Identification of variants in 94 Chinese patients with hereditary spherocytosis by next-generation sequencing.

Hereditary spherocytosis (HS) is the most common type of hereditary erythrocyte membrane disease and has varied phenotypic features and genetic patterns. We herein performed a retrospective study of 94 patients with HS and aimed to investigate the genetic variations and genotype-phenotype correlations using targeted next-generation sequencing. In 79/94 (84%) patients, 83 HS variants including 67 novel variants were identified. Pathogenic variants of SPTB, ANK1, SLC4A1, SPTA1, and EPB42 were found in 32/79(41%), 22/79(28%), 15/79 (19%), 8/79 (9%), and 3/79 (4%) of the patients respectively, revealing that SPTB is the most frequently mutated HS gene in Eastern China. Most SPTB and ANK1 gene variations were nonsense and frameshift variations. Missense variants were the main variant type of SLC4A1, SPTA1, and EPB42 genes. Interestingly, one SPTA1 variant (p. Arg1757Cys) showed an autosomal dominant inheritance pattern and one EPB42 variant (p. Gln377His) was apparent as a hotspot variation. Furthermore, genotype-phenotype analysis was performed among the five mutated gene groups. Besides the finding that patients with the SLC4A1 variant had the highest mean corpuscular hemoglobin levels, no clear correlations between genotype and phenotype were observed.

Integrative genomic and transcriptomic profiling reveals distinct molecular subsets in adult mixed phenotype acute leukemia.

Mixed phenotype acute leukemia (MPAL) is a subtype of leukemia in which lymphoid and myeloid markers are co-expressed. Knowledge regarding the genetic features of MPAL is lacking due to its rarity and heterogeneity. Here, we applied an integrated genomic and transcriptomic approach to explore the molecular characteristics of 176 adult patients with MPAL, including 86 patients with T-lymphoid/myeloid MPAL (T/My MPAL-NOS), 42 with Ph+ MPAL, 36 with B-lymphoid/myeloid MPAL (B/My MPAL-NOS), 4 with t(v;11q23), and 8 with MPAL, NOS, rare types. Genetically, T/My MPAL-NOS was similar to B/T MPAL-NOS but differed from Ph+ MPAL and B/My MPAL-NOS. T/My MPAL-NOS exhibited higher CEBPA, DNMT3A, and NOTCH1 mutations. Ph+ MPAL demonstrated higher RUNX1 mutations. B/T MPAL-NOS showed higher NOTCH1 mutations. By integrating next-generation sequencing and RNA sequencing data of 89 MPAL patients, we defined eight molecular subgroups (G1-G8) with distinct mutational and gene expression characteristics. G1 was associated with CEBPA mutations, G2 and G3 with NOTCH1 mutations, G4 with BCL11B rearrangement and FLT3 mutations, G5 and G8 with BCR::ABL1 fusion, G6 with KMT2A rearrangement/KMT2A rearrangement-like features, and G7 with ZNF384 rearrangement/ZNF384 rearrangement-like characteristics. Subsequently, we analyzed single-cell RNA sequencing data from five patients. Groups G1, G2, G3, and G4 exhibited overexpression of hematopoietic stem cell disease-like and common myeloid progenitor disease-like signatures, G5 and G6 had high expression of granulocyte-monocyte progenitor disease-like and monocyte disease-like signatures, and G7 and G8 had common lymphoid progenitor disease-like signatures. Collectively, our findings indicate that integrative genomic and transcriptomic profiling may facilitate more precise diagnosis and develop better treatment options for MPAL.

[Application of Bionano-DNA Full-Length Optical Mapping Technology in Genetic Diagnosis of Hemophilia A Carriers].

OBJECTIVE: To apply Bionano Saphyr visual full-length DNA optical mapping technology to the precise genetic diagnosis of hemophilia A carriers. METHODS: For 2 suspected F8 gene deficiency female carriers who could not be diagnosed by conventional next-generation sequencing technology, the full-length DNA optical mapping technology was used to detect and scan the sample X chromosome full-length visual haplotype characteristic map, which was compared with the normal haplotype. The gene structure variation information of the samples was obtained by compare with DNA atlas library. RESULTS: The average fluorescent marker length of the X chromosome DNA molecular where the F8 gene was located in the two samples was greater than 2.5 Mbp, and the average copy number was greater than 20×. After comparative analysis, one of the samples was a proximal inversion of intron 22 of the F8 gene, and another was an inversion of intron 22 accompanied by multiple deletions of large fragments. CONCLUSIONS: Bionano technology has a good detection rate for gene defects with large length and complex variation. In the absence of a proband or accurate genetic diagnosis results of the proband, the application of this technology to detect the heterozygous complex variant of the F8 gene is of great significance for the prenatal diagnosis and pre-pregnancy diagnosis of hemophilia carriers.

Treatments of Ph-like acute lymphoblastic leukemia: a real-world retrospective analysis from a single large center in China.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype of ALL. We retrospectively studied 70 cases with Ph-like ALL and here present the largest study of CAR-T cell treatment and haplo-HSCT for this leukemia. Median age was 26 years and median leukocyte count was 31.44 × 109/L. The proportion of patients receiving chemotherapy, KIs, CAR-T cells, and allo-HSCT was 19%, 30%, 46%, and 61%, respectively. The overall response rate was 62%, 73%, and 100% after one month of KI treatment combined with chemotherapy, CAR-T cell therapy, and allo-HSCT, respectively. Five-year DFS and OS were 35% and 51%, respectively. The five-year cumulative incidence of relapse and non-relapse mortality was 63% and 11%, respectively. Allo-HSCT was associated with a better DFS (p = 0.010) and OS (p = 0.000) by univariate analysis. In conclusion, allo-HSCT after KIs together with chemotherapy or CAR-T cell therapy is a safe and feasible treatment modality for Ph-like ALL.

A Recurrent Cryptic MED14-HOXA9 Rearrangement in an Adult Patient With Mixed-Phenotype Acute Leukemia, T/myeloid, NOS.

To define the fusion genes in T/myeloid mixed-phenotype acute leukemia (T/M MPAL), we performed transcriptome sequencing of diagnostic bone marrow samples from 20 adult patients. Our analysis identified a second instance of a recurrent MED14-HOXA9 chimeric gene resulting from the in-frame fusion of exon 23 of MED14 and exon 1 of HOXA9, the first in an adult patient. The MED14-HOXA9 fusion gene was detected in both the diagnostic and relapsed blasts with reverse transcription-polymerase chain reaction and Sanger sequencing. The patient received combined conventional chemotherapy but suffered relapse at 11 months and died of disease progression one year after the initial diagnosis. Our data suggest that MED14-HOXA9 is a cryptic recurrent aberration in T/M MPAL, which might indicate an aggressive clinical course and inferior outcome after conventional chemotherapy. Further studies will be carried out to reveal the effects of the MED14-HOXA9 fusion on the differentiation and proliferation of leukemia stem cells, as well as suitable treatment strategies for this emerging entity.

Efficacy of ruxolitinib in a patient with myelodysplastic/myeloproliferative neoplasm unclassifiable and co-mutated JAK2, SF3B1 and TP53.

Myelodysplastic/myeloproliferative neoplasm, unclassifiable (MDS/MPN-U) is a rare but heterogeneous subtype of MDS/MPN, with no specific genetic alterations and standard treatments. ASXL1, SRSF2, TET2, JAK2 and NRAS are commonly mutated in MDS/MPN-U. Double gene mutations could be detected in MDS/MPN-U, however, co-mutations of 3 and more genes in this disease entity are very rare. Here, we present a case of MDS/MPN-U with triple mutations involving JAK2, SF3B1, and TP53. After failure of traditional therapy including hydroxyurea and interferon-α, the patient received ruxolitinib monotherapy and achieved hematological response quickly. Though mutations in TP53 implied a poor prognosis in myeloid malignancies, this patient has maintained no AML transformation for 26 months since diagnosis. Further research on complex mutations in the pathogenesis and prognosis of MDS/MPN-U is warranted.

[ARMS-PCR combined with capillary electrophoresis can be a sensitive and quantitative method for detection of MYD88-L265P mutation in lymphoma].

OBJECTIVE: To investigate the feasibility of sensitive and quantitative detection of MYD88 gene L265P mutation in lymphoma patients by using ARMS-PCR combined with capillary electrophoresis. METHODS: ARMS-PCR amplified MYD88 gene was analyzed by capillary electrophoresis in ABI 3730 sequencer; Exon 5 of the same gene was sequenced bi-directionally as reported. RESULTS: The sensitivity of detection L265P mutations by the ARMS-PCR combined with capillary electrophoresis and direct sequencing was 0.2% and 5%, respectively, according to the detection of the gradient-diluted plasmid standards. The detection rate of 184 patients was 13.59% and 8.28%, respectively (p<0.001). Moreover, the former method can successfully detect the mutation ratio(R2=0.979), and the repeatabilities (CV=2.86%, 1.94%, 5.49%) are acceptable. CONCLUSION: ARMS-PCR combined with capillary electrophoresis can quantitatively detect the MYD88 gene L265P mutation, and the detection sensitivity is significantly higher than sanger sequencing. As a supplement to the latter, it can effectively lead to the earlier diagnose and monitoring of minimal residual disease.

[Genotype Analysis of Hemoglobinopathy in Chinese Jiangsu Population].

OBJECTIVE: To investigate the genotype distribution of hemoglobinopathy in Chinese Jiangsu population. METHOD: A total of 4115 samples were screened for hemaglobinopathy by using MCV combined with erythrocyte fragility tests and HPLC. Thalassemia genotypes were identified by Gap-PCR and Recerse Dot blot. PCR-DNA sequencing and PCR-elecrophoresis were used as supplement of PCR-RBD and for identifying the mutants of globin gene of abnormal hemoglobin. RESULTS: The positive screening rate was 6.10% (251/4115) in Chinese Jiangsu population, 232 cases received thalassemia genotype diagnosis and from them 195 people were positive. In all positive ones, α-thalassemia, ß-thalassemia, α-thalassemia combined with ß-thalassemia, SEA-HPFH and SEA-HPFH combined with ß-thalassemia were found respectively to be 31.28% (61/232), 66.15% (129/232), 1.54% (3/232), 0.43% (1/232) and 0.43% (1/232) of patients. The majority genotype of α-thalassemia was - - (SEA) and IVS-II-654 was the main genotype of ß-thalassemia, 11 cases of abnormal hemoglobin were found, including 3 cases of Hb E, 1 Hb Kenitra, 1 Hb Seattle, 1 Hb Saitama, 1 Hb Bushwick, 1 Hb Koln and 1 Hb M-Milwaukee-2. CONCLUSION: The main hemoglobinpathy is thalassemia in Chinese Jiangsu province and the HPLC play an important role in screening hemoglobinpathy. There is reference value of this study for genetic counseling and prenatal diagnosis.

T-SPOT.TB for detection of tuberculosis infection among hematological malignancy patients and hematopoietic stem cell transplant recipients.

The diagnosis of latent Mycobacterium tuberculosis infection (LTBI) is recommended in hematological malignancy patients and before hematopoietic stem cell transplantation (Guidelines for the prevention and management of infectious complications of solid organ transplantation, 2004). Compared to traditional methods such as tuberculin skin test (TST), T-SPOT.TB has been shown to be more specific. In the present study we enrolled 536 patients for whom T-SPOT.TB was performed, among which 295 patients also received the TST test. The agreement (79%) between T-SPOT.TB and TST was poor (?=0.274, P<0.001). The patients with positive T-SPOT.TB results numbered 62 (11.6%), in which only 20 (48.8%) of the 41 receiving the TST test had positive results. A majority of the patients with T-SPOT.TB positive results had some other evidence ofTB, such as TB history, clinical symptoms and an abnormal chest CT scan. Active TB was found in 9 patients, in which 2 had negative TST results. We followed up the patients and no one developed active TB. Our study suggested that the T-SPOT.TB may be more useful for screening LTBI and active TB in hematological malignancy patients and hematopoietic stem cell transplant recipients than the TST test.

[Expression of SET-NUP214 fusion gene in patients with T-cell acute lymphoblastic leukemia and its clinical significance].

This study was aimed to investigate the occurrence and clinical significance of the SET-NUP214 fusion gene in patients with T-cell acute lymphoblastic leukemia (T-ALL), analyse clinical and biological characteristics in this disease. RT-PCR was used to detect the expression of SET-NUP214 fusion gene in 58 T-ALL cases. Interphase FISH and Array-CGH were used to detect the deletion of 9q34. Direct sequencing was applied to detect mutations of PHF6 and NOTCH1. The results showed that 6 out of 58 T-ALL cases (10.3%) were detected to have the SET-NUP214 fusion gene by RT-PCR. Besides T-lineage antigens, expression of CD13 and(or) CD33 were detected in all the 6 cases. Deletions of 9q34 were detected in 4 out of the 6 patients by FISH. Array-CGH results of 3 SET-NUP214 positive T-ALL patients confirmed that this fusion gene was resulted from a cryptic deletion of 9q34.11q34.13. PHF6 and NOTCH1 gene mutations were found in 4 and 5 out of 6 SET-NUP214 positive T-ALL patients, respectively. It is concluded that SET-NUP214 fusion gene is often resulted from del(9)(q34). PHF6 and NOTCH1 mutations may be potential leukemogenic event in SET-NUP214 fusion gene.