Chronic neutrophilic leukemia with an associated V617F JAK2 tyrosine kinase mutation

Chronic neutrophilic leukemia (CNL) is a rare disease and can cause considerable diagnostic difficulty. Although the V617F JAK2 mutation has been described by several groups to be associated with classical myeloproliferative disorders (MPD), this same mutation has been detected with a low incidence in atypical MPD, such as CNL. Here we report the presence of the V617F mutation in a CNL patient, who is unusual for having survived for more than 96 months, with little disease progression. It remains to be established what role this mutation, which gives cells a proliferative advantage, might play in the pathogenesis and prognosis of rare atypical MPD.

Erythrocytosis due to a mutation in the erythropoietin receptor gene

Familial erythrocytosis, associated with high haemoglobin levels and low serum erythropoietin (Epo), has been shown to co-segregate with a sequence repeat polymorphism at the 5' region of the erythropoietin receptor (EpoR) in a large Finnish family. We have investigated the cause of erythrocytosis in an English boy. Sequencing of the cytoplasmic region of the EpoR detected a de novo transition mutation of G to A at nucleotide 6002. This mutation resulted in the formation of a stop codon at amino acid 439 with the loss of 70 amino acids from the carboxy terminus. The mutation (G6002A) has arisen independently in a Finnish family and de novo in this English boy. Patients with unexplained erythrocytosis and low serum Epo levels should be investigated for EpoR mutations.

A novel GUCY2D mutation, V933A, causes central areolar choroidal dystrophy

Purpose: To identify the genetic cause of central areolar choroidal dystrophy (CACD) in a large Northern Irish family.
Methods: We previously reported linkage of the locus for CACD in this family to an interval of approximately 5 cM on chromosome 17p13 flanked by polymorphic markers D17S1810 and CHLC GATA7B03. We undertook sequence capture, massively-parallel sequencing and computational alignment, base-calling and annotation to identify a causative mutation. Conventional sequencing was used to confirm the results.
Results: Deep sequencing identified a single-base substitution in guanylate cyclase 2D, membrane (retina-specific) (GUCY2D). The novel mutation segregated with the disease phenotype and resulted in substitution of valine by alanine at position 933, within the catalytic domain of the protein. It altered a motif that is strongly conserved in a large number of distantly related proteins across several species, and was predicted to have a damaging effect on protein activity.
Conclusion: Mutations in GUCY2D have previously been associated with dominant cone rod dystrophies (CORD6) and recessive forms of Leber's congenital amaurosis (LCA). This is the first report of GUCY2D mutation causing CACD and adds to our understanding of genotype-phenotype correlation in this heterogeneous group of choroidoretinal dystrophies.

Analysis of alteration of p75NTR processing and signalling by PS2 mutation and gamma-secretase inhibition

The presenilins (PSs) were identified as causative genes in cases of early-onset familial Alzheimer's disease (AD) and current evidence indicates that PSs are part of the gamma-secretase complex responsible for proteolytic processing of type I membrane proteins. p75NTR, a common neurotrophin receptor, was shown to be subject to gamma-secretase processing. However, it is not clear if the p75NTR downstream signal is altered in response to gamma-secretase cleavage, and further there is a possibility that AD-related PS mutations may affect this cleavage, resulting in pathogenic alterations in signal transduction. In this study, we confirmed that p75NTR downstream signalling is altered by PS2 mutation or gamma-secretase inhibition in SHSY-5Y cells. The activity of the small GTPase RhoA is strongly affected by these treatments. This study demonstrates that gamma-secretase and PS2 play an important role in regulating neurotrophin signal transduction and either mutation of PS2 or inhibition of gamma-secretase disturbs this function.

Autosomal dominant Alport syndrome caused by a COL4A3 splice site mutation

Alport syndrome (AS) is a clinically and genetically heterogeneous renal disorder, predominantly affecting the type IV collagen alpha 3/alpha 4/alpha 5 network of the glomerular basement membrane (GBM). AS can be caused by mutations in any of the three genes encoding these type IV collagen chains. The majority of AS families (85%) are X-linked (XL-AS) involving mutations in the COL4A5 gene. Mutations in the COL4A3 and COL4A4 genes cause autosomal recessive AS (AR-AS), accounting for approximately 14% of the cases. Recently, autosomal dominant AS (AD-AS) was linked to the COL4A3/COL4A4 locus in a large family.

Depletion of the ubiquitin-binding adaptor molecule SQSTM1/p62 from macrophages harboring cftr ΔF508 mutation improves the delivery of Burkholderia cenocepacia to the autophagic machinery

Cystic fibrosis is the most common inherited lethal disease in Caucasians. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), of which the cftr ?F508 mutation is the most common. ?F508 macrophages are intrinsically defective in autophagy because of the sequestration of essential autophagy molecules within unprocessed CFTR aggregates. Defective autophagy allows Burkholderia cenocepacia (B. cepacia) to survive and replicate in ?F508 macrophages. Infection by B. cepacia poses a great risk to cystic fibrosis patients because it causes accelerated lung inflammation and, in some cases, a lethal necrotizing pneumonia. Autophagy is a cell survival mechanism whereby an autophagosome engulfs non-functional organelles and delivers them to the lysosome for degradation. The ubiquitin binding adaptor protein SQSTM1/p62 is required for the delivery of several ubiquitinated cargos to the autophagosome. In WT macrophages, p62 depletion and overexpression lead to increased and decreased bacterial intracellular survival, respectively. In contrast, depletion of p62 in ?F508 macrophages results in decreased bacterial survival, whereas overexpression of p62 leads to increased B. cepacia intracellular growth. Interestingly, the depletion of p62 from ?F508 macrophages results in the release of the autophagy molecule beclin1 (BECN1) from the mutant CFTR aggregates and allows its redistribution and recruitment to the B. cepacia vacuole, mediating the acquisition of the autophagy marker LC3 and bacterial clearance via autophagy. These data demonstrate that p62 differentially dictates the fate of B. cepacia infection in WT and ?F508 macrophages.

The Interlaboratory Robustness Of Next-Generation Sequencing (IRON) Study Phase II: Deep-Sequencing Analyses Of Hematological Malignancies Performed In 8,867 Cases By An International Network Involving 27 Laboratories

Introduction: Amplicon deep-sequencing using second-generation sequencing technology is an innovative molecular diagnostic technique and enables a highly-sensitive detection of mutations. As an international consortium we had investigated previously the robustness, precision, and reproducibility of 454 amplicon next-generation sequencing (NGS) across 10 laboratories from 8 countries (Leukemia, 2011;25:1840-8).

Aims: In Phase II of the study, we established distinct working groups for various hematological malignancies, i.e. acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and multiple myeloma. Currently, 27 laboratories from 13 countries are part of this research consortium. In total, 74 gene targets were selected by the working groups and amplicons were developed for a NGS deep-sequencing assay (454 Life Sciences, Branford, CT). A data analysis pipeline was developed to standardize mutation interpretation both for accessing raw data (Roche Amplicon Variant Analyzer, 454 Life Sciences) and variant interpretation (Sequence Pilot, JSI Medical Systems, Kippenheim, Germany).

Results: We will report on the design, standardization, quality control aspects, landscape of mutations, as well as the prognostic and predictive utility of this assay in a cohort of 8,867 cases. Overall, 1,146 primer sequences were designed and tested. In detail, for example in AML, 924 cases had been screened for CEBPA mutations. RUNX1 mutations were analyzed in 1,888 cases applying the deep-sequencing read counts to study the stability of such mutations at relapse and their utility as a biomarker to detect residual disease. Analyses of DNMT3A (n=1,041) were focused to perform landscape investigations and to address the prognostic relevance. Additionally, this working group is focusing on TET2, ASXL1, and TP53 analyses. A novel prognostic model is being developed allowing stratification of AML into prognostic subgroups based on molecular markers only. In ALL, 1,124 pediatric and adult cases have been screened, including 763 assays for TP53 mutations both at diagnosis and relapse of ALL. Pediatric and adult leukemia expert labs developed additional content to study the mutation incidence of other B and T lineage markers such as IKZF1, JAK2, IL7R, PAX5, EP300, LEF1, CRLF2, PHF6, WT1, JAK1, PTEN, AKT1, IL7R, NOTCH1, CREBBP, or FBXW7. Further, the molecular landscape of CLL is changing rapidly. As such, a separate working group focused on analyses including NOTCH1, SF3B1, MYD88, XPO1, FBXW7 and BIRC3. Currently, 922 cases were screened to investigate the range of mutational burden of NOTCH1 mutations for their prognostic relevance. In MDS, RUNX1 mutation analyses were performed in 977 cases. The prognostic relevance of TP53 mutations in MDS was assessed in additional 327 cases, including isolated deletions of chromosome 5q. Next, content was developed targeting genes of the cellular splicing component, e.g. SF3B1, SRSF2, U2AF1, and ZRSR2. In BCR-ABL1-negative MPN, nine genes of interest (JAK2, MPL, TET2, CBL, KRAS, EZH2, IDH1, IDH2, ASXL1) have been analyzed in a cohort of 155 primary myelofibrosis cases searching for novel somatic mutations and addressing their relevance for disease progression and leukemia transformation. Moreover, an assay was developed and applied to CMML cases allowing the simultaneous analysis of 25 leukemia-associated target genes in a single sequencing run using just 20 ng of starting DNA. Finally, nine laboratories are studying CML, applying ultra-deep sequencing of the BCR-ABL1 tyrosine kinase domain. Analyses were performed on 615 cases investigating the dynamics of expansion of mutated clones under various tyrosine kinase inhibitor therapies.

Conclusion: Molecular characterization of hematological malignancies today requires high diagnostic sensitivity and specificity. As part of the IRON-II study, a network of laboratories analyzed a variety of disease entities applying amplicon-based NGS assays. Importantly, the consortium not only standardized assay design for disease-specific panels, but also achieved consensus on a common data analysis pipeline for mutation interpretation. Distinct working groups have been forged to address scientific tasks and in total 8,867 cases had been analyzed thus far.

Genetic screening protocol for familial hypercholesterolemia which includes splicing defects gives an improved mutation detection rate

Familial hypercholesterolemia (FH) is a common single gene disorder, which predisposes to coronary artery disease. In a previous study, we have shown that in patients with definite FH around 20% had no identifiable gene defect after screening the entire exon coding area of the low density lipoprotein receptor (LDLR) and testing for the common Apolipoprotein B (ApoB) R3500Q mutation. In this study, we have extended the screen to additional families and have included the non-coding intron splice regions of the gene. In families with definite FH (tendon xanthoma present, n = 68) the improved genetic screening protocol increased the detection rate of mutations to 87%. This high detection rate greatly enhances the potential value of this test as part of a clinical screening program for FH. In contrast, the use of a limited screen in patients with possible FH (n = 130) resulted in a detection rate of 26%, but this is still of significant benefit in diagnosis of this genetic condition. We have also shown that 14% of LDLR defects are due to splice site mutations and that the most frequent splice mutation in our series (c.1845 + 11 c > g) is expressed at the RNA level. In addition, DNA samples from the patients in whom no LDLR or ApoB gene mutations were found, were sequenced for the NARC-1 gene. No mutations were identified which suggests that the role of NARC-1 in causing FH is minor. In a small proportion of families (

Mutation screening and genotype:phenotype correlation in familial hypercholesterolaemia

The aim of this study was to develop a mutation screening protocol for familial hypercholesterolaemia (FH) patients and to assess genotype/phenotype effects in terms of pre-treatment lipid profiles and presentation of tendon xanthomata (TX). A total of 158 families with clinical definitions of possible (120) or definite (38) FH were studied using a tiered screening protocol. Mutations were identified in 52 families, 44 families showing 23 different LDLR gene defects and eight families showing the common Apo B100 gene defect R3500Q. LDLR defects were detected in various regions of the gene with 56% in the LDL binding domain (exons 2-6) and 37% in the EGF precursor homology domain (exons 7-14). The most common mutations were D461N(7), C210X(5), 932delA(5), and C163Y(4). Frameshift mutations accounted for 20% with nonsense 13%, mis-sense 35%, splice 3%, Apo B 13% and 2% large deletion, 13% of clinically definite FH remained undefined. In conclusion, DNA based diagnosis is possible in 79% (30/38) of clinically definite FH families and of the 120 possible FH families at the start of the screening program, 18% (22/120) now have defined mutations. Overall 60 families from the original 158 meet the clinical and/or genetic criteria for definite FH. Tendon xanthomata were present in only 58% (30/52) of genetically defined FH families, thus limiting its use as a strict diagnostic criteria. Families with low density lipoprotein receptor (LDLR) defects present with higher total and LDL cholesterol levels and a higher incidence of TX than do those with the common Apo B variant, and frameshift mutations appear to have the most severe presentation. Copyright (C) 1999 Elsevier Science Ireland Ltd.

The positive impact of cytological specimens for EGFR mutation testing in non-small cell lung cancer:a single South East Asian laboratory's analysis of 670 cases

To compare the rejection rates of non-small cell lung cancer (NSCLC) samples obtained by differing sampling methods for testing by Sanger sequencing for epidermal growth factor receptor (EGFR) mutations. To assess the association between unsatisfactory outcomes and the quantity of DNA extracted from cytological versus histological samples.