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.
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.
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.
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
SRSF2
wt (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
SF3B1
mut
patients is essential for a better prognostic evaluation.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Introduction: Conventional karyotype analysis is one of the most important diagnostic tools to determine the prognosis of acute myeloid leukemia (AML), in which more than 50% of cases are affected. ...However, the low sensitivity of this technique hampers the detection of small genetic alterations like Copy Number Variation (CNV) that could affect the pathophysiology and prognosis of the disease. Current modern genomic technologies based on next generation sequencing (NGS) are capable to detect CNV at low frequencies.
Objective: To analyse CNV of genes related to myeloid neoplasms profile in AML patients at diagnosis and evaluate their connection with the mutational profile, and its possible influence on the clinical-biological phenotype and prognosis of the disease.
Materials and methods: The CNV and mutational profile were analysed in samples from 380 AML patients, from PLATAFO-LMA reference centres (IMIBIC, Córdoba and Dr Negrín Las Palmas de Gran Canaria) by NGS, applying a panel of 30 genes (154 regions) related to myeloid neoplasms (Sophia Myeloid Solution®) on Ilumina Myseq platform.
Results: NGS detected CNV in at least one gene in 103 AML patients (27.1%). NGS detected 103 gains and 206 losses of genetic material. The median number of genes affected by CNV was 2 (range 1-12). When comparing with conventional karyotype information, CNV provided additional information in 51% of the cases.
The chromosomes 7, 11 and 21 were most affected with CNV, occurring in 79 (76.7%), 40 (38.8%) and 36 (35%) patients, respectively. The gains of genetic material occurred more frequently on chromosome 21 in U2AF1 and RUNX1 genes, in 13 patients each. The loss of genetic material in EZH2 and BRAF genes occurred mutually. Interestingly, we observed the tendency that patients with CNV (loss) in NPM1 gene had shorter overall survival compared to cases with NPM1 mutated and without CNV in this gene (1 month vs. 13 months, p = 0.072) (Figure 1). Moreover CNV (loss) in TP53 gene was associated with mutations in this gene, other than deletions (p <0.05).
In addition, NGS detected 390 mutations distributed in 29 genes in 103 AML with CNV. The median number of mutations was 3 (range 1-10) (Figure 2). Furthermore, only 2 patients did not have any mutation in genes analysed. The distribution and frequency of genes affected by CNV and by mutations was different (Figure 3).
Conclusions: The CNV of genes related to myeloid neoplasms are frequent in AML patients (27.1%) and provides additional information to the conventional karyotype in half of the cases. The loss of NPM1 gene could affect survival of AML patients. The use of NGS with CNV analysis provides important information on copy number alterations that are not detected by the karyotype, which could significantly affect the pathophysiology of AML and with potential clinical impact, especially in patients with normal karyotype.
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Hernández Rivas: Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees.
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
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
(n=2) and
(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.
Next-Generation Sequencing (NGS) has recently been introduced to efficiently and simultaneously detect genetic variations in acute myeloid leukemia (AML). 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 7 reference laboratories aimed to deliver molecular results to the clinics. We report the technical cross-validation results for NGS and clinical validation in 2960 AML samples.
Three cross-validation rounds (CVR) were performed to establish consensus parameters for NGS analysis and variant reporting. In the first CVR we evaluated the starting situation of the NGS studies. In the second CVR the laboratory network established minimum quality parameters and consensus recommendations to guarantee a valid NGS assay. The third CVR strengthened the established parameters and refined the clinical variant classification. The clinical validation was performed in 2960 samples from 2703 patients: 1530 male and 1173 female with a median age of 67.5 years old. 2522 samples were collected at diagnosis, 275 at relapse and 163 at refractory AML from October 2017 to October 2019. NGS analysis was performed according to already implemented protocols and only variants accomplishing the established quality control parameters were considered.
In the first CVR the error rate (ER) was 39% with a high variability in the studied genes. Then, 30 genes were agreed as key genes for AML pathogenesis: 8 were considered mandatory due to their implication in clinical guidelines, targeted therapy and risk stratification and the study of the remaining 22 was recommended based on panel availability (Table 1). In the second CVR the ER was reduced to 14.4% and the NGS quality metrics were 4032X of mean read depth and 98.3% of median uniformity. Therefore, the laboratory network established a minimum read depth of 500X and uniformity >85% as quality control parameters. Due to the high variability in the detection of low VAF variants (1 CVR: ER<5%: 86.1%; 2 CVR: ER<5%: 28.6%), VAF≥5% was established as a cut-off for variant reporting excepting variants with strong clinical evidence. In the third CVR the error rate for VAF≥5% variants was 10.9% achieving high concordance in variant detection and clinical classification.
8043 variants were reported in the 2960 samples, with 96.5% of patients showing at least 1 mutated gene. The mean number of variants per sample was 2.71 (range 0-9), the number of mutations in ≥65 years old patients (2.9 vs. 2.5, P<0.001) and in male patients (2.8 vs. 2.6, P=0.001) were significantly higher. In the global cohort, the most frequently mutated genes were FLT3 (24.9%), DNMT3A (23.9%) and NPM1 (23%). However, this genetic profile changed according to the disease stage, age and sex. NPM1 mutations were more frequent at diagnosis (P<0.001), IDH1 mutations (P=0.027) RUNX1 mutations (P=0.011) and WT1 mutations (P<0.001) were more frequent at relapse and mutations in KRAS (P=0.012) were more frequently detected in refractory AML (Fig 1). Younger patients (<65) had more mutations in FLT3 and NPM1 (P<0.001) while mutations in ASXL1, EZH2, IDH2, JAK2, RUNX1, SRSF2, TET2, TP53, U2AF1 and SF3B1 were associated to older age (P<0.05) (Fig 2). NPM1, FLT3 and DNMT3A were frequently mutated in female patients (P<0.001) while male patients had more mutations in ASXL1, JAK2, EZH2, RUNX1, SRSF2 and U2AF1 (P<0.01) (Fig 3).
At least 1 mutation in one of the 8 clinically relevant genes was detected in the 78.7% of patients: 39.8% of patients had targetable mutations (FLT3-ITD/TKD and IDH1/2 mutations) and 35.6% of patients, variants found in ASXL1, RUNX1 and TP53 were the only clinically relevant variant detected. The study of clonal evolution through paired-sample analysis showed that mutations in FLT3, KRAS, NRAS and PTPN11 were particularly unstable at relapse or refractoriness.
We show the development of the first national strategy for validation of NGS studies with centralized analysis in an AML cooperative group. We have developed a laboratory network with standardized protocols to ensure technical quality and equity in access to NGS studies. The unification of analysis and interpretation criteria represents a significant increase in the quality of diagnostic tests and translational research.
N/A-NI-AML-PETHEMA-007343, PI18/01340, PI19/00730, FI19/00059
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Ayala: Incyte Corporation: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Celgene: Honoraria. Tormo: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Perez-Simon: JANSSEN, TAKEDA, PFIZER, JAZZ, BMS, AMGEN, GILEAD: Other: honorarium or budget for research projects and/or participation in advisory boards and / or learning activities and / or conferences. Martínez-López: Janssen, BMS, Novartis, Incyte, Roche, GSK, Pfizer: Consultancy; Roche, Novartis, Incyte, Astellas, BMS: Research Funding. Montesinos: AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Agios: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Teva: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Forma Therapeutics: Consultancy; Glycomimetics: Consultancy; Tolero Pharmaceutical: Consultancy.
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.
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).
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.
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.
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