Next-generation sequencing reveals unique combination of mutations in cis of CSF3R in atypical chronic myeloid leukemia.

BACKGROUND: Atypical chronic myeloid leukemia (aCML) is a hematologic disorder characterized by leukocytosis with increased dysplastic neutrophils and their precursors. In CSF3R gene, the activation mutation including T618I is frequently reported in aCML but is rarely accompanied by truncation mutations. Herein, we report a unique aCML patient with two CSF3R mutations (T618I and Y779*) in the same DNA strand. METHODS: High-coverage next-generation sequencing for 40 genes related with myeloid leukemia was performed. Sanger sequencing was performed to confirm CSF3R mutations. To confirm whether two CSF3R mutations are in cis or not, TA cloning was used. Clinical information and bone marrow pathology were reviewed by two hematopathologists. RESULTS: In the patient diagnosed with aCML in bone marrow study, two CSF3R mutations, (T618I and Y779*) a SETBP1 mutation (G870S) and an U2AF1 mutation (Q157P), were identified by high-coverage next-generation sequencing. The two CSF3R mutations were confirmed to be located in the same DNA strand by TA cloning, indicating that the two mutations are harbored in one malignant clone. The SETBP1 mutation is known to be related with poor prognosis in aCML. Likewise, the patient was refractory to hydroxyurea and showed disease progression. Additionally, we discussed the potential therapeutic targets by reviewing the molecular profile of the patient. CONCLUSION: We believe that the accurate diagnosis and maximum therapeutic chance could be achieved by profiling the mutations and their characteristics.

Ethnicity-specific impact of HLA I/II genotypes on the risk of inhibitor development: data from Korean patients with severe hemophilia A.

Inhibitor development is the most serious complication in patients with hemophilia. We investigated association of HLA genotypes with inhibitor development in Korean patients with severe hemophilia A (HA). HLA genotyping was done in 100 patients with severe HA including 27 patients with inhibitors. The allele frequencies between inhibitor-positive and inhibitor-negative patients were compared. HLA class I alleles were not associated with the inhibitor status. In HLA class II, DRB1*15 [n = 100, odds ratio (OR) 0.217, P = 0.028] and DPB1*05:01 [OR 0.461, P = 0.026] were negatively associated with inhibitor development. In a subgroup of patients with intron 22 inversion, C*07:02 was positively associated with inhibitor development [n = 30, OR 5.500, P = 0.043]. In the subgroup of patients without intron 22 inversion, the negative association between DPB1*05:01 and inhibitor development was reinforced [n = 70, OR 0.327, P = 0.010], and positive association of DRB1*13:02 and DPB1*04:01 with inhibitor development was identified [OR 3.059, P = 0.037 for both]. Previously reported risk alleles were not consistently associated with inhibitor risk in our series. This study demonstrated the profile of HLA alleles associated with inhibitor risk in Korean patients with severe HA was different from that in patients of other ethnicities, which needs to be considered in risk assessment and management.

Paternal Somatic Mosaicism of a Novel Frameshift Mutation in ELANE Causing Severe Congenital Neutropenia.

Severe congenital neutropenia (SCN) is a bone marrow failure disease with an autosomal dominant inheritance from mutations in ELANE. Here, we report a 7-week-old Korean male with SCN. His elder sister died from pneumonia at 2 years. Direct sequencing of ELANE in the proband identified a heterozygous novel frameshift mutation: c.658delC (p.Arg220Glyfs20*). Family study involving his asymptomatic parents with normal cell counts revealed that his father had the same mutation, but at a lower burden than expected in a typical heterozygous state. Further molecular investigation demonstrated somatic mosaicism with ~18% mutant alleles. We concluded the proband inherited the mutation from his somatic mosaic father.

Distinct frequencies and mutation spectrums of genetic thrombophilia in Korea in comparison with other Asian countries both in patients with thromboembolism and in the general population.

Hereditary natural anticoagulant deficiencies are the major cause of genetic thrombophilia in Asia. Given the growing acknowledgment of the risk of venous thromboembolism in Asian populations, we investigated the frequency and mutation spectrums of natural anticoagulant deficiency in Korea. The group of patients consisted of consecutive patients with venous thromboembolism screened for thrombophilia. Genetic tests were performed on suspicion of natural anticoagulant deficiency. For the population group, >3,000 individuals were screened from routine check-ups, and those with a low level (<1(st) percentile) of natural anticoagulant underwent genetic tests. Mutations were detected by direct sequencing of PROC, PROS1, and SERPINC1, followed by additional multiplex ligation-dependent probe amplification for PROS1 and SERPINC1 for dosage mutations. Among 500 patients screened, 127 were suspected of having a natural anticoagulant deficiency, and this was genetically confirmed in 71: protein C deficiency in 36 (50.7%), antithrombin deficiency in 21 (29.6%), and protein S deficiency in 14 (19.7%). Among 3,129 individuals from the population who were screened, the frequency of natural anticoagulant deficiency was ~1.0%: antithrombin deficiency 0.49%, protein C deficiency 0.35%, and protein S deficiency 0.16%. Two PROC mutations causing type I protein C deficiency were prevalent (Arg211Trp and Met406Ile in patients and Arg211Trp in the population). Two SERPINC1 mutations causing type II antithrombin deficiency, Arg79Cys and Ser158Pro, were prevalent in the population group. This is the first study on the genetic epidemiology of natural anticoagulant deficiencies in Korea. The results demonstrated that the frequencies and spectrum of mutations underlying genetic thrombophilia in Korea are different not only from those in Caucasians but also those in other Asian populations.

Hereditary gene mutations in Korean patients with isolated erythrocytosis.

Most cases of erythrocytosis occur secondary to chronic tissue hypoxia or as a clonal disease such as polycythemia vera with somatic mutations in the Janus kinase 2 (JAK2) gene. Rarely, erythrocytosis is caused by hereditary gene mutations. This study investigated hereditary gene mutations in 38 unrelated Korean patients with isolated erythrocytosis without (1) JAK2 mutation and (2) secondary causes of erythrocytosis other than smoking history. Direct sequencing analyses were performed on six genes associated with hereditary erythrocytosis [HBB, exon 2 and exon 3 of HBA2, VHL, EGLN1 (previously PHD2), exon 12 of EPAS1 (previously HIF2A), and exons 5-8 of EPOR]. As a result, mutations were detected in five patients (three never smokers and two current smokers) out of 38 patients (13.2 %). The mutations detected in those five patients were EPOR:p.W439*, EPOR:p.G212C, HBB:p.H98Q (or conventionally H97Q, Hb Malmö [ß 97(FG4) His > Gln]), HBB:p.V138M (V137M), and EGLN1:p.L279Tfs43*, all in heterozygous state. No patient had mutations in HBA2, VHL, or in EPAS1. This study indicates that workup for hereditary gene mutations is needed for isolated erythrocytosis with or without smoking history.

Clinical significance of SF3B1 mutations in Korean patients with myelodysplastic syndromes and myelodysplasia/myeloproliferative neoplasms with ring sideroblasts.

Somatic mutations in the SF3B1 gene, a gene encoding the splicing factor 3B subunit 1, were recently reported in myelodysplastic syndromes (MDS), particularly in the presence of ring sideroblasts (RS). The authors investigated the prevalence and clinical significance of SF3B1 mutations in Korean patients with myeloid neoplasms with RS. The study subjects were 43 Korean patients with myeloid neoplasms. Twenty-nine patients (67 %) had 15 % or more RS (high-count RS [HC-RS]), and 14 (33 %) had RS less than 15 % (low-count RS [LC-RS]). Molecular genetic tests were performed to detect SF3B1 mutations by direct sequencing on bone marrow samples of the patients. SF3B1 mutations were detected in 55 % (16/29) of the HC-RS group: 3 RARS (3/3), 8 RCMD (8/16), 3 RARS-t (3/4), 1 RAEB (1/4), and 1 MDS-U (1/1). All mutations were previously reported mutations with K700E being the most common (63 % of mutation-positive cases). On the other hand, none (0 %) of the LC-RS group had SF3B1 mutation. The patients with SF3B1 mutations had higher platelet counts (p = 0.023), higher proportions of RS (p = 0.003), and lower proportions of bone marrow blasts (p = 0.026) than those without SF3B1 mutations. SF3B1 mutations showed a favorable survival implication (p = 0.025), but not in multivariate analysis (p = 0.178). This study confirmed that SF3B1 mutation is highly specific to the HC-RS phenotype in Korean patients with myeloid neoplasms with similar frequencies and distributions in previous findings and is associated with distinct hematologic features.

Gene mutation profiles and prognostic implications in Korean patients with T-lymphoblastic leukemia.

Genetic alterations implicated in the leukemogenesis of T cell acute lymphoblastic leukemia (T-ALL) have been identified in recent years. In this study, we investigated gene mutation profiles and prognostic implications in a series of Korean T-ALL patients. The study patients were 29 Korean patients with T-ALL; 13 adults (45 %) and 16 children (55 %; male-to-female ratio, 25:4). Clinical, hematologic, and cytogenetic findings were reviewed. We performed mutation analyses for NOTCH1, FBXW7, PHF6, and IL7R genes and survival analyses according to the mutational status. Gene mutations were identified in 66 % of the patients in our series (19/29). Eighteen patients (62 %) had NOTCH1/FBXW7 mutations. Sixteen patients (55 %) had NOTCH1 mutations including nine novel mutations, and eight patients (28 %) had known FBXW7 mutations. Eight patients (28 %; six males and two females) had PHF6 mutations including four novel mutations. Three patients (10 %) had IL7R mutations, which were all novel in-frame insertion or deletion-insertions. The gene mutation profile combined with cytogenetics and FISH study for the p16 gene detected genetic aberrations in 90 % of patients (26/29). There was no significant difference in the frequency of gene mutations between the pediatric and adult patients with T-ALL. Survival analyses suggested a favorable prognostic implication of NOTCH1 mutations in adult T-ALL. Gene mutation studies for NOTCH1, FBXW7, PHF6, and IL7R could detect genetic alterations in a majority of Korean T-ALL patients with novel mutations. We observed similar mutation profiles between adult and pediatric T-ALL, and a favorable prognostic implication of NOTCH1 mutations in adult T-ALL.

KIT D816 mutation associates with adverse outcomes in core binding factor acute myeloid leukemia, especially in the subgroup with RUNX1/RUNX1T1 rearrangement.

Core binding factor (CBF)-positive acute myeloid leukemia (AML) presents a favorable prognosis, except for patients with KIT mutation, especially D816 mutation. The current retrospective study attempted to validate a prognostic role of KIT mutation in 121 Korean patients with CBF AML. The study patients consisted of 121 patients with CBF AML (82 patients with RUNX1/RUNX1T1 [67.8 %] and 39 patients with CBFB/MYH11 [32.2 %]) recruited from eight institutions in Korea. All patients received idarubicin plus cytarabine or behenoyl cytosine arabinoside 3 + 7 induction chemotherapy. The KIT gene mutation status was determined by direct sequencing analyses. A KIT mutation was detected in 32 cases (26.4 %) in our series of patients. The KIT mutation was most frequent in exon 17 (n = 18, 14.9 %; n = 16 with D816 mutation), followed by exon 8 (n = 10, 8.3 %). The presence of KIT D816 mutation was associated with adverse outcomes for the event-free survival (p = 0.03) and for the overall survival (p = 0.02). The unfavorable impact of D816 mutation was more prominent when the analysis was confined to the RUNX1/RUNX1T1 subtype. The KIT mutation was detected in 26.4 % of Korean patients with CBF AML. The KIT D816 mutation demonstrated an unfavorable prognostic implication, particularly in the RUNX1/RUNX1T1 subtype.

Founder effects in two predominant intronic mutations of UNC13D, c.118-308C>T and c.754-1G>C underlie the unusual predominance of type 3 familial hemophagocytic lymphohistiocytosis (FHL3) in Korea.

Familial hemophagocytic lymphohistiocytosis (familial HLH or FHL) is a potentially fatal autosomal recessive disorder. Our previous study demonstrated that UNC13D mutations (FHL3) account for ~90 % of FHL in Korea with recurrent splicing mutation c.754-1G>C (IVS9-1G>C). Notably, half of the FHL3 patients had a monoallelic mutation of UNC13D. Deep intronic mutations in UNC13D were recently reported in patients of European descent. In this study, we performed targeted mutation analyses for deep intronic mutations and investigated on the founder effect in FHL3 in Korean patients. The study patients were 72 children with HLH including those with FHL3 previously reported to have a monoallelic UNC13D mutation. All patients were recruited from the Korean Registry of Hemophagocytic Lymphohistiocytosis. In addition to conventional sequencing of FHL2-4, targeted tests for c.118-308C>T and large intronic rearrangement mutations of UNC13D were performed. Haplotype analysis was performed for founder effects using polymorphic markers in the FHL3 locus. FHL mutations were detected in 20 patients (28 %). Seventeen patients had UNC13D mutations (FHL3, 85 %) and three had PRF1 mutations (FHL2, 15 %). UNC13D:c.118-308C>T was detected in ten patients, accounting for 38 % of all mutant alleles of UNC13D, followed by c.754-1G>C (26 %). Haplotype analyses revealed significantly shared haplotypes in both c.118-308C>T and c.754-1G>C, indicating the presence of founder effects. The deep intronic mutation UNC13D:c.118-308C>T accounts for the majority of previously missing mutations and is the most frequent mutation in FHL3 in Korea. Founder effects of two recurrent intronic mutations of UNC13D explain the unusual predominance of FHL3 in Korea.

Heterogeneous lengths of copy number mutations in human coagulopathy revealed by genome-wide high-density SNP array.

BACKGROUND: The recent advent of genome-wide molecular platforms has facilitated our understanding of the human genome and disease, particularly copy number aberrations. We performed genome-wide single nucleotide polymorphism-array in hereditary coagulopathy to delineate the extent of copy number mutations and to assess its diagnostic utility. DESIGN AND METHODS: The study subjects were 17 patients with hereditary coagulopathy from copy number mutations in coagulation genes detected by multiple ligation-dependent probe amplification. Eleven had hemophilia (7 hemophilia A and 4 hemophilia B) and 6 had thrombophilia (4 protein S deficiency and 2 antithrombin deficiency). Single nucleotide polymorphism-array experiments were performed using Affymetrix Genome-Wide Human SNP arrays 6.0. RESULTS: Copy number mutations were identified by single nucleotide polymorphism-array in 9 patients, which ranged in length from 51 Kb to 6,288 Kb harboring 2 to ~160 genes. Single nucleotide polymorphism-array showed a neutral copy number status in 8 patients including 7 with either a single-exon copy number mutation or duplication mutations of PROS1. CONCLUSIONS: This study revealed unexpectedly heterogeneous lengths of copy number mutations underlying human coagulopathy. Single nucleotide polymorphism-array had limitations in detecting copy number mutations involving a single exon or those of a gene with homologous sequences such as a pseudogene.

Gene mutations in the Ras pathway and the prognostic implication in Korean patients with juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a rare hematologic malignancy in children. Hyperactivation of the Ras pathway from gene mutations is known to be the key culprit in the development of JMML. In this study, we investigated Ras pathway mutations and prognostic implication in Korean patients with JMML. A total of 22 Korean patients with JMML were recruited from two institutions (19 boys and three girls; median age, 17 months; range, 1-74 months). Hematologic and cytogenetic findings were reviewed. Mutation analyses involved PTPN11, KRAS, NRAS, and CBL genes by direct sequencing analyses (selected exons except in CBL). Survival analysis was performed by the Kaplan-Meier method. Cytogenetic and/or gene mutations were detected in 18 patients out of 22 (82%). Four patients (18%) had chromosomal abnormalities, with monosomy 7 being the most common. Seventeen (77%) had gene mutations. PTPN11 mutations were detected in 13 patients (59%). The patient heterozygous for c.854T>C had Noonan syndrome. NRAS and KRAS mutations were detected in two patients (9%) and one patient (5%), respectively. A homozygous CBL mutation was detected in one patient (5%; c.1228-2A>G). All mutations detected were previously reported mutations. Survival analyses suggested an unfavorable prognostic implication of PTPN11 mutation, albeit without a statistical significance. Collectively, the results from molecular genetics study and survival analyses suggested a relatively higher frequency and unfavorable prognostic implication of PTPN11 mutations in Korean patients with JMML.

A novel mutation Gly603Arg of TMPRSS6 in a Korean female with iron-refractory iron deficiency anemia.

Iron-refractory iron deficiency anemia (IRIDA) is a rare hereditary form of IDA with autosomal recessive inheritance. IRIDA is characterized by hypochromic microcytic anemia unresponsive to oral iron treatment, low transferrin saturation, and a high level of iron-regulated hormone hepcidin. The genetic background of IRIDA is mutations in the TMPRSS6 gene encoding matriptase-2 (TMPRSS6) that prevent inactivation of hemojuvelin, an activator of hepcidin transcription. We herein report a Korean female with IRIDA who was compound heterozygous for two mutations in TMPRSS6: a novel missense mutation c.1807G>C (p.Gly603Arg) in the serine protease domain and a known splicing mutation c.863+1G>T (IVS6+1G>T).

Novel and recurrent mutations of ITGA2B and ITGB3 genes in Korean patients with Glanzmann thrombasthenia.

Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder caused by defective glycoprotein, αIIb and ß3, encoded by ITGA2B and ITGB3 genes, respectively. We herein describe four unrelated Korean patients with genetically confirmed GT. Two patients were homozygous for c.1913+5G>T (IVS11+5G>T) mutation of ITGB3 with a signature of founder effect. The other two patients were compound heterozygous for two mutations of ITGA2B: c.[2333A>C];[2975delA] (p.[Q778P];[E992Gfs*30]) and c.[1750C>T];[2333A>C] (p.[R584X];[Q778P]). The c.2975delA mutation was a novel frameshift mutation of ITGA2B. Although from a limited number of patients, these results suggests c.1913+5G>T of ITGB3 is a recurrent mutation in Korean patients with GT.

A novel nonsense mutation in the MPL gene in congenital amegakaryocytic thrombocytopenia.

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare autosomal recessive disorder characterized by thrombocytopenia from failure of megakaryopoiesis. CAMT is one of the bone marrow failure syndromes, and the disease progression may involve other lineages leading to pancytopenia. The genetic background of CAMT is mutations in the MPL gene encoding the thrombopoietin receptor. Here, we describe a Korean male with CAMT. Molecular genetic analyses by direct sequencing revealed that he was compound heterozygous for two nonsense mutations in MPL, Tyr63X (c.189C>A), and Arg357X (c.1069C>T), the latter being a novel mutation.

UNC13D is the predominant causative gene with recurrent splicing mutations in Korean patients with familial hemophagocytic lymphohistiocytosis.

BACKGROUND: Familial hemophagocytic lymphohistiocytosis is a fatal disease characterized by immune dysregulation from defective function of cytotoxic lymphocytes. Three causative genes have been identified for this autosomal recessive disorder (PRF1, UNC13D, and STX11). We investigated the molecular genetics of familial hemophagocytic lymphohistiocytosis in Korea. DESIGN AND METHODS: Pediatric patients who fulfilled the HLH-2004 criteria were recruited from the Korean Registry for Histiocytosis. Molecular genetic studies were performed on the patients' DNA samples by direct sequencing of all coding exons and flanking sequences of PRF1, UNC13D, and STX11. RESULTS: Forty patients were studied and familial hemophagocytic lymphohistiocytosis mutations were identified in nine; eight patients had UNC13D mutations (89%) and one had a mutation in PRF1. No patient had a STX11 mutation. Notably, four patients had only one UNC13D mutant allele, suggesting that the other mutation was missed by conventional direct sequencing. All UNC13D mutations were deleterious in nature. One known splicing mutation, c.754-1G>C, was recurrent, accounting for 58% of all the mutant alleles (7/12). Five UNC13D mutations were novel (p.Gln98X, p.Glu565SerfsX7, c.1993-2A>G, c.2367+1G>A, and c.2954+5G>A). The one patient with PRF1 mutation was homozygous for a frameshift mutation (p.Leu364GlufsX93), which was previously reported to be the most frequent PRF1 mutation in Japan. CONCLUSIONS: This is the first investigation on the molecular genetics of familial hemophagocytic lymphohistiocytosis in Korea. The data showed that UNC13D is the predominant causative gene in the Korean population. The identification of mutations missed by conventional sequencing would better delineate the mutation spectrum and help to establish the optimal molecular diagnostic strategy for familial hemophagocytic lymphohistiocytosis in Korea, which might need an RNA-based screening strategy.

A novel initiation codon mutation in the ribosomal protein S17 gene (RPS17) in a patient with Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by pure red cell aplasia, various congenital anomalies, and cancer predisposition. We report a novel mutation in the RPS17 gene in a Korean patient with DBA. The mutation occurred in the translation initiation codon, changing Atg to Gtg (c.1A>G), thus disrupting the natural start of the RPS17 protein biosynthesis. This is the third case of DBA from a RPS17 mutation in the literature and is the second case of a RPS17 mutation in the translation initiation codon, following c.2T>G.

Myelodysplastic Syndrome/Myeloproliferative Neoplasm with Ring Sideroblasts and Thrombocytosis with Cooccurrent SF3B1 and MPL Gene Mutations: A Case Report and Brief Review of the Literature.

BACKGROUND: Myelodysplastic syndrome/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T) is a new disease entity in the current WHO classification. Genetically, 60%-90% of cases have mutations in SF3B1, strongly associated with RS, and more than half of them cooccur with JAK2 V617F. This report describes the rare case of MDS/MPN-RS-T with SF3B1 mutation cooccurring with an MPL mutation. METHODS: We report a 79-year-old man who was referred because of generalized edema. Peripheral blood testing showed macrocytic anemia and thrombocytosis, and bone marrow analysis demonstrated dyserythropoiesis with RS and increased megakaryocytes. A molecular study was performed to detect SF3B1 mutations and recurrent mutations in MPN disease (JAK2 V617F/exon 12, CALR gene exon 9, and MPL gene exon 10 mutations). RESULTS: The molecular study revealed SF3B1 K666T and MPL W515R mutations, while BCR-ABL1 or JAK2 V617F/exon 12 and CALR mutations were all negative. CONCLUSION: This is a rare case of concomitant SF3B1 and MPL mutations in MDS/MPN-RS-T.

Congenital factor V deficiency from compound heterozygous mutations with a novel variant c.2426del (p.Pro809Hisfs*2) in the F5 gene: A case report.

INTRODUCTION: Congenital factor V deficiency (FVD) is a rare bleeding disorder characterized by low or undetectable plasma factor V (FV) levels leading to mild to severe bleeding symptoms. Currently, more than 100 mutations have been reported in F5. We herein report a patient with FVD from mutations in the F5 gene. PATIENT CONCERNS: A 52-year-old man with prolonged prothrombin time and activated partial thromboplastin time corrected by mixing test on preoperative screening. His past medical or family history was not remarkable. DIAGNOSIS: Factor assays revealed a markedly reduced FV activity at 7%. Other factors were not decreased. DNA sequencing analysis to detect F5 gene mutations showed the patient was compound heterozygous for c.286G>C (p.Asp96His) and c.2426del (p.Pro809Hisfs*2). Asp96His was previously described missense mutation and Pro809Hisfs*2 was a novel deleterious mutation. INTERVENTIONS: Fresh-frozen plasma was administered to supplement FV before surgery. OUTCOMES: Subsequent factor assays revealed temporarily increased FV activity at 33%. CONCLUSION: As was the case in our patient, genotype-phenotype correlations are poor in FVD, and molecular genetic test is necessary to confirm the diagnosis.