Woori Jang

Evaluation of MicroScan WalkAway and Vitek 2 for determination of the susceptibility of extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates to cefepime, cefotaxime and ceftazidime

OBJECTIVESnThe aim of this study was to evaluate the accuracies of these automated susceptibility test systems with cefepime, cefotaxime and ceftazidime using the new CLSI and EUCAST guidelines in extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae.nnnMETHODSnA total of 220 ESBL-producing clinical isolates were collected from 12 hospitals in Korea. Susceptibility testing for cefepime, cefotaxime and ceftazidime was performed by MicroScan WalkAway, Vitek 2 and the CLSI broth microdilution test. ESBL genotypes were determined by PCR amplification.nnnRESULTSnThe proportion of isolates classified as susceptible to cefepime and ceftazidime with the CLSI and EUCAST guidelines was 35.0% versus 2.3% for cefepime (P < 0.001) and 21.8% versus 8.2% for ceftazidime (P < 0.001), respectively, and the susceptible isolates were mainly the CTX-M-9 group or SHV-type ESBL producers. All of the isolates were resistant to cefotaxime. Against the total of 220 ESBL-producing isolates, using the CLSI (EUCAST) criteria, very major/major error rates of MicroScan and Vitek 2 were as follows: 1.9%/20.8% (1.8%/20.0%) and 27.4%/0% (12.2%/0%) for cefepime and 2.6%/8.3% (1.2%/0%) and 4.5%/0% (2.3%/0%) for ceftazidime, respectively. The very major error rates of MicroScan and Vitek 2 with cefotaxime were 0.9% and 1.4%, respectively. The errors were mainly major errors for MicroScan and very major errors for Vitek 2.nnnCONCLUSIONSnA substantial portion of ESBL-producing isolates were susceptible to cefepime and ceftazidime by using the CLSI and EUCAST breakpoints. Unfortunately, the error rates of the two automated susceptibility systems were not acceptable for cefepime and ceftazidime.

Mutational characteristics of ANK1 and SPTB genes in hereditary spherocytosis

The aim of this study was to describe the mutational characteristics in Korean hereditary spherocytosis (HS) patients. Relevant literatures including genetically confirmed cases with well‐documented clinical summaries and relevant information were also reviewed to investigate the mutational gene‐ or domain‐specific laboratory and clinical association. Twenty‐five HS patients carried one heterozygous mutation of ANK1 (n = 13) or SPTB (n = 12) but not in SPTA1, SLC4A1, or EPB42. Deleterious mutations including frameshift, nonsense, and splice site mutations were identified in 91% (21/23), and non‐hotspot mutations were dispersed across multiple exons. Genotype–phenotype correlation was clarified after combined analysis of the cases and the literature review; anemia was most severe in HS patients with mutations on the ANK1 spectrin‐binding domain (p < 0.05), and SPTB mutations in HS patients spared the tetramerization domain in which mutations of hereditary elliptocytosis and pyropoikilocytosis are located. Splenectomy (17/75) was more frequent in ANK1 mutant HS (32%) than in HS with SPTB mutation (10%) (p = 0.028). Aplastic crisis occurred in 32.0% of the patients (8/25; 3 ANK1 and 5 SPTB), and parvovirus B19 was detected in 88%. The study clarifies ANK1 or SPTB mutational characteristics in HS Korean patients. The genetic association of laboratory and clinical aspects suggests comprehensive considerations for genetic‐based management of HS.

Identification of small marker chromosomes using microarray comparative genomic hybridization and multicolor fluorescent in situ hybridization

BackgroundMarker chromosomes are small supernumerary chromosomes that cannot be unambiguously identified by chromosome banding techniques alone. However, the precise characterization of marker chromosomes is important for prenatal diagnosis and proper genetic counseling. In this study, we evaluated the chromosomal origin of marker chromosomes using a combination of banding cytogenetics and molecular cytogenetic techniques including diverse fluorescence in situ hybridization (FISH) assays and array comparative genomic hybridization (array CGH).ResultsIn a series of 2871 patients for whom cytogenetic analysis was requested, 14 cases with small supernumerary marker chromosomes (sSMCs) were identified. Nine sSMCs were mosaic, and five nonmosaic. Of the nine cases with known parental origins, four were identified as de novo, and four and one were maternally and paternally inherited, respectively. Six sSMCs were identified by FISH using centromeric probes; three sSMCs were derived from chromosome 15, including two heterochromatic sSMC(15)s and a large sSMC(15) spanning 15q11.1q13.1, and three sSMCs originated from chromosome 14 or 22. Array CGH revealed two cases with derivatives of chromosome 2 and whole chromosome painting multicolor-FISH (M-FISH) identified three cases with derivatives of chromosome 6, 16, and 19, respectively. One maker chromosome in Turner syndrome was characterized as sSMC(X) by preferential application of a centromeric probe for X-chromosome. In addition, one sSMC composed of genomic materials from chromosomes 12 and 18 was identified in parallel with parental karyotype analysis that revealed the reciprocal balanced translocation.ConclusionsThis report is the largest study on sSMCs in Korea and expands the spectrum of sSMCs that are molecularly characterized.

Mutational spectrum of Korean patients with corneal dystrophy.

Corneal dystrophy typically refers to a group of rare hereditary disorders with a heterogeneous genetic background. A comprehensive molecular genetic analysis was performed to characterize the genetic spectrum of corneal dystrophies in Korean patients. Patients with various corneal dystrophies underwent thorough ophthalmic examination, histopathologic examination, and Sanger sequencing. A total of 120 probands were included, with a mean age of 50 years (SD = 18 years) and 70% were female. A total of 26 mutations in five genes (14 clearly pathogenic and 12 likely pathogenic) were identified in 49 probands (41%). Epithelial–stromal TGFBI dystrophies, macular corneal dystrophy and Schnyder corneal dystrophy (SCD) showed 100% mutation detection rates, while endothelial corneal dystrophies showed lower detection rates of 3%. Twenty six non‐duplicate mutations including eight novel mutations were identified and mutations associated with SCD were identified genetically for the first time in this population. This study provides a comprehensive characterization of the genetic aberrations in Korean patients and also highlights the diagnostic value of molecular genetic analysis in corneal dystrophies.

T618I-Mutated Colony Stimulating Factor 3 Receptor in Chronic Neutrophilic Leukemia and Chronic Myelomonocytic Leukemia Patients who Underwent Allogeneic Stem Cell Transplantation

Dear Editor n nRecently, the mutations within the colony-stimulating factor 3 receptor gene (CSF3R) have been reported as a specific marker of chronic neutrophilic leukemia (CNL) and atypical CML (aCML) [1, 2]. The current WHO system classifies CNL as a BCR-ABL1-negative myeloproliferative neoplasm (MPN). However, in routine clinical practice, it is difficult to clearly distinguish CNL from aCML, chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia, and MDS/MPN, unclassifiable under the MDS/MPN umbrella [3]. Therefore, molecular characteristics (e.g., CSF3R mutation) need to be included in the WHO diagnostic criteria. Here, we describe two CSF3R T618I-mutated patients with CNL and CMML, respectively, who underwent allogeneic stem cell transplantation (allo-SCT). n nA 40-yr-old man presented in 2012 with marked neutrophilia. His total white blood cell count was 77.24×109/L with a differential of 80% neutrophils, 3% band forms, 3% metamyelocytes, 1% myelocytes, 1% promyelocytes, 1% blasts, 3% lymphocytes, and 10% monocytes (Fig. 1A). He had a 23-cm splenomegaly. His bone marrow (BM) aspirate and biopsy showed hypercellularity with granulocytic hyperplasia (Fig. 1B), with a normal karyotype and no evidence of BCR-ABL1, PDGFRA, or PDGFRB rearrangement when examined by FISH. Molecular studies demonstrated the absence of JAK2 V617F and BCR-ABL1 transcripts. CNL was diagnosed in accordance with the WHO diagnostic criteria, and the patient was treated with hydroxyurea. However, his neutrophilia persisted in the peripheral blood as anemia and thrombocytopenia developed. Repeated BM examinations showed hypercellular BM with granulocytic hyperplasia and decreased megakaryocytes, and a clonal chromosomal abnormality of 46,XY,der(18:21)(q10;q10),+21[7]/47,+21[5]/46,XY[8]. The CSF3R T618I mutation was detected (Fig. 2A). He then underwent myeloablative allo-SCT, using cells from an unrelated donor. At day 35 following the allo-SCT, BM examination showed 70% cellularity with 100% donor chimerism. The T618I mutation was not detected in the BM aspirates through sequencing (Fig. 2C). n n n nFig. 1 n nChronic neutrophilic leukemia; (A) peripheral blood showing neutrophilia with toxic granulation (Wright-Giemsa staining, ×1,000) and (B) bone marrow biopsy showing a markedly elevated myeloid:erythroid ratio. Chronic myelomonocytic leukemia (Hematoxylin ... n n n n n nFig. 2 n nSequencing of genomic DNA isolated from peripheral blood leukocytes, demonstrating the CSF3R c.1853C>T (p.Thr618Ile) mutation. The CSF3R T618I mutation was detected in patient #1 with CNL, pre-allo-SCT (A) and in patient #2 with CMML at diagnosis ... n n n nA 60-yr-old man who presented with recurrent purpura on his extremities was referred to our hospital. He had constitutional symptoms including fatigue and weight loss. A computed tomog raphy scan of the abdomen demonstrated 13.5-cm mild splenomegaly. His peripheral blood (Fig. 1C) and BM findings (Fig. 1D) were consistent with CMML-1. The patient had a normal karyotype with no evidence of BCR-ABL1, PDGFRA, or PDGFRB rearrangement following FISH examinations. Molecular studies demonstrated the absence of JAK2 V617F and BCR-ABL1 transcripts. The CSF3R T618I mutation was identified through direct sequencing (Fig. 2B). An initial azacitidine treatment failed to achieve any response. Thus, he underwent allo-SCT using cells from a sibling donor. At day 30 following the allo-SCT, a BM examination showed 30% cellularity with 97% donor chimerism. The CSF3R T618I mutation was not detected in the BM aspirates (Fig. 2D). n nSince high-frequency of CSF3R mutations in CNL (89%) and, to a lesser extent, in aCML (40%) were discovered [1, 4], Pardanani et al. [2] has confirmed that the CSF3R T618I mutation was detected exclusively in cases of WHO-defined CNL, with a mutational frequency of 83%. Thus, CSF3R T618I mutation should be a diagnostic criterion for CNL. n nPrevious studies showed discordant frequencies in the CSF3R mutations in CMML and aCML. Maxson et al. [1] reported that eight of 20 aCML (40%) cases exhibited CSF3R mutations, whereas Pardanani et al. [2] did not find the CSF3R mutation in any case of aCML. Although CSF3R mutations have been observed in 4% of patients with CMML, they are distinct from membrane proximal mutations; the T618I mutation was identified in a majority of patients with CNL [5]. However, we observed one CMML patient harboring the CSF3R T618I mutation. To incorporate CSF3R mutations into the diagnostic criteria of these disorders, the frequency, the location, and the specificity of the CSF3R mutations need to be determined. n nAlthough there is no current standard of care for CNL or for aCML, allo-SCT may be applicable to young patients with potential for blast transformation and progressive refractory neutrophilia [6,7,8,9]. In our two CNL and CMML patients harboring the CSF3R T618I mutation, and who had undergone allo-SCT, the CSF3R T618I mutation was not detected following allo-SCT. n nThis suggests a predictive role of this mutation in post-transplant relapse, supported by prior studies showing a correlation between post-transplant relapse and the persistence of the CSF3R T618I in aCML [10]. Thus, testing for the CSF3R mutation may lead to genetically informed therapy and useful diagnostic approach. The influence of the CSF3R mutations on genotype-phenotype associations, disease prognosis, and the efficacy of the therapeutic inhibition of CSF3R-related signaling needs to be clarified.

Two cases of concurrent development of essential thrombocythemia with chronic lymphocytic leukemia, one related to clonal B-cell lymphocytosis, tested by array comparative genomic hybridization

We present two cases of concurrent development of essential thrombocythemia (ET) with chronic lymphocytic leukemia (CLL) and one related to clonal B-cell lymphocytosis (CBL). Both patients were referred for lymphocytosis and thrombocytosis. A bone marrow biopsy revealed infiltration of small, mature lymphocytes and megakaryocytic hyperplasia. Flow cytometric immunophenotyping and immunoglobulin (IG) gene clonality tests revealed clonal B lymphocytes. Both patients were positive for the JAK2 V617F mutation in whole bone marrow aspirate. The JAK2 V617F mutation was present in isolated B lymphocytes of patient 1, but not patient 2. Cytogenetics were normal in both patients. An array comparative genomic hybridization (CGH) analyses of B cells revealed a gain of 4q28.3, which is reported in non-Hodgkin’s lymphoma, in patient 1, and deletion 22q11.22, which is associated with CLL, and a gain of Xp22.31 in patient 2. In both patients, B cells showed no myeloproliferative neoplasm (MPN)-specific genetic abnormalities. These results suggest that different oncogenic mechanisms in each cell lineage may underlie the concurrent development of ET and CLL (or CBL). Array CGH may be helpful in identifying the pathogenic mechanism in cases of concurrent development of lymphoid neoplasm and MPN.

Mean Corpuscular Volume (MCV) Values Reflect Therapeutic Effectiveness in Zidovudine-Receiving HIV Patients

An increase of the mean corpuscular volume (MCV) of erythrocytes and alterations in the lipid profiles have been described in HIV‐infected patients under combination of anti‐retroviral treatment (cART), particularly zidovudine (AZT).

Molecular analysis of myocilin and optineurin genes in Korean primary glaucoma patients.

To investigate the underlying genetic influences of primary glaucoma in Korea, molecular analysis was performed in 112 sporadic cases, and results compared with healthy controls. The myocilin (MYOC) and optineurin (OPTN) genes were directly sequenced in 112 unrelated patients, including 17 with primary open-angle glaucoma, 19 with juvenile open-angle glaucoma, and 76 with normal tension glaucoma. Healthy unrelated Korean individuals (n=100) were used as the non-selected population control. A total of three MYOC and four OPTN variants potentially associated with primary glaucoma were identified in 4 and 18 patients, respectively. A novel variant of MYOC, p.Leu255Pro, was predicted to be potentially pathogenic by in silico analysis. Another, p.Thr353Ile, has been previously reported. These two missense variants were detected in patients with a family history of glaucoma. Combined heterozygous variants p.[Thr123=;Ile288=] were identified in 2 of 112 (2%) patients but not in healthy controls. Among OPTN variants, a novel variant p.Arg271Cys was identified. Homozygous p.[Thr34=;Thr34=] (4/112, 4%), homozygous p.[Met98Lys;Met98Lys] (4/112, 4%), or combined heterozygous p.[Thr34=;Arg545Gln] (9/112, 8%) was significantly associated with the development of primary glaucoma [odds ratio (OR)=8.768, 95% confidence interval (CI)=1.972–38.988; relative risk=1.818, 95% CI=1.473–2.244; P=0.001]. The present study provides insight into the genetic or haplotype variants of MYOC and OPTN genes contributing to primary glaucoma. Haplotype variants identified in the present study may be regarded as potential contributing factors of primary glaucoma in Korea. Further studies, including those on additional genes, are required to elucidate the underlying pathogenic mechanism using a larger cohort to provide additional statistical power.

Comparison of Targeted Next-Generation and Sanger Sequencing for the BRCA1 and BRCA2 Mutation Screening

Dear Editor, n nHigh-throughput and cost-effective BRCA genetic screening is needed for application of pharmacogenetics in personalized HBOC therapy. Banerjee et al. [1] suggested that poly(ADP-ribose) polymerase (PARP) inhibitors show considerable promise for the treatment of cancers with BRCA1 or BRCA2 mutations. Secord et al. [2] demonstrated that compared to the global use of PARP inhibition, the BRCA1 and BRCA2 test for personalized PARP inhibition treatment may represent a de facto cost-reducing strategy. n nIn this study, results of targeted Next-generation sequencing (NGS) by Ion Torrent Personal Genome Machine (PGM, Life Technologies, Guilford, CT, USA) were compared with those of Sanger sequencing in seven HBOC patients harboring pathogenic variant or rare variant of BRCA1 and BRCA2 of uncertain clinical significance. n nAll enrolled subjects provided written informed consent for clinical and molecular analyses, and the study protocol was approved by the institutional review board (KC15SISE0263) of The Catholic University, Seoul, Korea. Seven HBOC patients harboring pathogenic or unclassified variants of BRCA1 and BRCA2 as confirmed by Sanger sequencing were studied. The Ion AmpliSeq BRCA1 and BRCA2 Panel (Life Technologies) consisting of 167 primer pairs in three primer pair pools was used for targeted NGS analysis. Sequencing on the Ion Torrent PGM was performed by using 500 flow runs that generated approximately 200 bp reads. Torrent Variant Caller 3.4 was applied for alignment and variant detection. The variant caller parameter setting was germline PGM high stringency (Table 1). Sequence data was visually confirmed with the Integrative Genomics Viewer (IGV) and any sequence, alignment, or variant call error artifacts were discarded. Non-synonymous variants called were evaluated by using ClinVar (http://www.ncbi.nlm.nih.gov/clinvar), the BIC database (http://research.nhgri.nih.gov/bic/), and the HGMD database (http://www.hgmd.cf.ac.uk/). Minor allele frequency (MAF) was determined from the 1000 Genomes Project database (http://www.1000genomes.org/). n n n nTable 1 n nOverride parameters selected for customizing hotspot calling of BRCA1 and BRCA2 variants in this study n n n nTechnical performance of the Ion AmpliSeq BRCA1/2 Panel showed 81% of template-positive ion sphere particle sample loading. The total read obtained was 4,541,406, with a mean read length of 137 bp. The mean sequencing depth for each region was ×494 and the average uniformity of coverage was 99%, which is percentage of target bases, covered by at least ×0.2 of the average base read depth. All variants located in all coding exons and in adjacent intronic regions of BRCA1 and BRCA2 identified by Sanger sequencing were detected by targeted NGS analysis. Direct sequencing of entire coding exons and flanking intronic sequences of BRCA1 and BRCA2 was performed on ABI 3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). n nOf seven patients, four carried deleterious BRCA1 or BRCA2 variants: two frameshift and two nonsense mutations. The p.Asn567Ilefs*5 and p.Arg1203* of BRCA1 have previously been reported in Turkish [3] and American [4] populations, respectively (Fig. 1A, B). The p.Ser1248Argfs*10 and p.Arg2494* of BRCA2 have been reported in German [5] and Finnish [6] populations, respectively (Fig. 1C, D). Meanwhile two BRCA1 and one BRCA2 out of nine non-synonymous variants are rare according to 1,000 Genome Project data (<1% population MAF). The p.Leu52Phe of BRCA1 and p.Val2109Ile of BRCA2 have been reported in the Koreans, and the p.Tyr856His of BRCA1 has been found in the Japanese populations. n n n nFig. 1 n nIdentification of deleterious mutations in BRCA1 or BRCA2 by Sanger sequencing and next-generation sequencing (NGS), as visualized by Sequencher Software (top) and Integrative Genomics Viewer (IGV, bottom), respectively. The deletion(s) or base change ... n n n nOf various NGS platforms, the PGM generates DNA sequencing reads by detecting ions released when deoxynucleotide triphosphates are incorporated into a growing DNA strand on a semiconductor device [7]. In particular, it has been documented that indel errors occurring in homopolymer DNA regions significantly affect the specificity of indel detection owing to the nature of sequencing chemistry of PGM [8]. In this study, sequence AGTG at position 3972_3975 of BRCA2 was given in NGS; however, the Human Genome Variation Society notation prescribes that on the forward strand it should be TGAG at position 3974_3977 as given in Sanger sequencing. n nWhile the protein-truncating variants (either frameshift, nonsense, or splice) are located generally in the coding exons or the flanking intronic sequences of BRCA genes, potentially deleterious alterations may also reside in the noncoding intronic sequences. For example, deep intronic mutation causing activation of a cryptic exon in BRCA2 has been reported in a French family with a history of breast cancer [9]. Thus, rare variants called with both NGS and Sanger sequencing should be considered for validation study, regardless of their location. n nIn conclusion, our study is a clear example that the quality of targeted NGS of a disease-specific subset of genes is equal to the quality of Sanger sequencing, and therefore it can be implemented reliably as a stand-alone diagnostic test demonstrated by Sikkema-Raddatz B et al. [10].

Identification of a novel de novo nonsense mutation of the NSD1 gene in monozygotic twins discordant for Sotos syndrome

INTRODUCTIONnSotos syndrome is a congenital overgrowth disorder characterized by facial gestalt, excessively rapid growth, acromegalic features and a non-progressive cerebral disorder with intellectual disability.nnnMETHODOLOGYnThe identical male twins showed somewhat different clinical, cognitive and behavioural phenotypes. Abnormal clinical manifestations including seizures, scoliosis, enlarged ventricles, and attention-deficit/hyperactivity disorder (ADHD) were found in the proband (first twin), but not in the sibling (second twin). We used diagnostic exome sequencing (DES) to identify a heterozygous de novo mutation of the NSD1 gene in monozygotic twins with Sotos syndrome.nnnRESULTSnDES revealed a novel nonsense mutation c.2596G>T (p.Glu866*) of the NSD1 gene in the proband, the first of monozygotic twins. Sanger sequencing analysis of the proband and his family members showed that this nonsense mutation was present in the proband and his sibling, but was absent in their parents, indicating that it occurred with de novo origin.nnnCONCLUSIONnThis finding expands the phenotypic spectrum associated with variable expression of the Sotos syndrome caused by NSD1 mutation, and it adds further support for postconceptual mutation, epigenetic change and/or an environmental factor involved in the cause of the Sotos syndrome.