Allele-specific Approach Research Articles

Advances and challenges in RAS signaling targeted therapy in leukemia.

RAS mutations are prevalent in leukemia, including mutations at G12, G13, T58, Q61, K117, and A146. These mutations are often crucial for tumor initiation, maintenance, and recurrence. While much is known about RAS function in the last 40 years, there is a substantial knowledge gap concerning the mutation-specific biological activities of RAS in cancer and the approaches needed to target specific RAS mutants effectively. The recent approval of KRASG12C inhibitors, adagrasib and sotorasib, has validated KRAS as a direct therapeutic target and demonstrated the feasibility of selectively targeting specific RAS mutants. Nevertheless, KRASG12C remains the only RAS mutant successfully targeted with FDA approved inhibitors for cancer treatment in patients, limiting its applicability for other oncogenic RAS mutants, such as G12D in leukemia. Despite these challenges, new approaches have generated optimism about targeting specific RAS mutations in an allele-dependent manner for cancer therapy, supporting by compelling biochemical and structural evidence, which inspires further exploration of RAS allele-specific vulnerabilities. This review will discuss recent advances and challenges in the development of therapies targeting RAS signaling, highlight emerging therapeutic strategies, and emphasize the importance of allele-specific approaches for leukemia treatment.

Comparison of Gene-Editing Approaches for Severe Congenital Neutropenia-Causing Mutations in the ELANE Gene.

Safety considerations for gene therapies of inherited preleukemia syndromes, including severe congenital neutropenia (CN), are paramount. We compared several strategies for CRISPR/Cas9 gene editing of autosomal-dominant ELANE mutations in CD34+ cells from two CN patients head-to-head. We tested universal and allele-specific ELANE knockout, ELANE mutation correction by homology-directed repair (HDR) with AAV6, and allele-specific HDR with ssODN. All strategies were not toxic, had at least 30% editing, and rescued granulopoiesis in vitro. In contrast to published data, allele-specific indels in the last exon of ELANE also restored granulopoiesis. Moreover, by implementing patient-derived induced pluripotent stem cells for GUIDE-Seq off-target analysis, we established a clinically relevant "personalized" assessment of off-target activity of gene editing on the background of the patient's genome. We found that allele-specific approaches had the most favorable off-target profiles. Taken together, a well-defined head-to-head comparison pipeline for selecting the appropriate gene therapy is essential for diseases, with several gene editing strategies available.

Survey of target site mutations linked with insecticide resistance in Italian populations of Aphis gossypii.

Aphis gossypii is a worldwide agricultural pest that causes high levels of economic losses by feeding and transmitting virus diseases. It is usually controlled by chemical insecticides, but this could lead to the selection of resistant populations. Several single nucleotide polymorphisms (SNPs) have been identified associated with insecticide resistance. Monitoring activities to detect the presence of such mutations in field populations can have an important role in insect pest management but, currently, no information on Italian strains is available. The presence of target site mutations conferring resistance to different insecticides was analysed in Italian field collected populations of A. gossypii with an allele specific approach (QSGG, Qualitative Sybr-Green Genotyping). Primers were designed to detect mutations in genes coding acetylcholinesterase (S431F), nicotinic acetylcholine receptor (R81T) and voltage-gated sodium channel (M918L and L1014F). S431F was widespread but with high variability across populations. R81T was detected for the first time in Italy but only in two populations. The L1014F mutation (kdr) was not found, while in the samples showing the M918L two different nucleotidic substitutions were detected. Mutant allele frequencies were, respectively, 0.70 (S431), 0.31 (M918) and 0.02 (R81). Further analysis on the voltage-gated sodium channel gene showed the presence of eight haplotypes and one non-synonymous mutation in the gene coding region. Multiple target-site mutations were detected within Italian populations. The combinations of genotypes observed in certain locations could affect negatively the control of this pest. Preliminary insights on the genetic structure in the Italian populations of A. gossypii were acquired. © 2024 Society of Chemical Industry.

Enhancing Myeloid Cancer Genomic Profiling through Comprehensive Whole Genome Sequencing Using the Isabl Gxt Heme Analytical Platform

Introduction: Genomic profiling in myeloid cancers is vital for disease classification, risk stratification, and treatment decisions. Whole genome sequencing (WGS) captures a complete range of biomarkers, overcoming conventional diagnostic constraints of multiple assays (Duncavage et al, 2021). Duncavage et al's seminal work demonstrated WGS as an alternative and sensitive approach to cytogenetics. Due to emerging evidence, NCCN integrated WGS in their AML testing guidelines (v2.2023). Given the importance of WGS in myeloid cancers, we developed Isabl GxT Heme, an analytical platform, that uses unmatched WGS data as input, assesses all mutation types across the entire genome (structural variants (SVs) and copy number variations (CNVs), small mutations) and delivers an automated report of clinically relevant findings in accordance to latest clinical guidelines. Here we use the Duncavage dataset to analytically validate this WGS approach for the Isabl platform. Methods:Data for 263 samples from Duncavage et al were processed using Isabl GxT Heme analytical framework. The cohort included 175 AML, 81 myelodysplastic syndrome (MDS), and 7 other myeloid malignancies, with a median coverage of 50x (19x - 90x). 2 samples were excluded due to low coverage (<20x). Sensitivity analysis for Isabl GxT Heme and ChromoSeq (reference test; Duncavage et al., 2021) involved comparing against clinical panel sequencing for single nucleotide variations (SNVs) and indels, and against cytogenetics for SVs and CNVs (restricted to clonal events). Results:Isabl GxT analytical workflow returned results within one day from FASTQ generation (<19 hours). Isabl GxT Heme and the reference test detected 100% of recurrent SVs (41/41) and clonal CNVs (67/67) that were reported by cytogenetics. Isabl GxT Heme sensitivity for SNVs and indels was 93% (219/236) and 93% (109/117) for all events called by panel sequencing. Sensitivities for the reference test were 85% and 92%, respectively. A total of 850 calls from 261 samples from the reference test were evaluated next (41 SVs, 205 SNVs, 113 Indels, and 491 CNVs). Isabl GxT Heme identified 100% of SVs, while sensitivity for SNVs was 99.5% (204), and that for indels was 98% (111). Isabl GxT Heme called 97% (475) of CNVs with clonality >20%. Our sensitivity in calling FLT3 internal tandem duplication (ITD) as compared to polymerase-chain-reaction (PCR)-based assay was 84% (27/32) as compared to the reference test at 87% (28/32). Of the 46 genes from the latest guidelines for myeloid disease classification and risk stratification (guideline genes; WHO-2022, IPSSM-2022), 12 were not reported by the targeted analytical approach of the reference test and panel sequencing. A total of 32 guideline related small mutations in 29 samples (11%) were called by Isabl GxT Heme alone . In 13/29 samples, the called events were deemed oncogenic/likely oncogenic by OncoKB (Chakravarty et al. 2017). Isabl GxT Heme's allele-specific approach detects copy-neutral loss of heterozygosity (cnLOH), identifying 7 samples (3%) with bi-allelic TP53 loss due to a small mutation and cnLOH. Additionally, 9 focal CNVs (<5Mbp) were detected in 6 samples (2%). Partial tandem duplication eventsinKMT2A(MLL PTD) in 4 AML and 2 MDS samples were uniquely reported by Isabl GxT Heme. 9 SVs were reported affecting guideline genes (Table 1) . Cytogenetics and panel sequencing information was available for 94 samples in this cohort, enabling an assessment of the validity of Isabl GxT Heme. Additional myeloid-relevant driver events not detected by panel sequencing/cytogenetics were found in 27% of patients by Isabl GxT Heme and 12% by the reference test. Isabl GxT Heme called events in 30% of patients as compared to the reference test (Fig 1). All events missed by Isabl GxT Heme were small mutations detected by panel sequencing of which 69% were absent in the aligned BAM file (11/16) and 31% (5/16) had a median variant allele fraction of 5%. Conclusion:Isabl GxT Heme represents a myeloid cancer profiling solution that reports all clinically relevant biomarkers in AML and MDS inclusive of TP53 allelic state, MLL PTD, disease defining and risk stratifying biomarkers across mutation types. Isabl GxT Heme had comparable sensitivity to the reference test and additionally detected myeloid-relevant driver events in 27% of patients. This work demonstrates the validity and added benefit of Isabl GxT Heme in characterizing myeloid cancers.

X chromosome inactivation in the human placenta is patchy and distinct from adult tissues.

SummaryIn humans, one of the X chromosomes in genetic females is inactivated by a process called X chromosome inactivation (XCI). Variation in XCI across the placenta may contribute to observed sex differences and variability in pregnancy outcomes. However, XCI has predominantly been studied in human adult tissues. Here, we sequenced and analyzed DNA and RNA from two locations from 30 full-term pregnancies. Implementing an allele-specific approach to examine XCI, we report evidence that XCI in the human placenta is patchy, with large patches of either maternal or paternal X chromosomes inactivated. Further, using similar measurements, we show that this is in contrast to adult tissues, which generally exhibit mosaic X inactivation, where bulk samples exhibit both maternal and paternal X chromosome expression. Further, by comparing skewed samples in placenta and adult tissues, we identify genes that are uniquely inactivated or expressed in the placenta compared with adult tissues, highlighting the need for tissue-specific maps of XCI.

Abstract 84: Characterization of oncogenic KRAS A59 alleles and their cooperation with the MAPK signaling pathway

Abstract The Kirsten rat sarcoma (KRAS) proto-oncogene regulates signaling pathways that control proliferation (e.g. MAPK pathway), metabolism and survival (e.g. AKT pathway), and apoptosis (e.g. Hippo pathway). Mutations in KRAS are found in 40% of colorectal cancers (CRC). However, there are no FDA-approved drugs for CRC that target this oncogene. One reason why developing drugs against KRAS is difficult is because a number of different oncogenic alleles have been identified. Thus, an allele-specific approach to target KRAS is necessary. In turn, this approach requires a detailed understanding of their structure and function to drive cell transformation and tissue dysregulation. Our goal is to characterize the function of KRAS A59T in CRC. These alleles are interesting for a number of reasons. First, A59T discriminates retroviral homologues of mammalian RAS from their cellular counterparts. Second, unlike other oncogenic alleles, the KRAS A59T protein undergoes autophosphorylation, the biological function of which is unknown. Third, patient data show that A59 alleles of KRAS frequently co-occur with other genetic alterations in the MAPK signaling pathway, which is uncommon for other KRAS alleles. We hypothesize that KRAS autophosphorylation promotes CRC in a manner that is independent, but cooperative, with the MAPK signaling pathway. To test this hypothesis, we first characterized the function of KRAS A59T and its phosphomimetic A59E using in silico and biochemical techniques, along with classic assays of cell transformation. Next, we generated a new genetically engineered mouse model, K-Ras+/LSL-A59E, in order to test how KRAS autophosphorylation cooperates with other oncogenic mechanisms of MAPK pathway activation to alter colon homeostasis and model colon tumorigenesis. Consistent with our hypothesis, KRAS autophosphorylation and A59E inhibit KRAS-effector interactions that activate the MAPK, AKT, and Hippo signaling pathways. How this change in function alters the ability of K-RasA59E, versus K-RasG12D, to promote hyperproliferation and tumor development in combination with N-RasQ61K and Apc loss in the mouse colon, is currently underway. Ultimately, these studies bring us closer to discovering allele-specific vulnerabilities of KRAS and provide a means to test less understood aspects of oncogenic KRAS signaling. Citation Format: Christian W. Johnson, Elizabeth M. Terrell, Christin E. Burd, Deborah Morrison, Kevin M. Haigis. Characterization of oncogenic KRAS A59 alleles and their cooperation with the MAPK signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 84.

Simple Nested Allele-Specific approach with penultimate mismatch for precise species and sex identification of tiger and leopard.

Accurate species and sex identification of non-invasive and forensic samples of the tiger and leopard is still confusing when using the allele-specific methods. We designed allele-specific methods with penultimate nucleotide mismatch in a nested manner for the exact identification and double-checking of forensic samples. The mismatch design is a novel concept in species and sex identification, making the allele-specific targeting precise. We developed three sets of markers, a 365bp outer and a 98bp inner marker for nested tiger species identification assay, 136bp leopard specific marker, and carnivore sex identification markers. We validated the method with tissue/blood forensic samples of various felids and herbivorous available in our lab and on known fecal samples from Vandalur Zoo. We also collected 37 scat samples at diverse stages of deterioration from the Mudumalai Tiger Reserve, Tamil Nadu, India. The 365bp targeted markers resulted in 70.2% (n = 22; 22/37) amplification success, while the 98bp FAM-labelled marker amplified 89% (n=33; 33/37) scat samples independently. The 136bp leopard markers answered four scat samples (11%) unrequited by the tiger specific markers. We evaluated species and the sex identification with these markers in another 190 non-invasive samples provided by the Mudumalai Tiger Reserve authorities. Among which 56.3% (n= 107) of samples were recognized as tiger (64 male and 43 female) and 38.9% (n= 74) as leopard (41 male and 33 female). The method supersedes any other previous methods in this regard by its high accuracy and simplicity.

A Comprehensive Haplotype-Targeting Strategy for Allele-Specific HTT Suppression in Huntington Disease

Huntington disease (HD) is a fatal neurodegenerative disorder caused by a gain-of-function mutation in HTT. Suppression of mutant HTT has emerged as a leading therapeutic strategy for HD, with allele-selective approaches targeting HTT SNPs now in clinical trials. Haplotypes associated with the HD mutation (A1, A2, A3a) represent panels of allele-specific gene silencing targets for efficient treatment of individuals with HD of Northern European and indigenous South American ancestry. Here we extend comprehensive haplotype analysis of the HD mutation to key populations of Southern European, South Asian, Middle Eastern, and admixed African ancestry. In each of these populations, the HD mutation occurs predominantly on the A2 HTT haplotype. Analysis of HD haplotypes across all affected population groups enables rational selection of candidate target SNPs for development of allele-selective gene silencing therapeutics worldwide. Targeting SNPs on the A1 and A2 haplotypes in parallel is essential to achieve treatment of the most HD-affected subjects in populations where HD is most prevalent. Current allele-specific approaches will leave a majority of individuals with HD untreated in populations where the HD mutation occurs most frequently on the A2 haplotype. We further demonstrate preclinical development of potent and selective ASOs targeting SNPs on the A2 HTT haplotype, representing an allele-specific treatment strategy for these individuals. On the basis of comprehensive haplotype analysis, we show the maximum proportion of HD-affected subjects that may be treated with three or four allele targets in different populations worldwide, informing current allele-specific HTT silencing strategies.

Sequence-Level Analysis of the Major European Huntington Disease Haplotype

Huntington disease (HD) reflects the dominant consequences of a CAG-repeat expansion in HTT. Analysis of common SNP-based haplotypes has revealed that most European HD subjects have distinguishable HTT haplotypes on their normal and disease chromosomes and that ∼50% of the latter share the same major HD haplotype. We reasoned that sequence-level investigation of this founder haplotype could provide significant insights into the history of HD and valuable information for gene-targeting approaches. Consequently, we performed whole-genome sequencing of HD and control subjects from four independent families in whom the major European HD haplotype segregates with the disease. Analysis of the full-sequence-based HTT haplotype indicated that these four families share a common ancestor sufficiently distant to have permitted the accumulation of family-specific variants. Confirmation of new CAG-expansion mutations on this haplotype suggests that unlike most founders of human disease, the common ancestor of HD-affected families with the major haplotype most likely did not have HD. Further, availability of the full sequence data validated the use of SNP imputation to predict the optimal variants for capturing heterozygosity in personalized allele-specific gene-silencing approaches. As few as ten SNPs are capable of revealing heterozygosity in more than 97% of European HD subjects. Extension of allele-specific silencing strategies to the few remaining homozygous individuals is likely to be achievable through additional known SNPs and discovery of private variants by complete sequencing of HTT. These data suggest that the current development of gene-based targeting for HD could be extended to personalized allele-specific approaches in essentially all HD individuals of European ancestry.

Genome-wide allele-specific analysis: insights into regulatory variation

Functional genomics is rapidly progressing towards the elucidation of elements that are crucial for the cis-regulatory control of gene expression, and population-based studies of disease and gene expression traits are yielding widespread evidence of the influence of non-coding variants on trait variance. Recently, genome-wide allele-specific approaches that harness high-throughput sequencing technology have started to allow direct evaluation of how these cis-regulatory polymorphisms control gene expression and affect chromatin states. The emerging data is providing exciting opportunities for comprehensive characterization of the allele-specific events that govern human gene regulation.

Allele-specific silencing of mutant huntingtin and ataxin-3 genes by targeting expanded CAG repeats in mRNAs

Many neurological disorders are caused by expanded trinucleotide repeats1, including Machado-Joseph Disease (MJD)2 and Huntington Disease (HD)3. MJD and HD are caused by expanded CAG repeats within the ataxin-3 (ATXN3) and huntingtin (HTT) genes. Inhibiting expression of ATXN3 or HTT are promising therapeutic strategies, but indiscriminant inhibition of wild-type and mutant alleles may lead to toxicity. We hypothesized that expanded triplet repeat mRNA might be preferentially recognized by complementary oligomers. We observe selective inhibition of mutant ataxin-3 and HTT protein expression by peptide nucleic acid (PNA) and locked nucleic acid (LNA) oligomers targeting CAG repeats. Duplex RNAs were less selective, suggesting an advantage for single-stranded oligomers. Inhibiting mutant HTT expression protected cultured striatal neurons from an HD mouse model against glutamate-induced toxicity. Antisense oligomers that discriminate between wild-type and mutant genes on the basis of repeat length offer new options for treating MJD, HD, and other hereditary diseases.

Kinetics of BCR-ABL Mutant Clones Determines Resistance in CML on Second Generation TKI Treatment.

BCR-ABL kinase domain (KD) mutations are the major mechanism of acquired imatinib resistance in patients (pts) with chronic myeloid leukemia (CML). Second generation tyrosine kinase inhibitors (TKI) are effective against most imatinib resistance mutations but treatment can be hampered by the emergence of secondary drug-resistant clones over time. Furthermore, individual KD mutations differ according to their in vitro transforming potency and tyrosine kinase activity which in vivo upon presence of multiple mutations may result in competition between different clones. Using a novel sensitive and quantitative monitoring approach we systematically investigated the kinetics of drug-resistant mutants on second generation TKI in order to identify patterns of dynamics and to understand mechanisms of polyclonal drug resistance. Fourty CML pts (24 m; median age 64 years, range 39–74) with resistance to imatinib in chronic phase (n=31), accelerated phase (n=7), or blast crisis (n=2) were treated with dasatinib (D, n=20) or nilotinib (N, n=20). Peripheral blood samples taken at baseline and after 3, 6, 9, and 12 months on second generation TKI therapy were subjected to standard genotyping performed by D-HPLC/sequencing and two high-sensitive allele-specific approaches (ligation PCR [L-PCR] and amplification refractory mutation system PCR [ARMS-PCR]) for a panel of 13 key mutations: G250E, Q252H, Y253F/H, E255K/V, V299L, T315I, F311I, F317L, M351T, E355G, F359V. All mutational findings obtained by at least two methods were subjected to quantitative monitoring of BCR-ABLmutant/GUS by ARMS-PCR allowing (i) a dynamical detection range of mutant BCR-ABL over 3 to 4 orders of magnitude and (ii) quantification of the mutant cell subset towards BCR-ABL/GUS ratio. We identified a total of 53 mutated clones in 28 imatinib resistant subjects, of which 46 were assessed quantitatively over time. The following patterns of kinetics were observed: I. A parallel decrease (>2 orders of magnitude) of BCR-ABLmutant/GUS and BCR-ABL/GUS ratio without further mutations emerging (monoclonal resistance, good molecular response, n=11). II. Persistence of a single mutated clone (change <0.5 orders of magnitude) upon treatment combined with a lack of molecular response indicating inadequate treatment (n=6). III. A divergent dynamics - stable, high BCR-ABL/GUS ratios paralleled by decrease of a primary mutation (n=11) - which in the further course is replaced by a secondary (n=11) or even a tertiary (n=4) clone (suboptimal response in all cases, polyclonal resistance). Identification of group III subjects was already possible after three months of treatment (in average 5 months before mutations appeared by sequencing). In four group III subjects, rapidly evolving, secondary clones [F317L (D)/T315I (N)] were replaced by a tertiary clone [T315I (n=2), V299L (n=1) or E255K (n=1)] in the further treatment course. Such a deselection of T315I upon emergence of other drug-resistant clones does not accommodate data derived from in vitro characterization of mutated clones where T315I always heads the list of resistant mutations towards TKI. Our data indicate that co-existing, drug-resistant clones may interfere according to mechanisms which are still poorly understood. Using systematic - in vivo - quantitative analysis of drug-resistant cell subsets we identified clonal interference as a co-factor influencing selective patterns of drug resistance. Understanding the mechanism behind such a phenomenon could eventually improve therapeutic options used for relapsing patients harboring multiple drug-resistant clones inclusive pan-resistant variants like the T315I.

A New Subtype of T-Cell Acute Leukemia in Very Young Children Is Defined by a Translocation Targeting the C-MYB Oncogene, and a Specific Gene Expression Signature.

Cytogenetics and expression studies have pointed out an increasing number of oncogenes in T-cell acute lymphoblastic leukemias (T-ALL), demonstrating the complexity of T-ALL oncogenesis and the requirement for a network of cooperative genomic events. Here, we identified two types of genomic alterations involving the C-MYB locus at 6q23 in human T-ALL, both associated with transcriptional deregulation. First, using a TCRB-based FISH screening, we found a new reciprocal translocation, t(6;7)(q23;q34), that juxtaposed the TCRB and C-MYB loci (n=6 cases). Breakpoints were fully characterized at the molecular level in 3 cases. Second, a genome wide copy-number analysis by array-CGH (customized 4K BAC/PAC arrays) identified short cryptic duplications which always include the C-MYB gene (MYBdup, n=11 out of 80 primary pediatric and adult T-ALL, 14%). Somatic origin of the genomic gain was demonstrated using paired leukemic and remission samples. The minimal region of gain was mapped to 230 Kb using 244K oligonucleotide a-CGH, and fiber-FISH demonstrated tandem duplication. Interestingly, these genomic events are reminiscent of the frequent myb retroviral insertions in murine leukemia. Expression analysis by RQ-PCR and microarrays data showed stronger C-MYB expression in the MYB-rearranged cases compared to other T-ALLs and normal controls (thymus, BM, and PBL). Moreover, allele-specific approaches showed a dramatically skewed allele expression in the TCRB-MYB cases, suggesting that the translocation-driven deregulated expression overcomes a cellular attempt to downregulate C-MYB. Considering that the C-MYB transcription factor has been involved at several key steps throughout normal thymic differentiation, these data strongly suggest that deregulation of C-MYB due to genomic events is oncogenic in T-ALL. Integrated analysis of clinical, genomic, and large-scale gene expression data showed that the MYB translocation defines a new T-ALL subtype. Strikingly, 5 out of 6 patients with the translocation had a very young age for T-cell leukemia (1.1, 1.3, 1.8, 2.5, and 2.9 year-old; median 2.2 versus 9.4 year-old for 355 pediatric T-ALL in the FRALLE 93/2000 pediatric trials, p<10−4). Gene expression profiling showed that these cases cluster in a new branch distinct from other subtypes and are characterized by a specific cell cycle/mitosis signature. By contrast, the MYBdup alteration was associated to previously defined T-ALL subtypes (TAL, TLX1/HOX11, TLX3/HOX11L2, MLL, CALM-AF10, and immature cases), suggesting an additional oncogenic event. NOTCH1 activating mutations and p16/CDKN2A/ARF deletions were found in both the TCRB-MYB and MYBdup cases, according to multi-step T-cell oncogenesis. This work is the first clear demonstration of the recurrent involvement of the C-MYB locus in a human neoplasia. Moreover the t(6;7) translocation defines a new T-ALL subtype associated with very young age for T-cell leukemia and a distinct biology.

The C-MYB locus is involved in chromosomal translocation and genomic duplications in human T-cell acute leukemia (T-ALL), the translocation defining a new T-ALL subtype in very young children

AbstractThe C-Myb transcription factor is essential for hematopoiesis, including in the T-cell lineage. The C-Myb locus is a common site of retroviral insertional mutagenesis, however no recurrent genomic involvement has been reported in human malignancies. Here, we identified 2 types of genomic alterations involving the C-MYB locus at 6q23 in human T-cell acute leukemia (T-ALL). First, we found a reciprocal translocation, t(6;7)(q23;q34), that juxtaposed the TCRB and C-MYB loci (n = 6 cases). Second, a genome-wide copy-number analysis by array-based comparative genomic hybridization (array-CGH) identified short somatic duplications that include C-MYB (MYBdup, n = 13 cases of 84 T-ALL, 15%). Expression analysis, including allele-specific approaches, showed stronger C-MYB expression in the MYB-rearranged cases compared with other T-ALLs, and a dramatically skewed C-MYB allele expression in the TCRB-MYB cases, which suggests that a translocation-driven deregulated expression may overcome a cellular attempt to down-regulate C-MYB. Strikingly, profiling of the T-ALLs by clinical, genomic, and large-scale gene expression analyses shows that the TCRB-MYB translocation defines a new T-ALL subtype associated with a very young age for T-cell leukemia (median, 2.2 years) and with a proliferation/mitosis expression signature. By contrast, the MYBdup alteration was associated with the previously defined T-ALL subtypes.

Allele-specific RNA interference for neurological disease

Suppressing the expression of toxic genes through RNAi holds great promise for the treatment of human disease. Allele-specific approaches have now been used to silence dominant toxic genes implicated in several neurological disorders. Here, we review strategies used to achieve allele-specific silencing in light of recent developments in the field of RNAi biology. In particular, new insights into siRNA and miRNA processing may be used to improve efficiency and specificity of RNAi therapy. We further discuss steps that can be taken to maximize the therapeutic benefits of this powerful technology.