A GP1BA Variant in a Czech Family with Monoallelic Bernard-Soulier Syndrome.

Bernard-Soulier syndrome (BSS) is a rare inherited disorder characterized by unusually large platelets, low platelet count, and prolonged bleeding time. BSS is usually inherited in an autosomal recessive (AR) mode of inheritance due to a deficiency of the GPIb-IX-V complex also known as the von Willebrand factor (VWF) receptor. We investigated a family with macrothrombocytopenia, a mild bleeding tendency, slightly lowered platelet aggregation tests, and suspected autosomal dominant (AD) inheritance. We have detected a heterozygous GP1BA likely pathogenic variant, causing monoallelic BSS. A germline GP1BA gene variant (NM_000173:c.98G > A:p.C33Y), segregating with the macrothrombocytopenia, was detected by whole-exome sequencing. In silico analysis of the protein structure of the novel GPIbα variant revealed a potential structural defect, which could impact proper protein folding and subsequent binding to VWF. Flow cytometry, immunoblot, and electron microscopy demonstrated further differences between p.C33Y GP1BA carriers and healthy controls. Here, we provide a detailed insight into its clinical presentation and phenotype. Moreover, the here described case first presents an mBSS patient with two previous ischemic strokes.

Genomic landscape of B-other acute lymphoblastic leukemia in an adult retrospective cohort with a focus on BCR-ABL1-like subtype.

INTRODUCTION: BCR-ABL1-like acute lymphoblastic leukemia (ALL) is a high-risk disease with a complex genomic background. Though extensively studied, data on the frequency and mutual associations of present mutations are still incomplete in adult patients. This retrospective study aims to map the genomic landscape of B-other ALL in a cohort of adult patients with a focus on the BCR-ABL1-like ALL subtype. METHODS: We analyzed bone marrow and peripheral blood samples of adult B-other ALL patients treated consecutively at three major Czech teaching hospitals. Samples were analyzed by cytogenetic methods, gene expression profiling, multiplex ligation-dependent probe amplification (MLPA), and next-generation sequencing (NGS). RESULTS: Fifty-eight B-other ALL patients (not BCR-ABL1, KMT2A-rearranged, ETV6-RUNX1, TCF3-PBX1, or iAMP21) were included in the study. Median follow-up was 23.8 months. Samples from 33 patients were available for a gene expression analysis, 48.9% identified as BCR-ABL1-like ALL. Of the BCR-ABL1-like ALL cases, 18.8% harbored IGH-CRLF2 and 12.5% P2RY8-CRLF2 fusion gene. We observed a higher MRD failure rate in BCR-ABL1-like than in non-BCR-ABL1-like ALL patients after the induction treatment (50.0 vs. 13.3%, p=.05). There was a trend to worse progression-free and overall survival in the BCR-ABL1-like group, though not statistically significant. Deletions in IKZF1 gene were found in 31.3% of BCR-ABL1-like cases. Patients with concurrent IKZF1 and CDKN2A/B, PAX5 or PAR1 region deletions (IKZF1plus profile) had significantly worse progression-free survival than those with sole IKZF1 deletion or IKZF1 wild-type (p=.02). NGS analysis was performed in 54 patients and identified 99 short variants in TP53, JAK2, NRAS, PAX5, CREBBP, NF1, FLT3, ATM, KRAS, RUNX1, and other genes. Seventy-five of these gene variants have not yet been described in B-cell precursor ALL to date. CONCLUSION: This study widens existing knowledge of the BCR-ABL1-like and B-other ALL genomic landscape in the adult population, supports previous findings, and identifies a number of novel gene variants.

Extensive, 3.8 Mb-Sized Deletion of 22q12 in a Patient with Bilateral Schwannoma, Intellectual Disability, Sensorineural Hearing Loss, and Epilepsy.

Introduction: In contrast with the well-known and described deletion of the 22q11 chromosome region responsible for DiGeorge syndrome, 22q12 deletions are much rarer. Only a few dozen cases have been reported so far. This region contains genes responsible for cell cycle control, chromatin modification, transmembrane signaling, cell adhesion, and neural development, as well as several cancer predisposition genes. Case Presentation: We present a patient with cleft palate, sensorineural hearing loss, vestibular dysfunction, epilepsy, mild to moderate intellectual disability, divergent strabism, pes equinovarus, platyspondylia, and bilateral schwannoma. Using Microarray-based Comparative Genomic Hybridization (aCGH), we identified the de novo 3.8 Mb interstitial deletion at 22q12.1→22q12.3. We confirmed deletion of the critical NF2 region by MLPA analysis. Discussion: Large 22q12 deletion in the proband encases the critical NF2 region, responsible for development of bilateral schwannoma. We compared the phenotype of the patient with previously reported cases. Interestingly, our patient developed cleft palate even without deletion of the MN1 gene, deemed responsible in previous studies. We also strongly suspect the DEPDC5 gene deletion to be responsible for seizures, consistent with previously reported cases.

Aggressive systemic mastocytosis with diffuse bone marrow 18F-FDG uptake.

Mastocytosis is a clonal hematopoietic disorder characterized by proliferation of abnormal mast cells in various organs including the skin, digestive system, lymph nodes, and bone marrow. We report on a 75-year-old woman presenting with abdominal pain, vomiting, diarrhoea, myalgia, and weight loss. Abdominal CT showed hepatosplenomegaly with heterogeneous splenic parenchyma, lymphadenopathy, and osteopenia with areas of osteosclerosis but no primary tumour. An 18F-FDG PET/CT revealed an overall low metabolic activity of the lesions with a diffuse bone marrow involvement raising suspicion of a haematological neoplasm. Subsequently, bone marrow and peripheral blood examinations confirmed the diagnosis of aggressive systemic mastocytosis.

MK-886 enhances tumour necrosis factor-alpha-induced differentiation and apoptosis.

We investigated the role of the 5-lipoxygenase (5-LOX) pathway of arachidonic acid metabolism in tumour necrosis factor-alpha (TNF-alpha)-induced differentiation of human leukemic HL-60 cells using MK-886, an inhibitor of 5-LOX activating protein. MK-886 augmented cell cycle arrest and differentiation induced by TNF-alpha; however, both effects were probably 5-LOX-independent, because a general LOX inhibitor, NDGA, had no effect. Apoptosis was significantly elevated after combined TNF-alpha and MK-886 treatment, which could be partially associated with changes of Mcl-1 protein expression. NF-kappaB signalling or activation of JNKs were not modulated by MK-886. Thus, in addition to apoptosis, MK-886 can enhance TNF-alpha-induced differentiation.

Molecular characterization of the rare translocation t(3;10)(q26;q21) in an acute myeloid leukemia patient.

BACKGROUND: In acute myeloid leukemia (AML), the MDS1 and EVI1 complex locus - MECOM, also known as the ecotropic virus integration site 1 - EVI1, located in band 3q26, can be rearranged with a variety of partner chromosomes and partner genes. Here we report on a 57-year-old female with AML who presented with the rare translocation t(3;10)(q26;q21) involving the MECOM gene. Our aim was to identify the fusion partner on chromosome 10q21 and to characterize the precise nucleotide sequence of the chromosomal breakpoint. METHODS: Cytogenetic and molecular-cytogenetic techniques, chromosome microdissection, next generation sequencing, long-range PCR and direct Sanger sequencing were used to map the chromosomal translocation. RESULTS: Using a combination of cytogenetic and molecular approaches, we mapped the t(3;10)(q26;q21) to the single nucleotide level, revealing a fusion of the MECOM gene (3q26.2) and C10orf107 (10q21.2). CONCLUSIONS: The approach described here opens up new possibilities in characterizing acquired as well as congenital chromosomal aberrations. In addition, DNA sequences of chromosomal breakpoints may be a useful tool for unique molecular minimal residual disease target identification in acute leukemia patients.

A method to identify new molecular markers for assessing minimal residual disease in acute leukemia patients.

Acute leukemias (AL) comprise a heterogeneous group of hematologic malignancies, and individual patient responses to treatment can be difficult to predict. Monitoring of minimal residual disease (MRD) is thus very important and holds great potential for improving treatment strategies. Common MRD targets include recurrent cytogenetic abnormalities and mutations in important hematological genes; unfortunately well-characterized targets are lacking in many AL patients. Here we demonstrate a technical approach for the identification and mapping of novel clone-specific chromosomal abnormalities down to the nucleotide level. We used molecular cytogenetics, chromosome microdissection, amplification of the microdissected material, and next-generation sequencing to develop PCR-based MRD assays based on unique breakpoint sequences.