Novel variants in GALE cause syndromic macrothrombocytopenia by disrupting glycosylation and thrombopoiesis.

Glycosylation is recognized as a key process for proper megakaryopoiesis and platelet formation. The enzyme uridine diphosphate (UDP)-galactose-4-epimerase, encoded by GALE, is involved in galactose metabolism and protein glycosylation. Here, we studied 3 patients from 2 unrelated families who showed lifelong severe thrombocytopenia, bleeding diathesis, mental retardation, mitral valve prolapse, and jaundice. Whole-exome sequencing revealed 4 variants that affect GALE, 3 of those previously unreported (Pedigree A, p.Lys78ValfsX32 and p.Thr150Met; Pedigree B, p.Val128Met; and p.Leu223Pro). Platelet phenotype analysis showed giant and/or grey platelets, impaired platelet aggregation, and severely reduced alpha and dense granule secretion. Enzymatic activity of the UDP-galactose-4-epimerase enzyme was severely decreased in all patients. Immunoblotting of platelet lysates revealed reduced GALE protein levels, a significant decrease in N-acetyl-lactosamine (LacNAc), showing a hypoglycosylation pattern, reduced surface expression of gylcoprotein Ibα-IX-V (GPIbα-IX-V) complex and mature ß1 integrin, and increased apoptosis. In vitro studies performed with patients-derived megakaryocytes showed normal ploidy and maturation but decreased proplatelet formation because of the impaired glycosylation of the GPIbα and ß1 integrin, and reduced externalization to megakaryocyte and platelet membranes. Altered distribution of filamin A and actin and delocalization of the von Willebrand factor were also shown. Overall, this study expands our knowledge of GALE-related thrombocytopenia and emphasizes the critical role of GALE in the physiological glycosylation of key proteins involved in platelet production and function.

Gut microbiota and its relationship with early vascular ageing in a Spanish population (MIVAS study).

BACKGROUND: Gut microbiota and its by-products are increasingly recognized as having a decisive role in cardiovascular diseases. The aim is to study the relationship between gut microbiota and early vascular ageing (EVA). METHODS: A cross-sectional study was developed in Salamanca (Spain) in which 180 subjects aged 45-74 years were recruited. EVA was defined by the presence of at least one of the following: carotid-femoral pulse wave velocity (cf-PWV), cardio-ankle vascular index (CAVI) or brachial-ankle pulse wave velocity (ba-PWV) above the 90th percentile of the reference population. All other cases were considered normal vascular ageing (NVA). MEASUREMENTS: cf-PWV was measured by SphygmoCor® System; CAVI and ba-PWV were determined by Vasera 2000® device. Gut microbiome composition in faecal samples was determined by 16S rRNA Illumina sequencing. RESULTS: Mean age was 64.4 ± 6.9 in EVA group and 60.4 ± 7.6 years in NVA (p < .01). Women in EVA group were 41% and 53% in NVA. There were no differences in the overall composition of gut microbiota between the two groups when evaluating Firmicutes/Bacteriodetes ratio, alfa diversity (Shannon Index) and beta diversity (Bray-Curtis). Bilophila, Faecalibacterium sp.UBA1819 and Phocea, are increased in EVA group. While Cedecea, Lactococcus, Pseudomonas, Succiniclasticum and Dielma exist in lower abundance. In logistic regression analysis, Bilophila (OR: 1.71, 95% CI: 1.12-2.6, p = .013) remained significant. CONCLUSIONS: In the studied Spanish population, early vascular ageing is positively associated with gut microbiota abundance of the genus Bilophila. No relationship was found between phyla abundance and measures of diversity.

GALE variants associated with syndromic manifestations, macrothrombocytopenia, bleeding, and platelet dysfunction.

GALE gene encodes the uridine diphosphate [UDP]-galactose-4-epimerase, which catalyzes the bidirectional interconversion of UDP-glucose to UDP-galactose, and UDP-N-acetyl-glucosamine to UDP-N-acetyl-galactosamine. In that way, GALE balances, through reversible epimerization, the pool of four sugars that are essential during the biosynthesis of glycoproteins and glycolipids. GALE-related disorder presents an autosomal recessive inheritance pattern, and it is commonly associated with galactosemia. Peripheral galactosemia generally associates with non-generalized forms or even asymptomatic presentations, while classical galactosemia may be related to complications such as learning difficulties, developmental delay, cardiac failure, or dysmorphic features. Recently, GALE variants have been related to severe thrombocytopenia, pancytopenia, and in one patient, to myelodysplastic syndrome.

What is the context? GALE gene encodes for the UDP-Galactose 4-Epimerase, an enzyme involved in the Leloir pathway of galactose catabolism and protein glycosylation.Homozygous or compound heterozygous GALE variants associate with the disorder known as galactosemia type III.Three types of galactosemia can be distinguished: the peripheral, the intermediate, and the generalized form, which associate with different clinical symptoms and GALE genetic variants.Peripheral form is considered benign, while the intermediate and the generalized form is associated with severe and syndromic manifestations, including learning difficulties, delayed growth, sensorineural hearing loss, and early-onset cataracts, among others.What is new? In the last few years, GALE variants have been linked to hematological manifestations, such as anemia, febrile neutropenia, and severe thrombocytopenia.To date, the only GALE variants described in patients presenting hematological disorders are GALE p.Arg51Trp, p.Lys78ValfsX32, p.Val128Met, p.Thr150Met, p.Leu223Pro, and p.Gly237Asp.The thrombocytopenia observed in GALE patients is associated with reduced GPIbα and ß1 integrin glycosylation and externalization to the megakaryocyte and platelet surface, disrupting the actin cytoskeleton remodeling.What is the impact? GALE is an essential protein for the correct megakaryocyte and platelet glycosylation.

Inherited Thrombocytopenia Caused by Variants in Crucial Genes for Glycosylation.

Protein glycosylation, including sialylation, involves complex and frequent post-translational modifications, which play a critical role in different biological processes. The conjugation of carbohydrate residues to specific molecules and receptors is critical for normal hematopoiesis, as it favors the proliferation and clearance of hematopoietic precursors. Through this mechanism, the circulating platelet count is controlled by the appropriate platelet production by megakaryocytes, and the kinetics of platelet clearance. Platelets have a half-life in blood ranging from 8 to 11 days, after which they lose the final sialic acid and are recognized by receptors in the liver and eliminated from the bloodstream. This favors the transduction of thrombopoietin, which induces megakaryopoiesis to produce new platelets. More than two hundred enzymes are responsible for proper glycosylation and sialylation. In recent years, novel disorders of glycosylation caused by molecular variants in multiple genes have been described. The phenotype of the patients with genetic alterations in GNE, SLC35A1, GALE and B4GALT is consistent with syndromic manifestations, severe inherited thrombocytopenia, and hemorrhagic complications.

TRAF3 alterations are frequent in del-3'IGH chronic lymphocytic leukemia patients and define a specific subgroup with adverse clinical features.

Interstitial 14q32 deletions involving IGH gene are infrequent events in chronic lymphocytic leukemia (CLL), affecting less than 5% of patients. To date, little is known about their clinical impact and molecular underpinnings, and its mutational landscape is currently unknown. In this work, a total of 871 CLLs were tested for the IGH break-apart probe, and 54 (6.2%) had a 300 kb deletion of 3'IGH (del-3'IGH CLLs), which contributed to a shorter time to first treatment (TFT). The mutational analysis by next-generation sequencing of 317 untreated CLLs (54 del-3'IGH and 263 as the control group) showed high mutational frequencies of NOTCH1 (30%), ATM (20%), genes involved in the RAS signaling pathway (BRAF, KRAS, NRAS, and MAP2K1) (15%), and TRAF3 (13%) within del-3'IGH CLLs. Notably, the incidence of TRAF3 mutations was significantly higher in del-3'IGH CLLs than in the control group (p < .001). Copy number analysis also revealed that TRAF3 loss was highly enriched in CLLs with 14q deletion (p < .001), indicating a complete biallelic inactivation of this gene through deletion and mutation. Interestingly, the presence of mutations in the aforementioned genes negatively refined the prognosis of del-3'IGH CLLs in terms of overall survival (NOTCH1, ATM, and RAS signaling pathway genes) and TFT (TRAF3). Furthermore, TRAF3 biallelic inactivation constituted an independent risk factor for TFT in the entire CLL cohort. Altogether, our work demonstrates the distinct genetic landscape of del-3'IGH CLL with multiple molecular pathways affected, characterized by a TRAF3 biallelic inactivation that contributes to a marked poor outcome in this subgroup of patients.

Co-occurrence of cohesin complex and Ras signaling mutations during progression from myelodysplastic syndromes to secondary acute myeloid leukemia.

Myelodysplastic syndromes (MDS) are hematological disorders at high risk of progression to secondary acute myeloid leukemia (sAML). However, the mutational dynamics and clonal evolution underlying disease progression are poorly understood at present. To elucidate the mutational dynamics of pathways and genes occurring during the evolution to sAML, next generation sequencing was performed on 84 serially paired samples of MDS patients who developed sAML (discovery cohort) and 14 paired samples from MDS patients who did not progress to sAML during follow-up (control cohort). Results were validated in an independent series of 388 MDS patients (validation cohort). We used an integrative analysis to identify how mutations, alone or in combination, contribute to leukemic transformation. The study showed that MDS progression to sAML is characterized by greater genomic instability and the presence of several types of mutational dynamics, highlighting increasing (STAG2) and newly-acquired (NRAS and FLT3) mutations. Moreover, we observed cooperation between genes involved in the cohesin and Ras pathways in 15-20% of MDS patients who evolved to sAML, as well as a high proportion of newly acquired or increasing mutations in the chromatin-modifier genes in MDS patients receiving a disease-modifying therapy before their progression to sAML.

Chronic lymphocytic leukemia patients with IGH translocations are characterized by a distinct genetic landscape with prognostic implications.

Chromosome 14q32 rearrangements/translocations involving the immunoglobulin heavy chain (IGH) are rarely detected in chronic lymphocytic leukemia (CLL). The prognostic significance of the IGH translocation is controversial and its mutational profile remains unknown. Here, we present for the first time a comprehensive next-generation sequencing (NGS) analysis of 46 CLL patients with IGH rearrangement (IGHR-CLLs) and we demonstrate that IGHR-CLLs have a distinct mutational profile with recurrent mutations in NOTCH1, IGLL5, POT1, BCL2, FBXW7, ZMYM3, MGA, BRAF and HIST1H1E genes. Interestingly, BCL2 and FBXW7 mutations were significantly associated with this subgroup and almost half of BCL2, IGLL5 and HISTH1E mutations reported were previously identified in non-Hodgkin lymphomas. Notably, IGH/BCL2 rearrangements were associated with a lower mutation frequency and carried BCL2 and IGLL5 mutations, while the other IGHR-CLLs had mutations in genes related to poor prognosis (NOTCH1, SF3B1 and TP53) and shorter time to first treatment (TFT). Moreover, IGHR-CLLs patients showed a shorter TFT than CLL patients carrying 13q-, normal fluorescence in situ hybridization (FISH) and +12 CLL, being this prognosis particularly poor when NOTCH1, SF3B1, TP53, BIRC3 and BRAF were also mutated. The presence of these mutations not only was an independent risk factor within IGHR-CLLs, but also refined the prognosis of low-risk cytogenetic patients (13q-/normal FISH). Hence, our study demonstrates that IGHR-CLLs have a distinct mutational profile from the majority of CLLs and highlights the relevance of incorporating NGS and the status of IGH by FISH analysis to refine the risk-stratification CLL model.

Spanish Guidelines for the use of targeted deep sequencing in myelodysplastic syndromes and chronic myelomonocytic leukaemia.

The landscape of medical sequencing has rapidly changed with the evolution of next generation sequencing (NGS). These technologies have contributed to the molecular characterization of the myelodysplastic syndromes (MDS) and chronic myelomonocytic leukaemia (CMML), through the identification of recurrent gene mutations, which are present in >80% of patients. These mutations contribute to a better classification and risk stratification of the patients. Currently, clinical laboratories include NGS genomic analyses in their routine clinical practice, in an effort to personalize the diagnosis, prognosis and treatment of MDS and CMML. NGS technologies have reduced the cost of large-scale sequencing, but there are additional challenges involving the clinical validation of these technologies, as continuous advances are constantly being made. In this context, it is of major importance to standardize the generation, analysis, clinical interpretation and reporting of NGS data. To that end, the Spanish MDS Group (GESMD) has expanded the present set of guidelines, aiming to establish common quality standards for the adequate implementation of NGS and clinical interpretation of the results, hoping that this effort will ultimately contribute to the benefit of patients with myeloid malignancies.

Transcriptomic analysis of patients with immune thrombocytopenia treated with eltrombopag.

In the last years, the use of thrombopoietin receptor agonists (TPO-RA), eltrombopag and romiplostim, has improved the management of immune thrombocytopenia (ITP). Moreover, eltrombopag is also active in patients with aplastic anemia and myelodysplastic syndrome. However, their mechanisms of action and signaling pathways still remain controversial. In order to gain insight into the mechanisms underlying eltrombopag therapy, a gene expression profile (GEP) analysis in patients treated with this drug was carried out. Fourteen patients with chronic ITP were studied by means of microarrays before and during eltrombopag treatment. Median age was 78 years (range, 35-87 years); median baseline platelet count was 14 × 109/L (range, 2-68 × 109/L). Ten patients responded to the therapy, two cases relapsed after an initial response and the remaining two were refractory to the therapy. Eltrombopag induced relevant changes in the hematopoiesis, platelet activation and degranulation, as well as in megakaryocyte differentiation, with overexpression of some transcription factors and the genes PPBP, ITGB3, ITGA2B, F13A1, F13A1, MYL9 and ITGA2B. In addition, GP1BA, PF4, ITGA2B, MYL9, HIST1H4H and HIST1H2BH, genes regulated by RUNX1 were also significantly enriched after eltrombopag therapy. Furthermore, in non-responder patients, an overexpression of Bcl-X gene and genes involved in erythropoiesis, such as SLC4A1 and SLC25A39, was also observed. To conclude, overexpression in genes involved in megakaryopoiesis, platelet adhesion, degranulation and aggregation was observed in patients treated with eltrombopag. Moreover, an important role regarding heme metabolism was also present in non-responder patients.

Molecular Diagnosis of Inherited Coagulation and Bleeding Disorders.

Diagnosis of inherited bleeding disorders (IBDs) remains challenging, especially in the case of inherited platelet disorders, due to the heterogeneity of the clinical and laboratory phenotype, the limited specificity of platelet function tests, and the large number of potential culprit genes. Unraveling the underlying molecular defect provides the definitive diagnosis of IBDs, facilitating prognosis and clinical care, which are especially important for severe clinical syndromes and those that may be associated with an increased risk of malignancy. Until recently, Sanger sequencing of candidate genes has been the only method of molecular diagnosis, but this approach is time-consuming and costly and requires phenotype-based identification of any obvious candidate gene(s). Nowadays, high-throughput sequencing (HTS) allows the simultaneous and rapid investigation of multiple genes at a manageable cost. This HTS technology that includes targeted sequencing of prespecified genes, whole-exome sequencing, or whole-genome sequencing, is revolutionizing the genetic diagnosis of human diseases. Through its extensive implementation in research and clinical practice, HTS is rapidly improving the molecular characterization of IBDs. However, despite the availability of this powerful approach, many patients still do not receive a diagnosis. As IBDs are complex and rare diseases, development of more advanced laboratory assays, improvements in bioinformatic pipelines, and the formation of multidisciplinary teams are encouraged to advance our understanding of IBDs.

Targeted genome editing in acute lymphoblastic leukemia: a review.

BACKGROUND: Genome editing technologies offers new opportunities for tackling diseases such as acute lymphoblastic leukemia (ALL) that have been beyond the reach of previous therapies. RESULTS: We show how the recent availability of genome-editing tools such as CRISPR-Cas9 are an important means of advancing functional studies of ALL through the incorporation, elimination and modification of somatic mutations and fusion genes in cell lines and mouse models. These tools not only broaden the understanding of the involvement of various genetic alterations in the pathogenesis of the disease but also identify new therapeutic targets for future clinical trials. CONCLUSIONS: New approaches including CRISPR-Cas9 are crucial for functional studies of genetic aberrations driving cancer progression, and that may be responsible for treatment resistance and relapses. By using this approach, diseases can be more faithfully reproduced and new therapeutic targets and approaches found.

Introducing high-throughput sequencing into mainstream genetic diagnosis practice in inherited platelet disorders.

Inherited platelet disorders are a heterogeneous group of rare diseases, caused by inherited defects in platelet production and/or function. Their genetic diagnosis would benefit clinical care, prognosis and preventative treatments. Until recently, this diagnosis has usually been performed via Sanger sequencing of a limited number of candidate genes. High-throughput sequencing is revolutionizing the genetic diagnosis of diseases, including bleeding disorders. We have designed a novel high-throughput sequencing platform to investigate the unknown molecular pathology in a cohort of 82 patients with inherited platelet disorders. Thirty-four (41.5%) patients presented with a phenotype strongly indicative of a particular type of platelet disorder. The other patients had clinical bleeding indicative of platelet dysfunction, but with no identifiable features. The high-throughput sequencing test enabled a molecular diagnosis in 70% of these patients. This sensitivity increased to 90% among patients suspected of having a defined platelet disorder. We found 57 different candidate variants in 28 genes, of which 70% had not previously been described. Following consensus guidelines, we qualified 68.4% and 26.3% of the candidate variants as being pathogenic and likely pathogenic, respectively. In addition to establishing definitive diagnoses of well-known inherited platelet disorders, high-throughput sequencing also identified rarer disorders such as sitosterolemia, filamin and actinin deficiencies, and G protein-coupled receptor defects. This included disease-causing variants in DIAPH1 (n=2) and RASGRP2 (n=3). Our study reinforces the feasibility of introducing high-throughput sequencing technology into the mainstream laboratory for the genetic diagnostic practice in inherited platelet disorders.

Mutations in TP53 and JAK2 are independent prognostic biomarkers in B-cell precursor acute lymphoblastic leukaemia.

BACKGROUND: In B-cell precursor acute lymphoblastic leukaemia (B-ALL), the identification of additional genetic alterations associated with poor prognosis is still of importance. We determined the frequency and prognostic impact of somatic mutations in children and adult cases with B-ALL treated with Spanish PETHEMA and SEHOP protocols. METHODS: Mutational status of hotspot regions of TP53, JAK2, PAX5, LEF1, CRLF2 and IL7R genes was determined by next-generation deep sequencing in 340 B-ALL patients (211 children and 129 adults). The associations between mutation status and clinicopathological features at the time of diagnosis, treatment outcome and survival were assessed. Univariate and multivariate survival analyses were performed to identify independent prognostic factors associated with overall survival (OS), event-free survival (EFS) and relapse rate (RR). RESULTS: A mutation rate of 12.4% was identified. The frequency of adult mutations was higher (20.2% vs 7.6%, P=0.001). TP53 was the most frequently mutated gene (4.1%), followed by JAK2 (3.8%), CRLF2 (2.9%), PAX5 (2.4%), LEF1 (0.6%) and IL7R (0.3%). All mutations were observed in B-ALL without ETV6-RUNX1 (P=0.047) or BCR-ABL1 fusions (P<0.0001). In children, TP53mut was associated with lower OS (5-year OS: 50% vs 86%, P=0.002) and EFS rates (5-year EFS: 50% vs 78.3%, P=0.009) and higher RR (5-year RR: 33.3% vs 18.6% P=0.037), and was independently associated with higher RR (hazard ratio (HR)=4.5; P=0.04). In adults, TP53mut was associated with a lower OS (5-year OS: 0% vs 43.3%, P=0.019) and a higher RR (5-year RR: 100% vs 61.4%, P=0.029), whereas JAK2mut was associated with a lower EFS (5-year EFS: 0% vs 30.6%, P=0.035) and a higher RR (5-year RR: 100% vs 60.4%, P=0.002). TP53mut was an independent risk factor for shorter OS (HR=2.3; P=0.035) and, together with JAK2mut, also were independent markers of poor prognosis for RR (TP53mut: HR=5.9; P=0.027 and JAK2mut: HR=5.6; P=0.036). CONCLUSIONS: TP53mut and JAK2mut are potential biomarkers associated with poor prognosis in B-ALL patients.

Multidimensional assessment of patient condition and mutational analysis in peripheral blood, as tools to improve outcome prediction in myelodysplastic syndromes: A prospective study of the Spanish MDS group.

The International Prognostic Scoring System and its revised form (IPSS-R) are the most widely used indices for prognostic assessment of patients with myelodysplastic syndromes (MDS), but can only partially account for the observed variation in patient outcomes. This study aimed to evaluate the relative contribution of patient condition and mutational status in peripheral blood when added to the IPSS-R, for estimating overall survival and the risk of leukemic transformation in patients with MDS. A prospective cohort (2006-2015) of 200 consecutive patients with MDS were included in the study series and categorized according to the IPSS-R. Patients were further stratified according to patient condition (assessed using the multidimensional Lee index for older adults) and genetic mutations (peripheral blood samples screened using next-generation sequencing). The change in likelihood-ratio was tested in Cox models after adding individual covariates. The addition of the Lee index to the IPSS-R significantly improved prediction of overall survival [hazard ratio (HR) 3.02, 95% confidence interval (CI) 1.96-4.66, P < 0.001), and mutational analysis significantly improved prediction of leukemic evolution (HR 2.64, 1.56-4.46, P < 0.001). Non-leukemic death was strongly linked to patient condition (HR 2.71, 1.72-4.25, P < 0.001), but not to IPSS-R score (P = 0.35) or mutational status (P = 0.75). Adjustment for exposure to disease-modifying therapy, evaluated as a time-dependent covariate, had no effect on the proposed model's predictive ability. In conclusion, patient condition, assessed by the multidimensional Lee index and patient mutational status can improve the prediction of clinical outcomes of patients with MDS already stratified by IPSS-R.

Wiskott-Aldrich syndrome in a child presenting with macrothrombocytopenia.

Wiskott-Aldrich syndrome (WAS) is a rare X-linked recessive disease resulting from variants in the WAS gene, characterized by a triad of immunodeficiency, eczema, and thrombocytopenia. Despite the fact that WAS is traditionally differentiated from immune thrombocytopenia (ITP) by small size of WAS platelets, in practice, microthrombocytopenia may occasionally not be present, and in certain cases, WAS patients exhibit some parallelism to ITP patients. We characterized one patient presenting with the classic form of the disease but increased mean platelet volume. Molecular studies revealed a novel hemizygous 1-bp deletion in WAS gene, c.802delC, leading to a frameshift and stop codon at amino acid 308 (p.Arg268Glyfs*40). Next-generation sequencing of a total of 70 additional genes known to harbor variants implicated in inherited platelet disorders did not identify additional defects. The pathogenesis of macrothrombocytopenia in this case is not known, but probably the coexistence of a still unidentified additional genetic variant might be involved.

The presence of genomic imbalances is associated with poor outcome in patients with burkitt lymphoma treated with dose-intensive chemotherapy including rituximab.

The introduction of Rituximab has improved the outcome and survival rates of Burkitt lymphoma (BL). However, early relapse and refractoriness are current limitations of BL treatment and new biological factors affecting the outcome of these patients have not been explored. This study aimed to identify the presence of genomic changes that could predict the response to new therapies in BL. Forty adolescent and adult BL patients treated with the Dose-Intensive Chemotherapy Including Rituximab (Burkimab) protocol (Spanish Programme for the Study and Treatment of Haematological Malignancies; PETHEMA) were analysed using array-based comparative genomic hybridization (CGH). In addition, the presence of TP53, TCF3 (E2A), ID3 and GNA13 mutations was assessed by next-generation sequencing (NGS). Ninety-seven per cent of the patients harboured genomic imbalances. Losses on 11q, 13q, 15q or 17p were associated with a poor response to Burkimab therapy (P = 0·038), shorter progression-free survival (PFS; P = 0·007) and overall survival (OS; P = 0·009). The integrative analysis of array-CGH and NGS showed that 26·3% (5/19) and 36·8% (7/19) of patients carried alterations in the TP53 and TCF3 genes, respectively. TP53 alterations were associated with shorter PFS (P = 0·011) while TCF3 alterations were associated with shorter OS (P = 0·032). Genetic studies could be used for risk stratification of BL patients treated with the Burkimab protocol.