Prevalence and clinical outcomes of germline variants among patients with myeloid neoplasms.

AIMS: Myeloid neoplasms (MNs) with germline predisposition have been recognised as a distinct entity. Emerging evidence suggests that sporadic myelodysplastic syndromes may also harbour undetected germline predispositions. We investigated germline alterations in a cohort of 122 adult Thai MNs. METHODS: MN patients were recruited and tested for germline variants using deep targeted next-generation sequencing. The germline variant was filtered using American College of Medical Genetics classifications and then evaluated for the association with clinical characteristics and outcomes. RESULTS: Our findings revealed pathogenic/likely pathogenic germline alterations in 12 (10%) of the patients. These germline lesions were commonly found in the DNA damage response pathway (n=6, 50%). We also identified novel deleterious FANCA A1219GfsTer59 variants in two patients diagnosed with secondary acute myeloid leukaemia (sAML) from aplastic anaemia and AML with myelodysplasia related. Among sAML, individuals with germline mutations had inferior overall survival compared with those with wild-type alleles (2 months vs 12 months) with HR 4.7 (95% CI 1.0 to 20), p=0.037. Therefore, the presence of pathogenic or likely pathogenic mutations may be linked to inferior survival outcomes. CONCLUSIONS: Our study highlighted that the prevalence of germline predisposition in Southeast Asian populations is comparable to that in Caucasians. This underscores the importance of germline genetic testing within the Asian population.

Stability of Hemoglobin Constant Spring Identified by Capillary Electrophoresis.

OBJECTIVE: Hemoglobin Constant Spring (HbCS) is often missed by routine hemoglobin analysis. The aim of this research was to study HbCS stability as identified by capillary electrophoresis (CE) to determine the specimen storage time limit. METHODS: The EDTA blood of 29 HbCS samples were kept at 4°C and analyzed every workday until CE could not detect HbCS or until 7 weeks after blood collection. The genotypes were confirmed by multiplex polymerase chain reaction. RESULTS: The median subject age was 27 years and 10 subjects were male. The HbCS levels were stable during the first 7 days but became undetectable in 5 cases (17.2%) after 1 week. All of them were heterozygous HbCS. Longer detection times were correlated with the higher baseline HbCS levels, with a correlation coefficient of 0.582 (P  ≤ 0.001). CONCLUSION: Routine hemoglobin typing and quantitation should be performed within 1 week after blood collection to detect low HbCS levels, especially in heterozygous HbCS.

Genetic mutations associated with blood count abnormalities in myeloid neoplasms.

INTRODUCTION: Myelodysplastic syndromes (MDS) predominantly present with varying degrees of cytopenia, while myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN) exhibit proliferative features. Genetic defects underlying different complete blood count (CBC) alterations remain to be defined. OBJECTIVE: We aimed to evaluate mutations and impacts on abnormal blood counts in MDS and MDS/MPN. METHOD: MDS and MDS/MPN patients were recruited and sequenced by targeted next-generation sequencing. Clinical parameters, especially CBC, were evaluated for the association with genetic abnormalities and clinical outcomes. RESULTS: A total of 168 patients with myeloid neoplasms were recruited (92 cases of low-risk MDS, 57 cases of high-risk MDS and 19 cases of MDS/MPN). Compared to low-risk MDS and MDS/MPN, patients with high-risk MDS were presented with more severe neutropenia with 17.5% showing absolute neutrophil counts (ANC) lower than 0.5 × 109/L. Patients with MDS/MPN more commonly harboured mutations and had a higher number of mutations per case than low-risk MDS (94.7% vs. 56.5%; p < 0.001 and 3 vs. 1; p < 0.001, respectively). Patients with SF3B1 mutations showed lower haemoglobin levels than wild-type (7.9 vs. 8.4 g/dL, p = 0.02), but were associated with normal platelet counts (286 vs. 93 × 109/L; p < 0.001). Patients with U2AF1 mutations were associated with more severe leukopenia than wild-type (3 vs. 4.18 × 109/L; p = 0.02). KRAS mutations were associated with monocytosis (p < 0.001). Multivariate analysis revealed high-risk MDS, MDS/MPN, severe neutropenia (ANC < 0.5 × 109/L), and mutations in ASXL1 and SETBP1 were associated with inferior survival outcomes. CONCLUSION: Certain mutations were related to more severe anaemia, lower white blood cell count or monocytosis in Asian MDS and MDS/MPN patients.

Excellent Prognosis of Low-Risk Myelodysplastic Syndromes (MDS) Without Detectable Myeloid-Related Mutations.

BACKGROUND: Unexplained cytopenia (UC) and low-risk myelodysplastic syndrome (MDS) are distinguished mainly by morphologic dysplasia, which sometimes shows inter-observer discrepancy. We hypothesized that gene mutations are strong prognostic factors for these low-risk patients. MATERIALS AND METHODS: We enrolled patients from 4 medical centers with unexplained cytopenia of at least 1 lineage. Diagnosis of low-risk MDS was made according to WHO 2016 classification and a revised international prognostic scoring system (R-IPSS) score of ≤ 3.5. DNA was extracted from bone marrow or blood and sequenced by targeted next generation sequencing (NGS). RESULTS: One hundred twenty-one patients were recruited: 25% with UC and 75% with low-risk MDS. Complete blood counts were similar, but low-risk MDS patients carried higher numbers of mutations (1 vs. 0; P = .04) than UC patients. Overall, the most frequent mutated genes were TET2 (14.6%), SF3B1 (12.2%), and ASXL1 (9.7%). Survival rates of low-risk MDS patients versus UC patients were not significantly different. UC patients and low-risk MDS patients without genetic abnormalities showed superior 5-year progression free survival compared to MDS patients with mutations (100% vs. 76.0%; P = .005). Overall, ASXL1 mutations were associated with decreased 4-year overall survival compared to wild-type (59% vs. 31%; P = .01). In a multivariate analysis, ASXL1 and DNMT3A mutations in low-risk MDS patients were associated with a higher risk of disease progression with hazard ratios of 7.88 (95% CI 1.76-35.32, P = .01) and 7.45 (95% CI 1.61-34.46, P = .01), respectively. CONCLUSION: Mutation detection is important for proper risk stratification of patients presenting with idiopathic cytopenia.

Comprehensive screening for coexisting heterozygous α0-thalassemia in hemoglobin E trait.

Objectives: A sensitive screening for the coexistence of α0-thalassemia and the hemoglobin E (Hb E) trait is important to identify at-risk couples for hydrops fetalis. However, previous cutoff values have shown a positive predictive value (PPV) of only 50% or less. This study aimed to define more specific indicators to reduce the need for DNA tests. Methods: Patients with Hb E trait, as diagnosed by high performance liquid chromatography (HPLC) and/or isoelectric focusing (IEF) techniques, were tested for α0-thalassemia and α+-thalassemia deletions using multiplex gap polymerase chain reaction. Iron deficiency anemia (IDA) were excluded using a red cell distribution width (RDW) of more than 14.5%. Results: From 390 specimens, suitable cutoff values showing a 100% sensitivity for detection of heterozygous α0-thalassemia were an Hb E level of less than 22% by HPLC, a mean corpuscular volume (MCV) of less than 72 fL, and a mean corpuscular hemoglobin (MCH) level of less than 22.5 pg. Comparable results were obtained in the validation cohort (N = 179). Using a combination of Hb E with either MCV or MCH cutoff points gave a PPV of 76.2% and 77.4%, respectively. Discussion: IDA was reported to interfere with Hb E level. In this study, we excluded IDA using RDW of more than 14.5% to enhance the test specificity. Conclusion: Lower cutoff screening values can be used to exclude α0-thalassemia in the Hb E trait yielding a higher specificity in a normal RDW condition. This can save the cost and labor of DNA testing.

Novel DDX41 variants in Thai patients with myeloid neoplasms.

Germline DDX41 mutations were recently reported to cause MDS/AML and donor-derived leukemia after transplantation. While previously described in Western countries, DDX41 variants have not been reported in a Southeast Asian population. We performed targeted sequencing of blood or bone marrow samples from 109 Thai patients with myeloid malignancies. Among the 109 patients (75 MDS, 8 MPN, 11 MDS/MPN and 15 AML), the most frequent mutations were in ASXL1 (17.4%), TET2 (16.5%) and SRSF2 (12.8%), respectively. DDX41 variants were detectable in six (5.5%) cases. Four patients exhibited three presumable germline DDX41 mutations: p.S21fs (n = 2), p.F235fs (n = 1), and p.R339H (n = 1). While p.S21fs was previously reported in myeloid neoplasm, the latter two variants have not been described. Two of these cases harbored concomitant probable germline/somatic DDX41 mutations (p.S21fs/p.R525H and p.R339H/p.K494T), while the other two patients carried only somatic mutations (p.R525H and p.F438L). The p.K494T and p.F438L variants have not been previously reported. In patients with DDX41 alterations, the diagnoses were MDS with excess blasts (4), secondary AML (1) and low-risk MDS (1). In conclusion, we identified DDX41 variants in Thai patients with myeloid malignancies in which these variants could be used to assess predisposition to MDS in Southeast Asia.

Natural anticoagulant deficiencies in Thais: A population-based study.

INTRODUCTION: In contrast to Caucasians, hereditary anticoagulant deficiencies are more common in Asians and mutations are heterogeneous among different countries. This study aimed to determine the prevalence and genetic basis of protein C and protein S deficiencies in Thai population. METHODS: Healthy volunteers were tested for protein C activity and free protein S antigen. Subjects with low values were requested for repeated testing and exclusion of acquired causes. Cases with persistently low levels were assayed for respective gene mutations using direct sequencing and multiplex ligation-dependent probe amplification (MPLA) when PROS1 point mutation was undetectable. RESULTS: For protein C activities (N = 5234), the values of men were lower than those of post-menopausal women (P < 0.001). In 17 of 18 subjects, there were 7 types of PROC mutations, 64.7% of which were p.R189W and 2 were previously unreported. Protein S levels (N = 5242) were highest in men, followed by post-menopausal and pre-menopausal women, respectively (P < 0.001). The repeatedly low protein S was mostly in female (88.2%). Among 29 subjects with protein S below the sex-specific 2.5 percentile cut-points, 4 types of mutations were found in 5 subjects (17.2%) with one previously unreported mutation. Free protein S levels were below 30 U/ml in all cases with mutations. The estimated prevalence of PROC and PROS1 mutations in Thai population was 0.69% and 0.22%, respectively. CONCLUSIONS: PROC mutations, especially p.R189W, are common in Thais. However, mildly depressed protein S levels were frequently found in women with undetectable PROS1 mutation.