Nonsense Changes Research Articles

Loss-of-function variants in ERF are associated with a Noonan syndrome-like phenotype with or without craniosynostosis.

Pathogenic, largely truncating variants in the ETS2 repressor factor (ERF) gene, encoding a transcriptional regulator negatively controlling RAS-MAPK signaling, have been associated with syndromic craniosynostosis involving various cranial sutures and Chitayat syndrome, an ultrarare condition with respiratory distress, skeletal anomalies, and facial dysmorphism. Recently, a single patient with craniosynostosis and a phenotype resembling Noonan syndrome (NS), the most common disorder among the RASopathies, was reported to carry a de novo loss-of-function variant in ERF. Here, we clinically profile 26 individuals from 15 unrelated families carrying different germline heterozygous variants in ERF and showing a phenotype reminiscent of NS. The majority of subjects presented with a variable degree of global developmental and/or language delay. Their shared facial features included absolute/relative macrocephaly, high forehead, hypertelorism, palpebral ptosis, wide nasal bridge, and low-set/posteriorly angulated ears. Stature was below the 3rd centile in two-third of the individuals, while no subject showed typical NS cardiac involvement. Notably, craniosynostosis was documented only in three unrelated individuals, while a dolichocephalic aspect of the skull in absence of any other evidence supporting a premature closing of sutures was observed in other 10 subjects. Unilateral Wilms tumor was diagnosed in one individual. Most cases were familial, indicating an overall low impact on fitness. Variants were nonsense and frameshift changes, supporting ERF haploinsufficiency. These findings provide evidence that heterozygous loss-of-function variants in ERF cause a "RASopathy" resembling NS with or without craniosynostosis, and allow a first dissection of the molecular circuits contributing to MAPK signaling pleiotropy.

Turkish population-based screening for first identified changes of BRCA1 and BRCA2 genes in breast and/or ovarian cancer patients

IntroductionIt is known that BRCA1 and BRCA2 genes’ mutation carriers are predisposed to breast and ovarian cancers and other organ cancers such as prostate, colon and cervix. In the previous study performed at X University, all coding exons of both genes were screened by denaturing gradient gel electrophoresis (DGGE). In addition to various nonsense, missense mutations, polymorphisms and intronic region changes, seven novel missense mutations, including H513L, H816P and S1517Y in BRCA1 and S326R, G258P, E2903K and N2742S in BRCA2, had been identified.MethodsTo determine whether these unclassified variants are pathogenic, DNA samples of 150 healthy individuals without a known cancer history in the family were screened in this study for these seven novel missense mutations. These DNA samples were recruited from archives of previous polymorphism studies. PCR performed DNA amplifications, and denaturing high-performance liquid chromatography (DHPLC) techniques did mutation screenings.ResultsPeak patterns suggestive of a change in DNA fragments were considered for sequencing analyses. Analyses revealed that none of the 150 DNA samples had any change in the seven screened fragments. As a result, it is assumed that these seven mutations might be novel pathogenic mutations described in the Turkish population.ConclusionIn conclusion, these carriers must be informed about the mutation and given appropriate genetic counseling by their physicians. In addition, genetic testing must be offered to high-risk individuals (men/women) in the family so that it would be possible for other family members to have genetic counseling and contribute to disease prevention. On the other hand, these findings would contribute to current literature with novel results and shed light on future research.

The pathogenic roles of the p.R130S prestin variant in DFNB61hearing loss.

DFNB61 is a recessively inherited nonsyndromic hearing loss caused by mutations in SLC26A5, the gene that encodes the voltage-driven motor protein, prestin. Prestin is abundantly expressed in the auditory outer hair cells that mediate cochlear amplification. Two DFNB61-associated SLC26A5 variants, p.W70X and p.R130S, were identified in patients who are compound heterozygous for these nonsense and missense changes (SLC26A5W70X/R130S ). Our recent study showed that mice homozygous for p.R130S (Slc26a5R130S/R130S ) suffer from hearing loss that is ascribed to significantly reduced motor kinetics of prestin. Given that W70X-prestin is nonfunctional, compound heterozygous Slc26a5R130S/- mice were used as a model for human SLC26A5W70X/R130S . By examining the pathophysiological consequences of p.R130S prestin when it is the sole allele for prestin protein production, we determined that this missense change results in progressive outer hair cell loss in addition to its effects on prestin's motor action. Thus, this study defines the pathogenic roles of p.R130S prestin and identifies a limited time window for potential clinical intervention. KEY POINTS: The voltage-driven motor protein, prestin, is encoded by SLC26A5 and expressed abundantly in cochlear outer hair cells (OHCs). The importance of prestin for normal hearing was demonstrated in mice lacking prestin; however, none of the specific SLC26A5 variants identified to date in human patients has been experimentally demonstrated to be pathogenic. In this study we used both cell lines and a mouse model to define the pathogenic role of compound heterozygous p.W70X (c.209G>A) and p.R130S (c.390A>C) SLC26A5 variants identified in patients with moderate to profound hearing loss. As in patients, mice carrying one copy of p.R130S Slc26a5showed OHC dysfunction and progressive degeneration, which results in congenital progressive hearing loss. This is the first functional study reporting pathogenic SLC26A5 variants and pointing to the presence of a therapeutic time window for potential clinical interventions targeting the affected OHCs before they are lost.

Molecular and clinical characteristics of POLE/POLD1 alterations among patients with colorectal cancer.

184 Background: POLE and POLD1 alterations (DNA polymerase ε and DNA polymerase δ) are key biomarkers of immune response for patients with MSS colorectal cancers (CRC). The clinical and molecular characteristics of POLE/POLD1 alterations are not yet well defined across CRCs. In this study, we investigated the clinical/molecular features of POLE and POLD1 alterations and its association with MSI-H CRC. Methods: All the patients and mutation data were selected from the cBioPortal database (https://www.cbioportal.org). All nonsynonymous mutations including missense, frameshift, nonsense, nonstop, splice site, and translation start site changes of POLE/POLD1 were considered. 21 studies were used in the analysis. 7179 data samples were obtained from 7179 patients. Tumors included were from all stages of CRCs. POLE/POLD1 mutations were from mutation tables, only protein-changing mutations were kept. MSI-H status was based on mutation data (presence of MLH1/PMS2/MSH2/MSH6 mutations). Results: Of 7179 patients with all stage CRCs, 6.1% (439) were found to harbor mutated POLE/POLD1 genes. Among those, 3446 patients had known MSI status and 14.9% (514/3446) had MSI-H disease. Notably, 3.3% patients with POLE/POLD1 mutations were found to have concurrent MSI-H disease (co-existence) and only 1.1% of patients with POLE/POLD1 mutations had MSS CRC. MSI status was unknown for 1.7% of patients with POLE/POLD mutant CRC. The mean age for patients with POLE/POLD1 + with or without MSI-H disease was 67. POLE/POLD mutations were exceedingly more common in colon than rectum regardless of MSI-H status (85% vs 15% overall and 88% vs 12% in MSI-H subgroup). No difference was seen among genders for the distribution of POLE/POLD1 mutations. Conclusions: POLE/POLD1 mutations frequently co-exist with MSI-H disease in CRC. Patients with MSS-CRC harboring POLE/POLD1 mutations represent a smaller subgroup of CRC (~1%). The incidence of POLE/POLD1 somatic mutations was more common among patients with colon cancer than those with rectal cancer.

Investigating Mutations of 288 Genes in a Family with Charcot-Marie-Tooth Disease by the Whole-Exome Sequencing Method in Khuzestan Province

Background: Genetic factors play an important role in the occurrence of many diseases, including skeletal diseases. Skeletal diseases are sometimes caused by a mutation in a gene that passes on to children according to the Mendelian inheritance pattern, and sometimes several genes are involved in this path and cause heterogeneous disease. The whole-exome sequencing (WES) technique can be a powerful tool in diagnosing this disease. Methods: Peripheral blood samples (5 mL) were collected from the individuals in EDTA tubes. After sequencing the patient's genome using the WES technique, the candidate genes were checked using the polymerase chain reaction (PCR) technique and Chromas software to confirm the variant in the family. Results: After studying the genome using the WES method and analyzing the results, mutation NM_001005373.4:c.1985C>T was identified by creating a nonsense change in the LRSAM1 gene. Conclusions: The data obtained by the WES technique can help diagnose the disease faster. In addition, these data can be used as background information in the development of a special panel to diagnose this disorder.

An integrative framework for clinical diagnosis and knowledge discovery from exome sequencing data

Non-silent single nucleotide genetic variants, like nonsense changes and insertion-deletion variants, that affect protein function and length substantially are prevalent and are frequently misclassified. The low sensitivity and specificity of existing variant effect predictors for nonsense and indel variations restrict their use in clinical applications. We propose the Pathogenic Mutation Prediction (PMPred) method to predict the pathogenicity of single nucleotide variations, which impair protein function by prematurely terminating a protein's elongation during its synthesis. The prediction starts by monitoring functional effects (Gene Ontology annotation changes) of the change in sequence, using an existing ensemble machine learning model (UniGOPred). This, in turn, reveals the mutations that significantly deviate functionally from the wild-type sequence. We have identified novel harmful mutations in patient data and present them as motivating case studies. We also show that our method has increased sensitivity and specificity compared to state-of-the-art, especially in single nucleotide variations that produce large functional changes in the final protein. As further validation, we have done a comparative docking study on such a variation that is misclassified by existing methods and, using the altered binding affinities, show how PMPred can correctly predict the pathogenicity when other tools miss it. PMPred is freely accessible as a web service at https://pmpred.kansil.org/, and the related code is available at https://github.com/kansil/PMPred.

Cis-Acting Splicing-Associated Variants Can Redefine the Molecular Signature of Genes Commonly Mutated in Acute Myeloid Leukemia

Introduction: Acute Myeloid Leukemia (AML) is a blood malignancy that occurs as a result of genomic alterations acquired in hematopoietic stem cells (HSCs). Several studies have recognized the importance of these alterations, including chromosomal rearrangements and single nucleotide variations (SNVs), for the classification and risk stratification of patients. However, none of these studies have thoroughly explored the functional impact of these lesions, potentially leading to the oversight or misinterpretation of certain driver mutations. In this study, we focused on the splicing process, as we aim to comprehensively characterize cis-acting splicing-associated variants (SAVs) in a large cohort of AML patients. Methods: We obtained recurrent driver gene variants (n= 3,847) from Table S5 reported by Papaemmanuil et al. and selected unique SNVs (n= 915) for our analysis. Among them, 628 (69%) were missense variants, 232 (25%) were nonsense variants, and 55 (6%) were located within splice sites. To assess their potential impact on splicing, we employed three splicing predictor tools (MaxEntScan, regSNP-splicing, and SpliceAI) and considered variants with two favorable predictions for further functional studies. Firstly, we attempted to locate the SNVs within the exomes of the TCGA-LAML (n= 149) and the BeatAML (n=342) datasets available in the Genomic Data Commons repository. If an SNV was identified, we obtained the RNA sequencing bam file from the corresponding patient's sample and utilized the rest of the cohort as a control for statistical analysis (p<0.05 for significance). In cases where the SNV was not found in these datasets, we employed the pSPL3-based splicing reporter minigene assay to study the variant. Results: The predictors unanimously identified all 55 splice-site affecting variants as true positives and reclassified 20 missense and nonsense variants as likely SAVs. Functional validation experiments confirmed that 11 of these variants (55%) indeed had an impact on splicing. Analysis of RNA sequencing data revealed interesting findings in commonly observed AML hotspots. For instance, in cases of U2AF1 c.470A>G and IDH1 c.394C>A, novel donor splice sites were created at distances of one (p=0.04522) and 34 nucleotides from the mutations (p=0.01878), respectively. Whereas, the TP53 c.395A>G change was found to activate a cryptic acceptor splice site located XX nucleotides away (p=0). Among private AML variants, minigene assays showed that eight variants resulted in aberrant splicing compared to the wild-type. Three variants led to exon skipping due to the loss of canonical donor splice sites ( EZH2 c.363G>A, FLT3 c.2523C>A, and TET2 c.3500G>A), four variants caused the activation of a novel donor splice site ( PTPN11 c.1508G>T, WT1 c.1157C>A, STAG2 c.545T>G, and KDM6A c.1516C>T), and one variant resulted in both exon skipping and the creation of a donor splice site ( RAD21 c.688G>A). Consequently, the predicted impact on the protein varied, with FLT3 and TET2 variants potentially leading to an in-frame deletion of the ATP-binding site and TET2-oxygenase domain, respectively, with the EZH2 variant likely resulting in an expression switch from the large to small isoform. The remaining SNVs were anticipated to trigger Nonsense-Mediated Decay or shorten proteins. Conclusions: This study revealed that approximately 7% of SNVs found in a large group of AML patients have the potential to impact splicing. Notably, 1% of these SNVs had been classified as missense or nonsense changes. These findings provide support for the significant role of mis-splicing in the development of leukemia and emphasize the importance of devising novel approaches to effectively characterize driver mutations.

Exome sequencing identifies genetic variants in anophthalmia and microphthalmia.

Anophthalmia and microphthalmia (A/M) are rare birth defects affecting up to 2 per 10,000 live births. These conditions are manifested by the absence of an eye or reduced eye volumes within the orbit leading to vision loss. Although clinical case series suggest a strong genetic component in A/M, few systematic investigations have been conducted on potential genetic contributions owing to low population prevalence. To overcome this challenge, we utilized DNA samples and data collected as part of the National Birth Defects Prevention Study (NBDPS). The NBDPS employed multi-center ascertainment of infants affected by A/M. We performed exome sequencing on 67 family trios and identified numerous genes affected by rare deleterious nonsense and missense variants in this cohort, including de novo variants. We identified 9 nonsense changes and 86 missense variants that are absent from the reference human population (Genome Aggregation Database), and we suggest that these are high priority candidate genes for A/M. We also performed literature curation, single cell transcriptome comparisons, and molecular pathway analysis on the candidate genes and performed protein structure modeling to determine the potential pathogenic variant consequences on PAX6 in this disease.

Mutational Analysis of Mucopolysaccharidosis in Iranian Patients

: Mucopolysaccharidosis (MPS) is a rare and heterogeneous metabolic disorder with wide phenotypic distributions throughout the world. This study aimed to determine the genetic polymorphisms, contributing to the most common types of MPS in 19 unrelated Iranian patients. The sequence of the coding region and exon‐intron boundaries of the MPS genes were analyzed by Sanger sequencing method. We used the biochemical and clinical characteristics of MPS subjects for genetic analysis. A novel IDUA variant (c.99T>C, p.H33H), a novel nonsense change (c.514C>T, p.R172*) in exon 5 of IDS gene, c.74G > A (p. p.R24H) in SGSH gene, and three variants including (c.607C>T (p.R203*), c.259G>C (p.A87P), and c.683G>A (p.R228Q)) in NAGLU (n-acetyl-alpha-glucosaminidase) were predicted as novel pathogenic mutations. In conclusion, this study broadened genotypic spectrum of Iranian MPS patients, facilitating the definition of disease-associated mutations, which help to provide a more effective approach in MPS carrier detection.

Functional analysis of TLK2 variants and their proximal interactomes implicates impaired kinase activity and chromatin maintenance defects in their pathogenesis

IntroductionThe Tousled-like kinases 1 and 2 (TLK1 and TLK2) are involved in many fundamental processes, including DNA replication, cell cycle checkpoint recovery and chromatin remodelling. Mutations in TLK2 were recently...

Abstract 4715: Characterization of potential co-drivers of pathogenesis in ALK positive ALCL

Abstract ALK positive Anaplastic large cell lymphoma (ALCL) is an aggressive non-Hodgkin lymphoma characterized by expression of the NPM/ALK fusion tyrosine kinase essential for neoplastic cells growth. When resistant or relapsed to front-line chemotherapy, ALK+ ALCL prognosis is very poor. Although ALK+ ALCL cells are mainly driven by the constitutively active NPM/ALK kinase, presence of additional mutations might affect the disease course and response to treatment, leading to different outcomes in different patients. We employed whole exome sequencing to characterize the co-mutational burden in ALK+ ALCL samples at diagnosis. Diagnosis biopsy and matched healthy control DNA samples from 24 ALK+ ALCL patients were sequenced. Somatic variants present at high frequency within the tumor (at least 25% variant allele frequency), which can be viewed as potential co-drivers, were annotated; the identified mutations were weighed using in silico tools to predict their oncogenic potential and their effects on protein function. All identified variants were confirmed by Sanger sequencing. Gene ontology and pathway analyses identified cell adhesion and actin cytoskeleton among the most enriched biological functions. Using the above-mentioned criteria, we found recurrent somatic variants in 7 genes including PPP1R9A, RUNX1T1, FAT4 and RBBP8. Here we report the initial characterization of the role of FAT4 in ALCL cells. FAT4 is a member of the cadherin superfamily with tumor suppressor functions. In our cohort, 3/24 patients (13%) were found to carry FAT4 mutations, including nonsense mutations and missense changes predicted to be deleterious, suggesting a loss of function effect. To characterize the biological consequences of these mutations, we generated FAT4 knockdown ALCL cell lines. FAT4-silenced cells grew faster and showed increased migratory properties compared to parental cells. They also differed from parental cells in terms of morphology, having bigger size and modulated F-actin rearrangement. Tumor suppressing activity of FAT4 has been linked to Wnt/β-catenin pathway; we found that FAT4-silenced cells had decreased levels of serine/threonine phosphorylated β-catenin and higher nuclear localization, which insinuates activation of Wnt/β-catenin signalling and henceforth, increased cellular proliferation. To explore therapeutic implications of FAT4 mutations, we analyzed the sensitivity of FAT4 silenced cells to chemotherapeutic agents. FAT4 knockdown cells were less sensitive to a CHOP combination treatment as compared to parental cells. No difference was seen in terms of sensitivity to crizotinib. Finally, we investigated the effects of RUNX1T1 somatic variants found in our study. Preliminary luciferase assay results showed that the variants significantly increase the transcriptional repression ability of RUNX1T1. Altogether, our data provides information about genes other than NPM-ALK that might play a role in the pathogenesis of ALCL. Citation Format: Luca Mologni, Geeta G. Sharma, Matteo Villa, Mario Mauri, Giulia Arosio, Cosimo Lobello, Hugo Larose, Alessandra Pirola, Silvia Bombelli, Nicoletta Cordani, Luca Massimino, Šárka Pospíšilová, Suzanne D. Turner, Giorgio Inghirami, Fabio Pagni, Rocco Piazza, Roberto Perego, Roberto Chiarle, Carlo Gambacorti-Passerini. Characterization of potential co-drivers of pathogenesis in ALK positive ALCL [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4715.

Genotype-phenotype correlation: Inheritance and variant-type infer pathogenicity in IQSEC2 gene

Pathogenic variants in the IQSEC2 gene including nonsense, frameshift, splice-alterations, deletions, and missense changes have been identified in individuals with X-linked mental retardation. Although highly variable, clinical features may include hypotonia, moderate to severe delayed psychomotor development, intellectual disability, speech deficits, refractory seizures, autistic features, and stereotypical movements. Females with de novo variants have been described with classical features. In contrast, the phenotype in carrier females identified through an affected male may range from asymptomatic to mild intellectual disability. We present male (N = 2) and female (N = 3) probands ascertained via diagnostic exome sequencing with distinct variant types in the IQSEC2 gene encompassing a spectrum of phenotypic severity with patient sex, variant type and inheritance hypothesized to drive disease penetrance and expressivity. All of these patients demonstrated epilepsy, global developmental delays, intellectual disability, and constipation. Our data support that de novo, truncating variants correlate with severe disease in both female and male patients harboring an IQSEC2 alteration. Missense variants in male and female patients may account for a milder disease overall, with more severe symptoms in males than females. We also present the first confirmed case of parental mosaicism, which has implications regarding counseling for recurrence risk. These data further delineate a genotype-phenotype correlation of IQSEC2 variation.

Molecular genetic study of 59 Chinese Oculocutaneous albinism families

Oculocutaneous albinism is an autosomal recessive disorder characterized by either a complete lack of or reduction in melanin biosynthesis in the skin, hair, and eyes. The aim of the present study was to identify the molecular basis for 59 Chinese OCA families. In this study, compound heterozygous or homozygous pathogenic variants were found in 53 families, 4 families possessed only one heterozygous variant, and the pathogenic variants of 2 families remain undiscovered by using Sanger sequencing, whole exome sequencing and multiplex ligation-dependent probe amplification. We have identified a total of 55 variants including 21 novel variants in TYR, OCA2, SLC45A2, SLC24A5, and HPS1. The 21 novel variants include 11 missense changes, 4 nonsense changes, 2 splice site changes, 1 frameshift and 3 gross deletions. Forty-six variants including 14 novel variants were segregated with the phenotype in 37 families. We conducted RT-PCR of the novel splicing site variant (c.399-14G > A) of HPS1 and verified that the variant would result in the inclusion of 12 bp of intronic material in exon 6 of HPS1. The results of platelet whole mount electron microscopy further confirmed the diagnosis of HPS1. These novel variants identified in our study expand the mutational spectrum of the disease, which contributes to prenatal diagnosis and genetic counselling.

Pathomechanisms of ATF6-Associated Cone Photoreceptor Diseases.

Activating transcription factor 6 (ATF6) is a key regulator of the unfolded protein response (UPR). In response to endoplasmic reticulum (ER) stress, ATF6 is transported from the ER to the Golgi apparatus where it is cleaved by intramembrane proteolysis, releasing its cytosolic fragment. The cleaved ATF6 fragment, which is a basic leucine zipper (bZip) transcription factor, translocates to the nucleus and upregulates the expression of ER protein-folding chaperones and enzymes. Mutations in ATF6 cause heritable forms of cone photoreceptor dysfunction diseases. These mutations include missense, nonsense, splice site, and deletion or duplication changes found across the entire ATF6. To date, there are 11 ATF6 mutations reported, and we classified them into three classes based on their functional defects that interrupt distinct steps in the ATF6 signaling pathway.

Novel GPR34 and CCR6 mutation and distinct genetic profiles in MALT lymphomas of different sites.

Mucosa-associated lymphoid tissue (MALT) lymphoma originates from a background of diverse chronic inflammatory disorders at various anatomic sites. The genetics underlying its development, particularly in those associated with autoimmune disorders, is poorly characterized. By whole exome sequencing of 21 cases of MALT lymphomas of the salivary gland and thyroid, we have identified recurrent somatic mutations in 2 G-protein coupled receptors (GPR34 and CCR6) not previously reported in human malignancies, 3 genes (PIK3CD, TET2, TNFRSF14) not previously implicated in MALT lymphoma, and a further 2 genes (TBL1XR1, NOTCH1) recently described in MALT lymphoma. The majority of mutations in GPR34 and CCR6 were nonsense and frameshift changes clustered in the C-terminal cytoplasmic tail, and would result in truncated proteins that lack the phosphorylation motif important for β-arrestin-mediated receptor desensitization and internalization. Screening of these newly identified mutations, together with previously defined genetic changes, revealed distinct mutation profiles in MALT lymphoma of various sites, with those of salivary gland characterized by frequent TBL1XR1 and GPR34 mutations, thyroid by frequent TET2, TNFRSF14 and PIK3CD mutations, and ocular adnexa by frequent TNFAIP3 mutation. Interestingly, in MALT lymphoma of the salivary gland, there was a significant positive association between TBL1XR1 mutation and GPR34 mutation/translocation (P=0.0002). In those of ocular adnexa, TBL1XR1 mutation was mutually exclusive from TNFAIP3 mutation (P=0.049), but significantly associated with IGHV3-23 usage (P=0.03) and PIK3CD mutation (P=0.009). These findings unravel novel insights into the molecular mechanisms of MALT lymphoma and provide further evidence for potential oncogenic co-operation between receptor signaling and genetic changes.

P.26 - COL6A genes transcriptomic by RNAseq and fluidic card tools

Mutations in collagen VI A1, A2, and A3 genes cause collagen VI related myopathies, ranging from the severe Ullrich congenital muscular dystrophy (UCMD) to the milder Bethlem myopathy (BM). COL6A1 gene produces a single transcript. COL6A2 gene is transcribed in multiple alternatively spliced mRNAs that differ in the 5'-untranslated region as well as in the 3'-coding and noncoding sequences. COL6A3 transcription is even more complex with various N-terminal exons subjected to multiple alternative splicing. The pattern of isoforms representation in muscle and their functional role has been fairly investigated. Furthermore, COL6A genes mutations affecting splicing are a frequent cause of UCMD-BM phenotypes (around 20% of cases), with genotype-phenotype correlations often unclear. In these cases, non-sense mediated decay of aberrant transcript represents a frequent event, often hampering the detection of mutated transcripts. We have compared two different approaches, RNAseq and a TaqMan® low-density array for Real-Time transcription analysis, for the ability to profile COL6A genes isoforms and to detected mutated transcripts resulting for splicing mutations. RNA-seq was performed on three COL6A mutated patients, carrying either a small in-frame deletion, a mutation expected to induce NMD (homozygous nonsense change) and a canonical splicing mutation. In parallel, the ability of the fluidic card tool to detect splicing mutations and to profile COL6A genes isoforms was also tested in patients and control muscle samples. The RNA-seq turned out to be the optimal strategy, able to pick-up low quantity COL6A genes mRNAs derived both from physiological alternative splicing and from pathogenic mutations. We constructed a RNA-seq based map of COL6A genes isoforms expression in normal and affected muscles.

Rapid molecular diagnostics of severe primary immunodeficiency determined by using targeted next-generation sequencing

Primary immunodeficiency diseases (PIDDs) are inherited disorders of the immune system. The most severe form, severe combined immunodeficiency (SCID), presents with profound deficiencies of Tcells, Bcells, or both at birth. If not treated promptly, affected patients usually do not live beyond infancy because of infections. Genetic heterogeneity of SCID frequently delays the diagnosis; a specific diagnosis is crucial for life-saving treatment and optimal management. We developed a next-generation sequencing (NGS)-based multigene-targeted panel for SCID and other severe PIDDs requiring rapid therapeutic actions in a clinical laboratory setting. The target gene capture/NGS assay provides an average read depth of approximately 1000×. The deep coverage facilitates simultaneous detection of single nucleotide variants and exonic copy number variants in one comprehensive assessment. Exons with insufficient coverage (<20× read depth) or high sequence homology (pseudogenes) are complemented by amplicon-based sequencing with specific primers to ensure 100% coverage of all targeted regions. Analysis of 20 patient samples with low T-cell receptor excision circle numbers on newborn screening or a positive family history or clinical suspicion of SCID or other severe PIDD identified deleterious mutations in 14 of them. Identified pathogenic variants included both single nucleotide variants and exonic copy number variants, such as hemizygous nonsense, frameshift, and missense changes in IL2RG; compound heterozygous changes in ATM, RAG1, and CIITA; homozygous changes in DCLRE1C and IL7R; and a heterozygous nonsense mutation in CHD7. High-throughput deep sequencing analysis with complete clinical validation greatly increases the diagnostic yield of severe primary immunodeficiency. Establishing a molecular diagnosis enables early immune reconstitution through prompt therapeutic intervention and guides management for improved long-term quality of life.

Quantitative Gait Analysis Using a Motorized Treadmill System Sensitively Detects Motor Abnormalities in Mice Expressing ATPase Defective Spastin.

The hereditary spastic paraplegias (HSPs) are genetic conditions in which there is progressive axonal degeneration in the corticospinal tract. Autosomal dominant mutations, including nonsense, frameshift and missense changes, in the gene encoding the microtubule severing ATPase spastin are the most common cause of HSP in North America and northern Europe. In this study we report quantitative gait analysis using a motorized treadmill system, carried out on mice knocked-in for a disease-associated mutation affecting a critical residue in the Walker A motif of the spastin ATPase domain. At 4 months and at one year of age homozygous mutant mice had a number of abnormal gait parameters, including in stride length and stride duration, compared to heterozygous and wild-type littermates. Gait parameters in heterozygous animals did not differ from wild-type littermates. We conclude that quantitative gait analysis using the DigiGait system sensitively detects motor abnormalities in a hereditary spastic paraplegia model, and would be a useful method for analyzing the effects of pharmacological treatments for HSP.

ASXL1 and ASXL2 Mutations in Childhood AML Are Strongly Associated with t(8;21) but Do Not Independently Impact on Prognosis: A Report from the Children's Oncology Group and NCI/COG Target Initiative

Increasing use of high-throughput sequencing technologies has enabled the identification of novel somatic gene alterations in cancers, with important implications in understanding the mechanisms of transformation and potentially in improving therapeutic decision-making. In contrast to many solid tumors, the mutational spectrum of acute myeloid leukemia (AML) has proven bland, with relatively few recurrent somatic mutations per tumor. This result appears more pronounced in pediatric AML, where even mutation of genes commonly mutated in adult disease, such as IDH1/2 or DNMT3A, appear at much lower frequencies. Alterations of ASXL1, a regulator of the polycomb-repressive complex, have been identified in adults with myelodysplastic syndromes, myeloproliferative syndromes, and AML. Mutations in the related gene family member, ASXL2, were recently associated with t(8;21) AML in adults and children. In this study, we evaluated the prevalence and prognostic impact of mutations of these epigenetic regulator genes in pediatric AML patients treated on COG studies AAML03P1 and AAML0531.ASXL1 and ASXL2 (ASXL1/2) were sequenced from genomic DNA isolated by directed capture of the coding sequences of each gene followed by Illumina paired-end sequencing in a total of 769 diagnostic AML specimens. We identified ASXL1/2 mutations in 44/769 pediatric AML cases (5.7%), with ASXL2 altered more frequently (61% of mutated cases) than ASXL1. Consistent with prior reports, the majority of mutations (80%) were nonsense changes involving exons 11-12 of either gene. ASXL1 and ASXL2 mutations were mutually exclusive, with no case demonstrating concurrent mutation of both genes.In comparison of the clinical covariates of cases with ASXL1/2 mutation against those without, there were no differences in age, gender, or ethnicity; however we found an increased proportion of ASXL1/2 mutations in patients of Asian race, constituting 15% of those with mutated ASXL1/2 vs. 5% of those without (p=0.018). Patients possessing ASXL1/2 mutations tended to present with lower peripheral WBC and marrow blast counts (median 20K vs 30.8K, p=0.067 and 49% vs 70%, p=0.003, respectively).In agreement with recent reports, we identified a striking positive association of ASXL1/2 alterations and t(8;21), which was present in 68% of cases with ASXL1/2 vs 12% of those without (p<0.001). More than 25% (30/113) of patients with t(8;21) had a concomitant ASXL1/2 mutation. We similarly found an inverse association with other major cytogenetic subgroups-inv(16): 2% vs 14%, p=0.024; normal cytogenetics: 5% vs 25%, p<0.001; MLL-rearranged: 2% vs 21%, p=0.003. In the evaluation of concurrent molecular changes, ASXL1/2 mutations were inversely associated with NPM1 mutation, with no ASXL1/2 mutated case having a concurrent NPM1 mutation compared to a 9% NPM1 mutation rate in those without alterations of ASXL1/2 (p=0.041). There were no significant associations with FLT3-ITD, CEBPA or KIT mutation status.We found no differences in the clinical outcomes of patients with ASXL1/2 mutations in comparison to those without. Complete remission rates were similar for both groups at the end of induction courses 1 and 2 (84 vs 75%, p=0.2 and 91 vs 89%, p=1.0, respectively). 5-year EFS and OS was 59 ± 15% vs 50 ± 4% (p=0.305) and 65 ± 15% vs. 66 ± 4% (p=0.851). Although prior reports have suggested the possibility of enhanced relapse risk with ASXL1/2 mutations, the 5-year RR for pediatric patients treated in these trials was similar at 31 ± 16% vs 40 ± 4% (p=0.407). Given the co-occurrence of ASXL1/2 mutations and t(8;21), we also evaluated outcomes of t(8;21) patients (n=113) with or without ASXL1/2 mutations, however no significant differences in 5-year OS, EFS, or RR were observed in this subset analysis.This work demonstrate that mutations of ASXL1 or ASXL2 are highly prevalent in childhood t(8;21) AML. Although there was no effect on treatment outcomes, this concomitance suggests the possibility of discrete cooperating lesions, even within the group of CBF-translocated AML, which may be amenable to specifically-directed therapy DisclosuresNo relevant conflicts of interest to declare.

KLF2 mutation is the most frequent somatic change in splenic marginal zone lymphoma and identifies a subset with distinct genotype.

To characterise the genetics of splenic marginal zone lymphoma (SMZL), we performed whole exome sequencing of 16 cases and identified novel recurrent inactivating mutations in Kruppel-like factor 2 (KLF2), a gene whose deficiency was previously shown to cause splenic marginal zone hyperplasia in mice. KLF2 mutation was found in 40 (42%) of 96 SMZLs, but rarely in other B-cell lymphomas. The majority of KLF2 mutations were frameshift indels or nonsense changes, with missense mutations clustered in the C-terminal zinc finger domains. Functional assays showed that these mutations inactivated the ability of KLF2 to suppress NF-κB activation by TLR, BCR, BAFFR and TNFR signalling. Further extensive investigations revealed common and distinct genetic changes between SMZL with and without KLF2 mutation. IGHV1-2 rearrangement and 7q deletion were primarily seen in SMZL with KLF2 mutation, while MYD88 and TP53 mutations were nearly exclusively found in those without KLF2 mutation. NOTCH2, TRAF3, TNFAIP3 and CARD11 mutations were observed in SMZL both with and without KLF2 mutation. Taken together, KLF2 mutation is the most common genetic change in SMZL and identifies a subset with a distinct genotype characterised by multi-genetic changes. These different genetic changes may deregulate various signalling pathways and generate cooperative oncogenic properties, thereby contributing to lymphomagenesis.