at the fusion junction producing a tumor-specific epitope target for cellular or humoral immunotherapy. We have previously shown that vaccination with a peptide that spans the EGFRvIII fusion junction (PEPvIII-KLH/CDX-110) is an efficacious immunotherapy in syngeneic murine models. In this review, we summarize our results in GBM patients targeting this mutation in multiple, multi-institutional Phase II immunotherapy trials. These trials demonstrated that a selected population of GBM patients who received vaccines targeting EGFRvIII had an unexpectedly long survival time. Further therapeutic strategies and potential pitfalls of using this approach are discussed.

Prevalence and prognostic implications of CEBPA mutations in pediatric acute myeloid leukemia (AML): a report from the Children's Oncology Group

AbstractCEBPA mutations have been associated with improved outcome in adult acute myeloid leukemia (AML). We evaluated the prevalence and prognostic significance of CEBPA mutations in 847 children with AML treated on 3 consecutive pediatric trials. Two types of CEBPA mutations—N-terminal truncating mutations and in-frame bZip-domain mutations—were detected in 38 (4.5%) of 847 patients tested; 31 (82%) of 38 patients with mutations harbored both mutation types. Mutation status was correlated with laboratory and clinical characteristics and clinical outcome. CEBPA mutations were significantly more common in older patients, patients with FAB M1 or M2, and patients with normal karyotype. Mutations did not occur in patients with either favorable or unfavorable cytogenetics. Actuarial event-free survival at 5 years was 70% versus 38% (P = .015) with a cumulative incidence of relapse from complete remission of 13% versus 44% (P = .007) for those with and without CEBPA mutations. The presence of CEBPA mutations was an independent prognostic factor for improved outcome (HR = 0.24, P = .047). As CEBPA mutations are associated with lower relapse rate and improved survival, CEBPA mutation analysis needs to be incorporated into initial screening for risk identification and therapy allocation at diagnosis. The clinical trials in this study are registered at http://www.clinicaltrials.gov under NCT00002798 and NCT00070174.

Defects in cell spreading and ERK1/2 activation in fibroblasts with lamin A/C mutations

In-frame mutations in nuclear lamin A/C lead to a multitude of tissue-specific degenerative diseases known as the ‘laminopathies’. Previous studies have demonstrated that lamin A/C-null mouse fibroblasts have defects in cell polarisation, suggesting a role for lamin A/C in nucleo-cytoskeletal-cell surface cross-talk. However, this has not been examined in patient fibroblasts expressing modified forms of lamin A/C. Here, we analysed skin fibroblasts from 3 patients with Emery–Dreifuss muscular dystrophy and from 1 with dilated cardiomyopathy. The emerin–lamin A/C interaction was impaired in each mutant cell line. Mutant cells exhibited enhanced cell proliferation, collagen-dependent adhesion, larger numbers of filopodia and smaller cell spread size, compared with control cells. Furthermore, cell migration, speed and polarization were elevated. Mutant cells also showed an enhanced ability to contract collagen gels at early time points, compared with control cells. Phosphotyrosine measurements during cell spreading indicated an initial temporal lag in ERK1/2 activation in our mutant cells, followed by hyper-activation of ERK1/2 at 2 h post cell attachment. Deregulated ERK1/2 activation is linked with cardiomyopathy, cell spreading and proliferation defects. We conclude that a functional emerin–lamin A/C complex is required for cell spreading and proliferation, possibly acting through ERK1/2 signalling.

Distinct and novel SLC26A4/Pendrin mutations in Chinese and U.S. patients with nonsyndromic hearing loss

Mutations of the human SLC26A4/PDS gene constitute the most common cause of syndromic and nonsyndromic hearing loss. Definition of the SLC26A4 mutation spectrum among different populations with sensorineural hearing loss is important for development of optimal genetic screening services for congenital hearing impairment. We screened for SLC26A4 mutations among Chinese and U.S. subjects with hearing loss, using denaturing HPLC (DHPLC) and direct DNA sequencing. Fifty-two of 55 Chinese subjects with deafness accompanied by enlargement of the vestibular aqueduct (EVA) exhibited at least one mutant SLC26A4 allele, whereas SLC26A4 mutations were found in only 2 of 116 deaf Chinese patients without EVA. The spectrum of SLC26A4 mutations differed among Chinese and U.S. subjects and included 10 previously unreported SLC26A4 variants: 4 in the Chinese population (p.E303Q, p.X329, p.X467, p.X573) and 6 in the U.S. population (p.V250A, p.D266N, p.F354S, p.D697A, p.K715N, p.E737D). Among the seven novel in-frame missense mutations, five encoded SLC26A4 proteins with substantially reduced Cl(-)/anion exchange activity as expressed and measured in Xenopus oocytes, but four of these were sufficiently active to allow study of anion selectivity. The only mutant polypeptide exhibiting complete loss of anion exchange function, p.E303Q, was expressed at or near the oocyte surface at near-wild-type levels. Two variants, p.F354S and p.E737D, displayed selective reduction in relative rate of Cl(-)/HCO(3)(-) exchange compared with similarly measured rates of Cl(-)/Cl(-) and Cl(-)/I(-) exchange. Our data show that mutation analysis of the SLC26A4 gene is of high diagnostic yield among subjects with deafness and bilateral EVA in both China and the U.S. However, the pathogenicity of monoallelic SLC26A4 gene variants in patients with hearing loss remains unclear in many instances.

Expression of Mutant p53 Proteins Implicates a Lineage Relationship between Neural Stem Cells and Malignant Astrocytic Glioma in a Murine Model

Recent studies have identified genes and core pathways that are altered in human glioblastoma. However, the mechanisms by which alterations of these glioblastoma genes singly and cooperatively transform brain cells remain poorly understood. Further, the cell of origin of glioblastoma is largely elusive. By targeting a p53 in-frame deletion mutation to the brain, we show that p53 deficiency provides no significant growth advantage to adult brain cells, but appears to induce pleiotropic accumulation of cooperative oncogenic alterations driving gliomagenesis. Our data show that accumulation of a detectable level of mutant p53 proteins occurs first in neural stem cells in the subventricular zone (SVZ) and that subsequent expansion of mutant p53-expressing Olig2(+) transit-amplifying progenitor-like cells in the SVZ-associated areas initiates glioma formation.