Clinical, biological, and prognostic implications of SF3B1 co-occurrence mutations in very low/low- and intermediate-risk MDS patients.

SF3B1 is a highly mutated gene in myelodysplastic syndrome (MDS) patients, related to a specific subtype and parameters of good prognosis in MDS without excess blasts. More than 40% of MDS patients carry at least two myeloid-related gene mutations but little is known about the impact of concurrent mutations on the outcome of MDS patients. In applying next-generation sequencing (NGS) with a 117 myeloid gene custom panel, we analyzed the co-occurrence of SF3B1 with other mutations to reveal their clinical, biological, and prognostic implications in very low/low- and intermediate-risk MDS patients. Mutations in addition to those of SF3B1 were present in 80.4% of patients (median of 2 additional mutations/patient, range 0-5). The most frequently mutated genes were as follows: TET2 (39.2%), DNMT3A (25.5%), SRSF2 (10.8%), CDH23 (5.9%), and ASXL1, CUX1, and KMT2D (4.9% each). The presence of at least two mutations concomitant with that of SF3B1 had an adverse impact on survival compared with those with the SF3B1 mutation and fewer than two additional mutations (median of 54 vs. 87 months, respectively: p = 0.007). The co-occurrence of SF3B1 mutations with specific genes is also linked to a dismal prognosis: SRSF2 mutations were associated with shorter overall survival (OS) than SRSF2wt (median, 27 vs. 75 months, respectively; p = 0.001), concomitant IDH2 mutations (median OS, 11 [mut] vs. 75 [wt] months; p = 0.001), BCOR mutations (median OS, 11 [mut] vs. 71 [wt] months; p = 0.036), and NUP98 and STAG2 mutations (median OS, 27 and 11 vs. 71 months, respectively; p = 0.008 and p = 0.002). Mutations in CHIP genes (TET2, DNMT3A) did not significantly affect the clinical features or outcome. Our results suggest that a more comprehensive NGS study in low-risk MDS SF3B1mut patients is essential for a better prognostic evaluation.

Networking for advanced molecular diagnosis in acute myeloid leukemia patients is possible: the PETHEMA NGS-AML project

Next-Generation Sequencing has recently been introduced to efficiently and simultaneously detect genetic variations in acute myeloid leukemia. However, its implementation in the clinical routine raises new challenges focused on the diversity of assays and variant reporting criteria. To overcome this challenge, the PETHEMA group established a nationwide network of reference laboratories aimed to deliver molecular results in the clinics. We report the technical cross-validation results for next-generation sequencing panel genes during the standardization process and the clinical validation in 823 samples of 751 patients with newly diagnosed or refractory/relapse acute myeloid leukemia. Two cross-validation rounds were performed in seven nationwide reference laboratories in order to reach a consensus regarding quality metrics criteria and variant reporting. In the pre-standardization cross-validation round, an overall concordance of 60.98% was obtained with a great variability in selected genes and conditions across laboratories. After consensus of relevant genes and optimization of quality parameters the overall concordance rose to 85.57% in the second cross-validation round. We show that a diagnostic network with harmonized next-generation sequencing analysis and reporting in seven experienced laboratories is feasible in the context of a scientific group. This cooperative nationwide strategy provides advanced molecular diagnostic for acute myeloid leukemia patients of the PETHEMA group.

Chronic graft-versus-host disease could ameliorate the impact of adverse somatic mutations in patients with myelodysplastic syndromes and hematopoietic stem cell transplantation.

Somatic mutations in patients with myelodysplastic syndromes (MDS) undergoing allogeneic hematopoietic stem cell transplantation (HSTC) are associated with adverse outcome, but the role of chronic graft-versus-host disease (cGVHD) in this subset of patients remains unknown. We analyzed bone marrow samples from 115 patients with MDS collected prior to HSCT using next-generation sequencing. Seventy-one patients (61%) had at least one mutated gene. We found that patients with a higher number of mutated genes (more than 2) had a worse outcome (2 years overall survival [OS] 54.8% vs. 31.1%, p = 0.035). The only two significant variables in the multivariate analysis for OS were TET2 mutations (p = 0.046) and the development of cGVHD, considered as a time-dependent variable (p < 0.001), correlated with a worse and a better outcome, respectively. TP53 mutations also demonstrated impact on the cumulative incidence of relapse (CIR) (1 year CIR 47.1% vs. 9.8%, p = 0.006) and were related with complex karyotype (p = 0.003). cGVHD improved the outcome even among patients with more than 2 mutated genes (1-year OS 88.9% at 1 year vs. 31.3%, p = 0.02) and patients with TP53 mutations (1-year CIR 20% vs. 42.9%, p = 0.553). These results confirm that cGVHD could ameliorate the adverse impact of somatic mutations in patients with MDS with HSCT.

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.

Identification of novel variants in ten patients with Hermansky-Pudlak syndrome by high-throughput sequencing.

Background: Hermansky-Pudlak syndrome (HPS) is a rare inherited platelet disorder characterized by bleeding diathesis, oculocutaneous albinism (OCA) and a myriad of often-serious clinical complications. Methods: We established the clinical and laboratory phenotype and genotype of six unrelated pedigrees comprising ten patients with clinical suspicion of HPS; including platelet aggregation, flow cytometry, platelet dense granule content, electron microscopy and high-throughput sequencing (HTS). Results: The clinical presentation showed significant heterogeneity and no clear phenotype-genotype correlations. HTS revealed two known and three novel disease-causing variants. The Spanish patients carried a homozygous p.Pro685Leufs17* deletion (n = 2) in HPS4, or the novel p.Arg822* homozygous variant (n = 1) in HPS3. In the case of two Turkish sisters, a novel missense homozygous HPS4 variant (p.Leu91Pro) was found. In two Portuguese families, genetic studies confirmed a previously reported nonsense variant (p.Gln103*) in DTNBP1 in three patients and a novel duplication (p.Leu22Argfs*33) in HPS6 in two unrelated patients. Conclusions: Our findings expand the mutational spectrum of HPS, which may help in investigating phenotype-genotype relationships and assist genetic counselling for affected individuals. This approach is a proof of principle that HTS can be considered and used in the first-line diagnosis of patients with biological and clinical manifestations suggestive of HPS. Key messages We established the relationships between the clinical and laboratory phenotype and genotype of six unrelated pedigrees comprising ten patients with clinical suspicion of HPS. Molecular analysis is useful in confirming the diagnosis and may offer some prognostic information that will aid in optimizing monitoring and surveillance for early detection of end-organ damage. This approach is a proof of principle that HTS can be considered and used in the first-line diagnosis of patients with biological and clinical manifestations suggestive of HPS.

Sitosterolemia: Diagnosis, Metabolic and Hematological Abnormalities, Cardiovascular Disease and Management.

Sitosterolemia is a recessive inherited metabolic disorder of unknown prevalence, characterized by increased levels of plasma plant sterols. It is caused by 28 and 31 variants in ABCG5 and ABCG8 genes, respectively, and is characterized by a predisposition to hyperabsorption and accumulation of toxic levels of plant sterols in plasma. Its clinical picture is extremely heterogeneous. The main clinical features are tendinous and cutaneous xanthomas, arthritis or arthralgia, premature cardiovascular disease and atherosclerosis. These characteristics are shared with familial hypercholesterolemia (FH), making it possible for sitosterolemia to be misdiagnosed as homozygous FH, especially in pediatric patients. In such cases, a specific chromatography-based laboratory method is essential to differentiate sitosterol and cholesterol. Hematological abnormalities (hemolytic anemia and macrothrombocytopenia) may be present in 25-35% of patients, in whom it is usually associated with the main clinical features, as occurs in the 70% of the cases. In this context, the peripheral blood smear is essential and reveals giant platelets and stomatocytes. Only 21 causative variants in ABCG5/ABCG8 are associated with macrothrombocytopenia. Most physicians still do not recognize these hematological abnormalities or relate them to sitosterolemia. Patients may suffer long-term misdiagnosis of immune thrombocytopenia and be at high risk of receiving harmful therapies or of not benefitting from a low-cholesterol diet and/or from the gold standard treatment with ezetimibe. This drug reduces the levels of plasma plant sterols, provokes regression of xanthomas, and can alleviate hematological abnormalities. Finally, to identify genetic defects, recent advances in high-throughput sequencing, especially in the use of targeted sequencing of pre-specified genes, have begun to be incorporated in the first-line approach in the field of genetic disorders.

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.

A two-step approach for sequencing spliceosome-related genes as a complementary diagnostic assay in MDS patients with ringed sideroblasts.

Our study aimed to analyze the presence of mutations in SF3B1 and other spliceosome-related genes in myelodysplastic syndromes with ringed sideroblasts (MDS-RS) by combining conventional Sanger and next-generation sequencing (NGS) methods, and to determine the feasibility of this approach in a clinical setting. 122 bone marrow samples from MDS-RS patients were studied. Initially, exons 14 and 15 of the SF3B1 gene were analyzed by Sanger sequencing. Secondly, they were studied by NGS covering besides SF3B1, SRSF2, U2AF1 and ZRSR2 genes. An 86% of all patients showed mutations in the SF3B1 gene. Six of them, which were not identifiable by conventional sequencing in the first diagnostic step, were revealed by NGS. In addition, 19.5% of cases showed mutations in other splicing genes: SRSF2, U2AF1, and ZRSR2. Furthermore, 8.7% of patients had two mutations in SF3B1, SF3B1 and SRSF2, and SF3B1 and U2AF1, while 5.7% showed no mutations in the four spliceosome-related genes analyzed. The combined use of conventional Sanger and NGS allows the identification of mutations in spliceosome-related genes in almost all MDS patients with RS. This two-step approach is affordable and could be useful as a complementary technique in cases with an unclear diagnosis.

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.

Application of a molecular diagnostic algorithm for haemophilia A and B using next-generation sequencing of entire F8, F9 and VWF genes.

Currently, molecular diagnosis of haemophilia A and B (HA and HB) highlights the excess risk-inhibitor development associated with specific mutations, and enables carrier testing of female relatives and prenatal or preimplantation genetic diagnosis. Molecular testing for HA also helps distinguish it from von Willebrand disease (VWD). Next-generation sequencing (NGS) allows simultaneous investigation of several complete genes, even though they may span very extensive regions. This study aimed to evaluate the usefulness of a molecular algorithm employing an NGS approach for sequencing the complete F8, F9 and VWF genes. The proposed algorithm includes the detection of inversions of introns 1 and 22, an NGS custom panel (the entire F8, F9 and VWF genes), and multiplex ligation-dependent probe amplification (MLPA) analysis. A total of 102 samples (97 FVIII- and FIX-deficient patients, and five female carriers) were studied. IVS-22 screening identified 11 out of 20 severe HA patients and one female carrier. IVS-1 analysis did not reveal any alterations. The NGS approach gave positive results in 88 cases, allowing the differential diagnosis of mild/moderate HA and VWD in eight cases. MLPA confirmed one large exon deletion. Only one case did have no pathogenic variants. The proposed algorithm had an overall success rate of 99 %. In conclusion, our evaluation demonstrates that this algorithm can reliably identify pathogenic variants and diagnose patients with HA, HB or VWD.

Deregulation of genes related to iron and mitochondrial metabolism in refractory anemia with ring sideroblasts.

The presence of SF3B1 gene mutations is a hallmark of refractory anemia with ring sideroblasts (RARS). However, the mechanisms responsible for iron accumulation that characterize the Myelodysplastic Syndrome with ring sideroblasts (MDS-RS) are not completely understood. In order to gain insight in the molecular basis of MDS-RS, an integrative study of the expression and mutational status of genes related to iron and mitochondrial metabolism was carried out. A total of 231 low-risk MDS patients and 81 controls were studied. Gene expression analysis revealed that iron metabolism and mitochondrial function had the highest number of genes deregulated in RARS patients compared to controls and the refractory cytopenias with unilineage dysplasia (RCUD). Thus mitochondrial transporters SLC25 (SLC25A37 and SLC25A38) and ALAD genes were over-expressed in RARS. Moreover, significant differences were observed between patients with SF3B1 mutations and patients without the mutations. The deregulation of genes involved in iron and mitochondrial metabolism provides new insights in our knowledge of MDS-RS. New variants that could be involved in the pathogenesis of these diseases have been identified.