The Significance of the Next Generation Targeted Sequencing in the Precise Diagnosis of Pediatric Acquire Aplastic Anemia and Inherited Bone Marrow Failure Syndromes

Abstract Background Diagnosis of inherited bone marrow failure syndromes (IBMFs) depend on classic clinical manifestation including early onset, physical anomalies, family history of cancer and/or bone marrow failure and chromosome breakage testing (MMC and/or DEB), mutation analyses and bone marrow chromosome analyses. At present, more than 70 pathogenic gene mutations had been identified. However, in some patients, physical anomalies is absent or delayed, and were misdiagnosed as acquired aplastic anemia(AA). Genetic analysis is very important to establish a precise diagnosis, predict cancer risk, direct treatment and genetic counseling. In this study, we focus on the application of next generation targeted sequencing in precise diagnosis of pediatric acquired AA/IBMFs, and the association between genetic abnormalities and clinical and laboratory characteristics. Methods We designed a targeted sequencing assay to test a panel of 417 genes. The panel contain reported gene associated with IBMFs and other diseases need be differentiated. Pediatic patients (≤14 year old) with suspected diagnosis of AA/IBMFs were enrolled. Peripheral blood (PB) DNA was used to genetic analysis and oral epithelia cells or PB DNA from their parents were used to identify somatic mutations and unreported polymorphism. All the results were validated by Sanger sequencing. Results The average coverage of targeted region was 98.15%, and the average sequencing depth was 315.9×. Totally, 283 patients were enrolled, including 176 clinically diagnosed acquired AA, 51 Fanconi anemia (FA), 8 dyskeratosis congenital(DKC), 30 Diamond-Blanckfan anemia(DBA), 15 congenital neutropenia(CN), and 3 congenital thrombocytopenia. Totally, 19% subjects had IBMFs related genetic mutations. In the patients who were clinically diagnosed as acquired AA patients, about 7% had IBMFs related disease-causing genetic mutations. Finally, 7 patients were genetically diagnosed as FA, 2 were DKC, 1 was WAS and 1 was SDS. In patients who were clinically diagnosed as FA, 33.4% had FANC related gene mutations. Telomere associated gene mutations were detected in 75% of clinical diagnosed DKC. For patients clinically diagnosed as DBA and CN, 36.7% and 20% were detected disease-causing mutations. After genetic screening, 2 patients who had been diagnosed as FA were modified as WAS and 1 DBA was modified as SDS. Only 26% genetic diagnosed IBMFs patients had family history of bone marrow failure, leukemia, tumor or physical anomalies. Compared with acquired AA, patients with genetic diagnosed FA were more likely to have physical anomalies of short stature and development retardation, Cafe au lait spots and finger or toe malformation(P<0.001).However, 46% patients with IBMFs did not have any type of physical anomalies. Moreover, there were only 24% patients with genetic diagnosed IBMFs had positive results of MMC induced chromosome breakage test or SCGE, and both the examinations could not differentiate subtype of IBMFs. FANCD2 mono-ubiquitination test were performed recently. However, even in the genetically confirmed FA, the positive rate was only 18% (2/11). And, there were positive results in some acquired AA patients. For FA patients with definitely genetic mutations, 62.5%(15/24) were compound heterozygous mutations,37.5%(9/24) were homozygous mutations. Mutational frequencies of FANC were: FANCA 65%, FANCD2 23%, FANCG 9%, FANCI 9% and FANCB 4%. For the mutated type, the frequencies of missense, frameshift, nonsense, splicing mutation were 42%, 26%, 16%, 16%. In our study, there were 4 undetermined patients met the clinical diagnostic criteria of FA, and having heterozygous damaged mutations in FANC genes. Conclusion In conclusion, our IBMFs associated genes targeted sequencing assay is an effective strategy for precise diagnosis of bone marrow failure diseases, especially for those without family history or physical anomalies. However, nearly half of the clinically diagnosed IBMFs patients in our study were not detected the disease-causing mutations. This may be due to the mutations in the intron area, or large fragment deletion, which cannot be detected by targeted sequencing. And the novel gene involved in IBMFs need further study. Disclosures No relevant conflicts of interest to declare.

Download Full-text

correspondence: Identification of TINF2 gene mutations in adult Japanese patients with acquired bone marrow failure syndromes

British Journal of Haematology ◽

10.1111/j.1365-2141.2010.08278.x ◽

2010 ◽

Vol 150 (6) ◽

pp. 725-727 ◽

Cited By ~ 4

Author(s):

Hiroki Yamaguchi ◽

Koiti Inokuchi ◽

Junko Takeuchi ◽

Hayato Tamai ◽

Yoshio Mitamura ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Failure ◽

Gene Mutations ◽

Japanese Patients ◽

Bone Marrow Failure Syndromes

Download Full-text

Whole Exome Sequencing Shows a Paucity Of Somatic Gene Mutations In Pediatric Idiopathic Bone Marrow Failure Syndrome

Blood ◽

10.1182/blood.v122.21.3708.3708 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3708-3708

Author(s):

Atsushi Narita ◽

Hideki Muramatsu ◽

Kenichi Yoshida ◽

Yusuke Okuno ◽

Asahito Hama ◽

...

Keyword(s):

Bone Marrow ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Somatic Mutations ◽

Bone Marrow Failure ◽

Gene Mutations ◽

Bone Marrow Failure Syndrome ◽

Cell Lineages ◽

Bone Marrow Failure Syndromes ◽

Whole Exome

Abstract Introduction Pancytopenia accompanied by a severe decrease in bone marrow (BM) cellularity in children can be due to a broad variety of underlying disorders. Appropriate classification of bone marrow failure syndrome in children is challenging, particularly in relation to the morphological distinction between aplastic anemia (AA), refractory cytopenia of childhood (RCC), and refractory cytopenia with multilineage dysplasia (RCMD). The goal of this study was to characterize the molecular pathogenesis of these conditions by identifying the full spectrum of gene mutations in 29 patients with these disorders through the use of exome sequencing. Patient and Methods Diagnosis of AA, RCC, or RCMD was made on basis of the 2008 World Health Organization (WHO) classification criteria. AA patients exhibited no morphologically dysplastic changes in any of their hematopoietic cell lineages, while RCC patients had<10% dysplastic changes in two or more cell lineages or >10% in one cell lineage. Patients classified as RCMD exhibited >10% of the dysplastic changes in two or more cell lineages. Blood and BM samples were obtained from 29 children (16 boys and 13 girls) with AA (n = 8), RCC (n = 11), or RCMD (n = 10). The median age at diagnosis was 11 years (range, 2–15 years). Exome capture from paired DNA (non-T cells/CD3+ lymphocyte) was performed using SureSelect® Human All Exon V3 (Agilent Technologies, Santa Clara, CA) covering 50 Mb of the coding exons, followed by massive parallel sequencing using HiSeq 2000 (Illumina, San Diego, CA) according to the manufacturer’s protocol. Candidate somatic mutations were detected through our pipeline for whole exome sequencing (genomon: http://genomon.hgc.jp/exome/index.html). All candidate somatic nucleotide changes were validated by Sanger sequencing. Results Exome sequencing pipeline identified a total of 193 non-synonymous somatic mutations or indels candidates among the 29 patients (range, 2–15 per patient). After validation by Sanger sequencing, one nonsense, 11 missense, and two frame-shift mutations were confirmed as non-silent somatic mutations. The average numbers of mutations per sample were not significantly different when comparing morphological diagnostic groups (0.50 in AA, 0.36 in RCC, 0.60 in RCMD). Of these validated genes, BCOR (n = 2) and CSK (n = 2) mutations were recurrent genetic events. BCOR is a frequent mutational target in myelodysplastic syndrome, whereas CSK somatic mutations were not reported in human cancers. BCOR mutations were found both in AA (c.472delA:p.S158fs; patient 13) and in RCMD (c.G3856T:p.E1286X; patient 39). Both patients with CSK mutations were classified as RCC (c.G994A:p.D332N; patient 23 and 27). When comparing the clinical outcomes of patients with somatic mutations (n = 7) versus those without somatic mutations (n = 22), response rate to immunosuppressive therapy at 6 months (50% vs. 50%), 5-year clonal evolution rate (95% confidential interval) [0% (0% - 0%) vs. 6% (0% - 26%)], and the 5-year overall survival rate (95% confidential interval) [100% (100% - 100%) vs. 95% (70% - 99%)] were not significantly different. Conclusion Whole exome sequencing analysis was used for gene mutational profiling of patients with idiopathic bone marrow failure syndromes; i.e., AA, RCC, and RCMD. Although BCOR and CSK somatic mutations were recurrently identified, idiopathic bone marrow failure syndromes in children are characterized by a paucity of gene mutations, irrespective of morphological diagnosis. These findings suggest that morphological diagnosis based on WHO classification system does not discriminate the mutational profile and pathogenesis of bone marrow failure in children. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Frequency and natural history of inherited bone marrow failure syndromes: the Israeli Inherited Bone Marrow Failure Registry

Haematologica ◽

10.3324/haematol.2009.018119 ◽

2010 ◽

Vol 95 (8) ◽

pp. 1300-1307 ◽

Cited By ~ 35

Author(s):

H. Tamary ◽

D. Nishri ◽

J. Yacobovich ◽

R. Zilber ◽

O. Dgany ◽

...

Keyword(s):

Bone Marrow ◽

Natural History ◽

Bone Marrow Failure ◽

Bone Marrow Failure Syndromes ◽

History Of

Download Full-text

Heterozygous RTEL1 Variants Are Associated with Bone Marrow Failure and Abnormal Clinical Phenotype

Blood ◽

10.1182/blood.v128.22.1043.1043 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1043-1043

Author(s):

Shreyans Gandhi ◽

Jie Jiang ◽

Mariam Ibanez ◽

Isabelle Callebaut ◽

Judith CW Marsh ◽

...

Keyword(s):

Bone Marrow ◽

Family History ◽

Bone Marrow Failure ◽

3D Structure ◽

Telomere Maintenance ◽

The Other ◽

Helicase Domain ◽

Cell Transplant ◽

Variable Regions ◽

History Of

Abstract Introduction Heterozygous RTEL1 mutations have recently been described in familial pulmonary fibrosis (PF) but are not known to be associated with cytopenias or bone marrow failure (BMF), in contrast to heterozygous mutations in other telomere maintenance genes TERT, TERC and TINF2. Constitutional BMF syndromes typically present with less severe pancytopenia and it is often unclear if they have hypocellular MDS (hypoMDS) or non-severe AA (NSAA) morphologically. Methods We screened 284 patients with idiopathic AA or uncharacterised BMF and 172 patients with MDS or acute myeloid leukemia (AML) for TL and RTEL1 variants, and for the other currently known telomere gene complex (TGC) mutations, after excluding patients with Fanconi anemia, DBA or other known inherited BMF syndrome. TL was measured using a monochrome multiplex quantitative PCR method on peripheral blood mononuclear cells. Illumina Nextera-amplicon sequencing was used to screen exons of the DC genes (DKC1, TERC, TERT, RTEL1, CTC1, NHP10, NOP2, USB1, WRAP53, TINF2, PARN and ACD) by MiSeq platform. Constitutional DNA was also analysed in 10 patients (skin 9, buccal swab 1) with RTEL1 variants. A targeted gene panel of 24 genes of an Illumina Tru-Seq Custom Amplicon workflow and platform was used to identify genes frequently mutated in MDS/AML. Impact of mutations was predicted based on 3D structure information from comparative modelling for the helicase domain, comprising the HD1 and HD2 subdomains, a Fe-S cluster and an ARCH domain, and for two harmonin-like (HML) domains and a RING finger domain, located in the C-terminal regulatory region of RTEL1. Results Heterozygous RTEL1 variants were identified in 20 (4.4%) patients. RTEL1 variant allele frequency (VAF) was 45-70% consistent with heterozygous inheritance in all cases. TL was short in 18 (90%) patients, being < 1st centile in 15 and <10th centile in 3. 2 patients had normal TL, <20th centile and >50th centile, respectively. Median age was 35 years (range 18-73). 15/20 (75%) had a hypocellular BM (7 hypoMDS, 5 non-severe AA, 3 ICUS), and 1 each with RAEB1, RAEB2, CMML1, AML and isolated macrocytosis. 3 patients had abnormal karyotype: +8 (hypoMDS), -Y,+1,del(1) (hypoMDS), del7q (RAEB1). 2 other patients with hypoMDS had somatic mutations: U2AF1 (30% VAF) with ASXL1 (27% VAF); U2AF1 (43% VAF). Lung abnormalities were early PF (1), interstitial lung disease (1), and abnormal lung function with reduced TLCO (1) and an obstructive picture (1). Liver fibrosis with portal hypertension and varices and reticulate skin pigmentation were present in the patient with ILD, 2 patients had dystrophic nails, and 1 unexplained mild hepato-splenomegaly. 2 patients had familial MDS, 5 had a family history of cancers affecting first-degree relatives, and 2 had skeletal and cartilage anomalies, associated with learning difficulties in 1 patient. 8/15 patients with hypocellular BM required no treatment (5 hypoMDS and 3 NSAA), one hypoMDS had CR with ciclosporin and another underwent successful unrelated donor stem cell transplant; for NSAA, 2 received ATG with CSA, with PR followed by relapse in one, the other was lost to follow up, and 1 was androgen responsive. 16/20 (80%) patients are alive; 3 patients with RAEB or AML died of progressive disease and 1 patient with ICUS and severe constitutional features died from lymphoma 10 years after presentation. Mutations were spread throughout the entire RTEL1 sequence (summarised in Figure). 3D structure analysis predicted the missense RTEL1 mutations would result in disturbance of the FeS cluster and/or interfere with DNA binding, destabilisation of the HD1, HD2 or the ARCH sub-domains of the helicase domain, or destabilisation of inter-domain interactions. One HML1 mutation occurred in a loop opposite the putative ligand binding site and the rest in the variable regions outside the conserved domains. RTEL1 variants were associated with TERT mutations in 4 patients, of which 3 were known pathogenetic and 1 novel TERT mutation with low telomerase activity on TRAP assay confirming its pathogenetic nature. Conclusions We show for the first time that heterozygous RTEL1 mutations occur in 4.4% of patients, most commonly in young patients with a hypocellular BM, and often a family history of BMF/malignancy, and less often with high risk MDS/AML. Abnormal clinical features were present in a third of patients, some similar to but others distinct from dyskeratosis congenita. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Perforin Gene Mutations in Patients with Acquired Aplastic Anemia.

Blood ◽

10.1182/blood.v108.11.998.998 ◽

2006 ◽

Vol 108 (11) ◽

pp. 998-998

Author(s):

Elena E. Solomou ◽

Federica Gibellini ◽

Stephen J. Chanock ◽

Daniela Malide ◽

Maria Berg ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Nk Cells ◽

Nk Cell ◽

Bone Marrow Failure ◽

Gene Mutations ◽

Activated T Cells ◽

Hematopoietic Stem ◽

Bone Marrow Failure Syndromes ◽

Prf1 Gene

Abstract Perforin is a cytolytic protein expressed mainly in activated cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In T and NK cells perforin is stored in cytoplasmic granules and is essential for killing via non-Fas-mediated mechanisms. Perforin regulates the translocation of granzyme B from cytotoxic cells into target cells; after entering the target-cell granzyme B migrates to the target cell nucleus to participate in triggering apoptosis. Functional perforin is essential for normal CTL and NK cell function; without perforin CTL and NK cells show reduced or no cytolytic effect. Inherited perforin mutations account for 20–40% of familial hemophagocytic lymphohistiocytosis, an autosomal recessive fatal disease of early childhood characterized by uncontrolled accumulation of activated T cells and macrophages in many organs, increased Th1 cytokines and absent functional perforin. Acquired aplastic anemia (AA), the paradigm of immune mediated bone marrow failure syndromes, is characterized by hematopoietic stem cell destruction by activated T cells and Th1 cytokines. We examined whether mutations in Prf1 occur in AA; peripheral blood DNA samples from 75 patients and 302 controls were analyzed. Three novel nonsynonymous Prf1 mutations among five unrelated patients (ages: 21, 31, 33, 75, and 77 years old), not present in controls, were discovered; two polymorphisms were also identified (H300H, A274A). The mutations were in the coding region of Prf1 gene. In exon 2, arginine was replaced by histidine in one patient (CGT/CAT, R4H) and in 3 patients the same A91V mutation was identified (GCG/GTG, alanine to valine substitution). In exon 3, serine was replaced by isoleucine (S388I; AGC/ATC) in one patient. Germ-line origin of the Prf1 mutations was established by their presence also in DNA from buccal mucosa obtained from affected AA patients. Four of five patients with mutations showed some hemophagocytosis in the bone marrow examination when first diagnosed, but there were no other typical features of hemophagocytic syndrome such as hepatosplenomegaly or altered liver function tests. None of the patients with Prf1 mutations experienced hematologic recovery with immunosuppressive treatment. Perforin protein levels in all patients carrying mutations were very low or absent. By confocal microscopy, CD8 cells from patients with Prf1 mutations had complete absence of perforin granules (perforin and cathepsin D showed the expected pattern of co-localization in controls’ cytotoxic granules). NK cell killing efficiency from patients carrying mutations in a standard Cr51-release cytolytic assay was significantly decreased compared to controls. Prf1 gene mutations may be related to a more severe phenotype of AA associated with marrow hemophagocytosis and failure to respond to immunosuppression. Mutations in the immune regulatory mechanisms identified in young children can manifest in adults without typically associated clinical findings or a suggestive family history. Mechanistically, Prf1 gene mutations help explain the aberrant proliferation and activation of cytotoxic T cells that are destructive of hematopoietic stem cells in AA and may be useful as predictive factors for responses to immunosuppressive treatments and the decision to rapidly undertake stem cell replacement. Prf1 gene mutations are genetic risk factors for bone marrow failure syndromes.

Download Full-text

Development of Paroxysmal Nocturnal Hemoglobinuria in Children with Aplastic Anemia

Blood ◽

10.1182/blood.v128.22.1499.1499 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1499-1499 ◽

Cited By ~ 1

Author(s):

Atsushi Narita ◽

Hideki Muramatsu ◽

Yusuke Okuno ◽

Yuko Sekiya ◽

Kyogo Suzuki ◽

...

Keyword(s):

Bone Marrow ◽

Aplastic Anemia ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Bone Marrow Failure ◽

Subsequent Development ◽

Poor Quality ◽

Targeted Sequencing ◽

Hematopoietic Stem ◽

Bone Marrow Failure Syndromes

Abstract Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a nonmalignant clonal disease of hematopoietic stem cells resulting from a somatic mutation in the PIGA gene. PNH frequently manifests in association with aplastic anemia (AA), in which PIGA mutations are believed to enable escape from the immune-mediated destruction by pathogenic T cells. Recent studies using next-generation sequencing have revealed that frequent somatic PIGA mutationsin AA patients are associated with a better response to IST and prognosis (Yoshizato et al N Engl J Med. 2015; 373: 35-47). However, clinical PNH is a progressive and life-threatening disease driven by chronic hemolysis that leads to thrombosis, renal impairment, poor quality of life, and death. Large studies in adults have reported that clinical PNH developed in 10%-25% of AA patients; however; the frequency of clinical PNH in children with AA has rarely been described. Here we aimed to elucidate the pathological link between PNH and AA in children. Methods: In total, 57 children (35 boys and 22 girls) diagnosed with acquired AA at our hospital between 1992 and 2010 were retrospectively studied. Patients who underwent hematopoietic stem cell transplantation as first-line treatment within 1 year after AA diagnosis and those with clinical PNH at AA diagnosis were excluded. Flow cytometry (FCM) was used to detect PNH CD13+/CD55−/CD59− granulocytes and PNH glycophorin A+/CD55−/CD59− red blood cells (RBCs). Clinical PNH was defined as the presence of intravascular hemolysis and ≥5% PNH granulocytes or PNH RBCs. Minor PNH clones were defined as those with >0.005% PNH granulocytes or >0.010% PNH RBCs. We performed targeted sequencing of bone marrow samples from patients with clinical PNH that were obtained at 2 time points: at AA diagnosis and after PNH development. The panel of 184 genes for targeted sequencing included most of the genes known to be mutated in inherited bone marrow failure syndromes and myeloid cancers, as well as PIGA. Results: The median patient age at AA diagnosis was 9.3 (1.2-17.8) years, and the median follow-up period was 123 (2-228) months. A total of 43 patients were screened for PNH clones by FCM after AA diagnosis, and 21 of these with minor PNH clones were identified. The median percentages of PNH granulocytes and PNH RBCs were 0.001% (0.000%-4.785%) and 0.000% (0.000%-3.829%), respectively. During follow-up, 5 patients developed clinical PNH after adolescence (15-22 years of age). The median time between AA diagnosis and PNH development was 4.9 (3.3-7.9) years. All clinical PNH patients were treated with IST for AA, and complete and partial response after 6 months were achieved in 1 and 4 patients, respectively. Gross hemoglobinuria was present in all clinical PNH patients, but thrombosis was not observed. The size of PNH clones varied greatly among patients: PNH granulocytes and PNH RBCs were 42.96% (10.04%-59.50%) and 48.87% (15.02%-90.80%), respectively. Oral cyclosporine A and intravenous eculizumab were administered to 3 and 1 patients, respectively; all patients showed sustained response as indicated by improvement in gross hemoglobinuria and normal blood counts after treatment. The remaining 1 patient underwent bone marrow transplantation from the HLA-identical mother and was alive without any complications. Overall, the 10-year probability of developing clinical PNH was 10.2% (95%CI, 3.6-20.7). Among 43 patients screened for PNH clones at AA diagnosis, the 10-year cumulative clinical PNH incidence was significantly higher in patients with minor PNH clones than in those without minor PNH clones at AA diagnosis [29% (95% CI, 10%-51%) vs. 0% (95% CI, 0%-0%); p = 0.015]. Among all clinical PNH patients, a total of 8 somatic PIGA mutations were detected (missense, 2; splice site, 2; and frameshift, 4). However, PIGA mutations were not detected at AA diagnosis even in patients who subsequently developed clinical PNH. Conclusion: In our cohort, the percentage of patients who eventually developed clinical PNH was comparable to that reported in adults in a previous study. Furthermore, the current study showed that the presence of minor PNH clones at AA diagnosis was a risk factor for the subsequent development of clinical PNH, although the clones were not detected by targeted sequencing. Thus, pediatric AA patients with PNH clones at AA diagnosis should undergo long-term periodic monitoring for potential clinical PNH development. Disclosures Kojima: SANOFI: Honoraria, Research Funding.

Download Full-text

Distinct EBV and CMV reactivation patterns following antibody-based immunosuppressive regimens in patients with severe aplastic anemia

Blood ◽

10.1182/blood-2006-09-045625 ◽

2006 ◽

Vol 109 (8) ◽

pp. 3219-3224 ◽

Cited By ~ 86

Author(s):

Phillip Scheinberg ◽

Steven H. Fischer ◽

Li Li ◽

Olga Nunez ◽

Colin O. Wu ◽

...

Keyword(s):

Bone Marrow ◽

Aplastic Anemia ◽

Bone Marrow Failure ◽

Immunosuppressive Treatment ◽

Viral Reactivation ◽

Bone Marrow Failure Syndromes ◽

History Of ◽

Polymerase Chain ◽

Cmv Disease ◽

Cmv Reactivation

Abstract The natural history of EBV and CMV reactivation and the potential for serious complications following antibody-based immunosuppressive treatment for bone marrow failure syndromes in the absence of transplantation is not known. We monitored blood for EBV and CMV reactivation by polymerase chain reaction (PCR) weekly in 78 consecutive patients (total of 99 immunosuppressive courses) with aplastic anemia. Four regimens were studied: (1) HC, horse ATG/cyclosporine; (2) HCS, horse ATG/CsA/sirolimus; (3) RC, rabbit ATG/CsA; and (4) CP, alemtuzumab. There were no cases of EBV or CMV disease, but EBV reactivation occurred in 82 (87%) of 94 and CMV reactivation in 19 (33%) of 57 seropositive patients after starting immunosuppression. The median peak EBV copies were higher in the RC group when compared with HC, HCS, and alemtuzumab (P < .001). The median duration of PCR positivity for EBV was higher in the RC group compared with HC, HCS, and alemtuzumab (P = .001). Subclinical reactivation of both EBV and CMV is common and nearly always self-limited in patients with bone marrow failure receiving immunosuppression; different regimens are associated with different intensity of immunosuppression as measured by viral load and lymphocyte count; and viral reactivation patterns differ according to immunosuppressive regimens.

Download Full-text

The Patient With Transfusion-Dependent Anemia: Diagnosis and Directed Management With Targeted Next Generation Sequencing and Copy Number Analysis

Blood ◽

10.1182/blood.v122.21.3419.3419 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3419-3419

Author(s):

Vincent P Schulz ◽

Yelena Maksimova ◽

Kimberly Lezon-Geyda ◽

Patrick G Gallagher

Keyword(s):

Bone Marrow ◽

Next Generation Sequencing ◽

Bone Marrow Failure ◽

Gene Mutations ◽

Next Generation ◽

Targeted Next Generation Sequencing ◽

Functional Studies ◽

Bone Marrow Failure Syndromes ◽

In Trans ◽

Generation Sequencing

Abstract Management of the patient with transfusion-dependent anemia (TDA) is complex. Diagnosis is frequently difficult as numerous disorders may lead to TDA, including bone marrow failure syndromes, congenital dyserythropoietic anemias, or inherited hemolytic anemias. Assigning the diagnosis may be problematic as transfused blood or reticulocytosis confound diagnostic testing, or, mutant erythrocytes are so unstable, they are rapidly destroyed. Complications of chronic transfusion include iron overload, infection risk, alloimmunization, cost, and inconvenience. TDA is an excellent candidate for targeted next generation sequencing. There is significant genotypic variability and many of the associated genetic loci are very large, making traditional sequencing strategies cumbersome. We studied 21 patients with TDA using genome-wide targeted exon capture followed by high-throughput next generation DNA sequencing (whole-exome sequencing, WES) using a NimbleGen SeqCap EZ Exome v2.0 solution-based capture system followed by next-generation sequencing on a HiSeq 2000 with paired-end sequencing at 75bp read length. The male:female ratio was 13:8. Age at referral ranged from 2 months to 14 years. All patients were transfusion dependent by 6 months of age. Working diagnoses included possible marrow failure syndrome, congenital dyserythropoietic anemia, and possible enzyme or membrane defect. Variant analyses were performed using the GATK pipeline. Targeted filtering and annotation of protein changing variants in 154 erythrocyte disease genes were performed using the ANNOVAR algorithm. Variants were assessed by mutation prediction and conservation programs including PolyPhen2, Sift, LRT, and Mutation Taster. Variants were also assessed for occurrence and frequency Thousand Genomes, Exome Sequencing Project, dbSNP, on line and local mutation databases, and PubMed. Copy number variants were assessed by ExomeCount and visual inspection. Potential disease-associated variants were validated by Sanger sequencing of DNA from the proband and parents. Interpretation was made using historical, clinical, laboratory and genetic data. The most common diagnosis was hereditary spherocytosis due to alpha spectrin gene (SPTA1) mutations, found in 7 patients. Two patients had deleterious mutations in both SPTA1 alleles; one with nonsense mutations in trans died of liver failure associated with iron overload, the other with nonsense and splicing mutations in trans remains transfusion dependent. One patient homozygous for an SPTA1 missense mutation in a highly conserved, functionally important amino acid had a sibling homozygous for the same mutation die in the perinatal period due to complications of anemia. Finally, one patient with SPTA1 nonsense and missense mutations in trans became transfusion independent post splenectomy. Ten patients had defects in erythrocyte metabolism. Mutations in the pyruvate kinase gene PKLR were found in 6 patients; two of these patients had deletions in the PKLR gene locus suggested by WES and confirmed by Gap PCR and MLPA. Three patients had bi-allelic mutations in the glucose phosphate isomerase gene and one had bi-allelic mutations in the hexokinase gene. Homozygosity was found in 4 of 10 patients with metabolic gene mutations. A single patient had beta thalassemia major with homozygous beta-globin gene mutations. Confirmatory functional studies are underway in three patients. Two TD patients had bone marrow findings suggestive of hypoplastic anemia; one had a missense mutation in a highly conserved residue of RPS7, recently associated with Diamond Blackfan anemia; the other had a deleterious mutation in FANCI predicted to function as a dominant negative. Functional studies are underway in a third patient with likely deleterious, membrane-linked variants. Application of targeted WES to TDA allows precise diagnosis to guide appropriate therapy, e.g. splenectomy or transplant; it allows genetic counseling of associated family members, and permits diagnosis and expectant management of future pregnancies. Targeted WES is an excellent tool for application to monogenic hematologic diseases where genotypic variability, i.e. mutations in numerous genes, leads to the same clinical phenotype. Examples include bone marrow failure syndromes, hemolytic anemia, congenital neutropenia, and immunodeficiency syndromes. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Comprehensive Identification Of Germline Alterations In Telomerase Complex Genes By Whole Exome Sequencing Of MDS and Related Myeloid Neoplasms

Blood ◽

10.1182/blood.v122.21.522.522 ◽

2013 ◽

Vol 122 (21) ◽

pp. 522-522

Author(s):

Swapna Thota ◽

Sarah McMahon ◽

Bartlomiej Przychodzen ◽

Thomas LaFramboise ◽

Hideki Makishima ◽

...

Keyword(s):

Bone Marrow ◽

Family History ◽

Research Funding ◽

Somatic Mutations ◽

Germ Line ◽

Bone Marrow Failure ◽

Telomere Maintenance ◽

Myeloid Neoplasms ◽

History Of ◽

The Mean

Abstract In addition to classical familiar forms of bone marrow failure, some cases of aplastic anemia (AA) have been linked to inherited germ line polymorphism/mutations of telomerase machinery, leading to excessive telomere shortening. Germline telomere maintenance machinery mutations have been also been found in a proportion of acute myeloid leukemia (AML) and Myelodysplastic syndromes (MDS) patients (pts). However, the molecular pathogenesis of adult MDS and AML is complex and determination of genetic risk factors in addition to established familial and congenital syndromes has been difficult. To date targeted sequencing has been used for mutational screens with the inherent limitations of limited exome coverage, empiric bias and labor intensity. New generation (NGS) whole genome approaches prioritize somatic mutations as initial discovery targets, but the availability of sequenced cohorts allows also for detection of germline lesions both in a targeted and an unbiased fashion. Using NGS we studied 136 pts (mean age, 68.8 years, range 41-85) with MDS and related myeloid neoplasms for the presence of non-synonymous polymorphisms (SNV), which could affect telomerase machinery. These genes included TERT, DKC1, SMG6, NOP10, POT1, WRAP53, NHP2, GAR1, TINF2. No somatic defects of the telomerase complex were detected. We focused on novel sequence alterations or those described in available databases with a population allelic frequency of less than 5%. We identified 45 non-synonymous germline sequence alterations in 39 cases (32%). Most frequent SNV were found in TERT (n=15), DKC1 (n=7), SMG6 (n=6), NOP10 (n=4), POT1 (n=4), WRAP53 (n=4), while observations of NHP2 (n=3), GAR1 (n=1), TINF2 (n=1) were less prevalent. These variants were distributed in an almost mutually exclusive manner. Out of 3 variants in TERT, p.H412Y (n=3) and p.A279T (n=9) were reported to be pathogenic in bone marrow failure syndromes. In addition, p.A999T found in 8 cases in our cohort could also be pathogenic since it is less frequent in healthy controls. Similarly, p.441_442del (n=1), located in the N-terminal region, is a completely novel germline variant not detected in 6500 samples publicly available in ESP6500. In the pAML cohort (TCGA; n=197), the observations of germline variants for these telomerase complex genes were SMG6 (n=21), POT1 (n=19), NHP2 (n=1), NOP10 (n=1) GAR1 (n=1). Next, we analyzed clinical characteristics, including treatment responsiveness as assessed per modified 2006 IWG response criteria. The mean age of the 39 patients with germline telomerase machinery alterations was 67 years, 24% (9/39) were younger (age<60 years) compared to 12% (12/97) of wild type (WT; p=.12). Of note, 58% of these cases had a family history of solid tumors including breast, gastrointestinal and prostate and 8% (3/36) had a family history of myeloid malignancies. 41% (16/39) of the telomerase mutants had higher-risk MDS/sAML at presentation compared to 23% in WT cases (23/97; p=.19). A higher percentage of mutants also had complex cytogenetics compared to WT (35% vs. 13%; p=.01). Response rates to common therapies, including hypomethylating agents were similar, but we noted that none of the carrier cases (n=16) treated with lenalidomide showed therapeutic responses (0% vs. 37%). The mean overall survival of the carrier cases was lower compared to the WT (36 vs. 39 months, p=.10). When we studied cases with telomerase alterations for the presence of coinciding somatic mutations, using a targeted deep sequencing panel of the 100 most common mutations acquired in pts with germline telomerase complex alterations, we found most common the acquisition of DNMT3 (18% vs. 6%, p.10) and cohesin mutations (13% vs. 4%,p=.11). In sum, unbiased NGS sequencing approaches in MDS and related myeloid neoplasms allowed for identification of genetic germline alterations in telomerase maintenance machinery at higher rates than previously detected using targeted screening approaches, suggesting that such genetic defects may more frequently than previously thought contribute to cryptic and likely complex genetic predisposition to these diseases. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding.

Download Full-text

Clonal Hematopoiesis and Genetic Mutations in Individual Patients with Acquired Bone Marrow Failure Diseases

Blood ◽

10.1182/blood-2019-125357 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 937-937

Author(s):

Baixin Ye ◽

Xinhua Du ◽

Li Yan ◽

Qingping Gao ◽

Chunrui Li ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Failure ◽

Gene Mutations ◽

Clonal Expansion ◽

Genetic Mutations ◽

Individual Patient Level ◽

Clonal Hematopoiesis ◽

Patient Level ◽

Diagnosis And Prognosis ◽

Undetermined Significance

Clonal hematopoiesis (CH) is characterized by clonal expansion of hematopoietic stem/progenitor cells with the capability of multilineage differentiation and can be detected through the biomarker of genetic mutations. Bone marrow failure (BMF) diseases including idiopathic cytopenia of undetermined significance (ICUS), aplastic anemia (AA), paroxysmal nocturnal hemoglobinuria (PNH) and myelodysplasia syndrome (MDS) are reported to partially coexist with CH. However, till now, the CH and genetic mutations in acquired BMF diseases are still not very clear. Therefore, we designed a panel that contains more than 500 candidate genes to perform a targeted sequencing of bone marrow cells isolated from 48 BMF patients, taking their mucosal cells as controls. In total, 75 somatic mutations and two germline mutations (PTCH1 c.1177G>A, CHEK2 c.1240C>T) were profiled in 30 patients, including 5 ICUS (also termed CCUS (clonal cytopenia of undetermined significance)), 14 AA, 3 PNH and 7 MDS patients. 67.53% missense mutations, 10.39% nonsense mutations, 10.39% frameshift mutations, 1.30% splicing mutations, 7.79% cds-del mutations, 2.60% CNV (copy number variations) can be found in our sequenced mutations. In comparison, the genetic mutation burden is relatively lower in AA and ICUS but higher in PNH and MDS, suggesting that the extent of CH possibly correlated with disease progression. Genetic mutations in our assay also implicated insightful views in the pathogenesis of BMF diseases. We can functionally divide genetic mutations into several types such as signaling transduction (21.6%), transcriptional regulation (19.6%), epigenetic regulation (15.5%), cell cycle regulation (7.2%), DNA repair (3.1%), cohesion complex (2.1%), immunity (3.1%), RNA splicing (4.1%) and others (3.1%). Specifically, we found a serial of novel genetic mutations. For example, the mutations in extracellular membrane receptors such as Notch (Notch1 c.4759A>C , Notch2 c.4819C>T, Notch3 c.3592A>C), CXCR4(c.598C>T), IGF1(c.341C>T), MPL(c.611C>T) suggest bone marrow environment is possibly associated with BMF diseases. In detail, Notch, IGF1, CXCR4 and MPL-mediated signaling are critical for HSC niche regulation, suggesting niche signals serve as a promoting factor for acquired BMF pathogenesis, which is consistent with previous reports. Also, we found the immunity-related mutations in HLA-A(c.750_751delGG) and CD58(c.475T>G), which are closely related with specific T cell recognition, implicated that clonal hematopoiesis and evolution to malignancies can be partially attributed to immune evasion possibly. The mutation ERBB4 (c.3424C>T), a membrane receptor which binds to and is activated by nerve cell released neuregulins, implicated that nerve fiber might involve in BMF. Additionally, we also found novel mutations in epigenetic factors such as KDM5C (c.265delA) and CHD3 (cds-del c.222GCC [7>6]) in PNH and MDS patients. Interestingly, LRP1B (c.13771G>A) that plays an important role in lipid metabolism was found in PNH patient firstly. These gene mutations provide clues for potential biomarkers for BMF diagnosis and prognosis and need more functional investigations. To systemically observe the clonal hematopoiesis at individual patient-level, we delineated the mutational composition in 30 individual BMF patients. We found that 56.7% patients have more than two mutations (eg. MDS 85.71%, PNH 100%, CCUS 60% and AA 35.7%), suggesting that multiple gene mutations-driven clonal expansion constitutes a complex mutational composition and their interaction possibly influences disease progression. At individual patient-level, mutational composition better promotes our understanding on BMF pathogenesis and clinical diagnosis. For example, an MDS patient bearing several mutations in ASXL (non-sense mutation, c.3187C>T, VAF: 18.2%), DNMT3A (missense mutation, c.2644C>T, VAF: 3.01%) and RUNX1 (missense, c.602G>A, VAF: 2.08%) suggested a complex pathological process and poor prognosis. Therefore, mutational composition structure can be revealed in individual acquired BMF patients, promoting precise diagnosis and prognosis. In conclusion, our study not only found a serial of novel genetic mutations that need further investigations, but also provided potential clues for the identification of biomarkers in acquired BMF diseases. Figure Disclosures No relevant conflicts of interest to declare.

Download Full-text