Analysis of genetic variants in myeloproliferative neoplasms using a 22-gene next-generation sequencing panel

Background The Philadelphia (Ph)-negative myeloproliferative neoplasms (MPNs), namely essential thrombocythaemia (ET), polycythaemia vera (PV) and primary myelofibrosis (PMF), are a group of chronic clonal haematopoietic disorders that have the propensity to advance into bone marrow failure or acute myeloid leukaemia; often resulting in fatality. Although driver mutations have been identified in these MPNs, subtype-specific markers of the disease have yet to be discovered. Next-generation sequencing (NGS) technology can potentially improve the clinical management of MPNs by allowing for the simultaneous screening of many disease-associated genes. Methods The performance of a custom, in-house designed 22-gene NGS panel was technically validated using reference standards across two independent replicate runs. The panel was subsequently used to screen a total of 10 clinical MPN samples (ET n = 3, PV n = 3, PMF n = 4). The resulting NGS data was then analysed via a bioinformatics pipeline. Results The custom NGS panel had a detection limit of 1% variant allele frequency (VAF). A total of 20 unique variants with VAFs above 5% (4 of which were putatively novel variants with potential biological significance) and one pathogenic variant with a VAF of between 1 and 5% were identified across all of the clinical MPN samples. All single nucleotide variants with VAFs ≥ 15% were confirmed via Sanger sequencing. Conclusions The high fidelity of the NGS analysis and the identification of known and novel variants in this study cohort support its potential clinical utility in the management of MPNs. However, further optimisation is needed to avoid false negatives in regions with low sequencing coverage, especially for the detection of driver mutations in MPL.

Phenotypic heterogeneity in individuals with MECOM variants in 2 families

MECOM encodes the transcriptional regulators, EVI1 and MDS1-EVI1, from two distinct transcription start sites. EVI1 plays important roles in hematopoiesis and stem cell self-renewal. Recently, our group and others revealed that individuals with MECOM variants present diverse hematological and skeletal defects, including radioulnar synostosis (RUS). In the present study, we analyzed two families suspected with MECOM-associated syndrome. In family 1, a MECOM splicing variant (c.2285+1G>A) was identified in an individual with bone marrow failure (TRS4) without RUS and her mother, who had mild leukocytopenia, thrombocytopenia, and bilateral RUS. A copy neutral loss of heterozygosity decreasing the variant allele frequency was observed in the bone marrow of TRS4 and the peripheral blood leukocytes of her mother. However, TRS4 remained transfusion-dependent. In family 2, a MECOM variant (c.2208-4A>G), which was predicted to cause a cryptic acceptor site that results in a 3-base insertion (an insertion of Ser) in the mRNA, was identified in the proband, with bone marrow failure; this variant was also observed in her brother and father, both of whom have skeletal malformations, but no cytopenia. RT-PCR using leukocytes revealed a transcript with a 3-bp insertion in the proband, her brother, and the father, suggesting that the transcript variant with a 3-bp insertion is independent of blood phenotype. Collectively, these results suggest the presence of intrafamilial clinical heterogeneity in both families with MECOM splicing variants. Somatic genetic event may complicate the understanding of clinical variability among family members.

Inherited human Apollo deficiency causes severe bone marrow failure and developmental defects

Inherited bone marrow failure syndromes (IBMFS) represent a group of disorders typified by impaired production of one or several blood cell types. The telomere biology disorders dyskeratosis congenita (DC) and its severe variant Høyeraal-Hreidarsson (HH) syndrome are rare IBMFS characterized by bone marrow failure, developmental defects, and various premature aging complications associated with critically short telomeres. Here we identified biallelic variants in the gene encoding the 5'-to-3' DNA exonuclease Apollo/SNM1B in three unrelated patients presenting with a DC/HH phenotype consisting of early onset hypocellular bone marrow failure, B and NK lymphopenia, developmental anomalies, microcephaly and/or intrauterine growth retardation. All three patients carry a homozygous or compound heterozygous (in combination with a null-allele) missense variant affecting the same residue L142 (L142F or L142S) located in the catalytic domain of Apollo. Apollo-deficient cells from patients exhibited spontaneous chromosome instability and impaired DNA repair that was complemented by CRISPR/Cas9-mediated gene correction. Furthermore, patients' cells showed signs of telomere fragility that were however not associated with global reduction of telomere length. Unlike patients' cells, human Apollo KO HT1080-cell lines showed strong telomere dysfunction accompanied by excessive telomere shortening, suggesting that the L142S and L142F Apollo variants are hypomorphic. Collectively, these findings define human Apollo as a genome caretaker and identify biallelic Apollo variants as a genetic cause of a hitherto unrecognized severe IBMFS combining clinical hallmarks of DC/HH with normal telomere length.

BMT for Myelodysplastic Syndrome: When and Where and How

Myelodysplastic syndromes (MDS) are a diverse group of hematological malignancies distinguished by a combination of dysplasia in the bone marrow, cytopenias and the risk of leukemic transformation. The hallmark of MDS is bone marrow failure which occurs due to selective growth of somatically mutated clonal hematopoietic stem cells. Multiple prognostic models have been developed to help predict survival and leukemic transformation, including the international prognostic scoring system (IPSS), revised international prognostic scoring system (IPSS-R), WHO prognostic scoring system (WPSS) and MD Anderson prognostic scoring system (MDAPSS). This risk stratification informs management as low risk (LR)-MDS treatment focuses on improving quality of life and cytopenias, while the treatment of high risk (HR)-MDS focuses on delaying disease progression and improving survival. While therapies such as erythropoiesis stimulating agents (ESAs), erythroid maturation agents (EMAs), immunomodulatory imide drugs (IMIDs), and hypomethylating agents (HMAs) may provide benefit, allogeneic blood or marrow transplant (alloBMT) is the only treatment that can offer cure for MDS. However, this therapy is marred, historically, by high rates of toxicity and transplant related mortality (TRM). Because of this, alloBMT is considered in a minority of MDS patients. With modern techniques, alloBMT has become a suitable option even for patients of advanced age or with significant comorbidities, many of whom who would not have been considered for transplant in prior years. Hence, a formal transplant evaluation to weigh the complex balance of patient and disease related factors and determine the potential benefit of transplant should be considered early in the disease course for most MDS patients. Once alloBMT is recommended, timing is a crucial consideration since delaying transplant can lead to disease progression and development of other comorbidities that may preclude transplant. Despite the success of alloBMT, relapse remains a major barrier to success and novel approaches are necessary to mitigate this risk and improve long term cure rates. This review describes various factors that should be considered when choosing patients with MDS who should pursue transplant, approaches and timing of transplant, and future directions of the field.

Single-Cell RNA-Seq of Bone Marrow Cells in Aplastic Anemia

Aplastic anemia (AA) is an autoimmune disease characterized by peripheral blood pancytopenia and bone marrow failure. Recently, a research study verified bone marrow failure of AA patients resulting from hematopoietic stem and progenitor cell (HSPC) attack by active T cells. Nonetheless, whether B cells, as one of the important immune cells, destruct the hematopoiesis is still unclear. Here, a large-scale single-cell transcriptomic sequencing of 20,000 bone marrow cells from AA patients and healthy donors was performed. A total of 17 clusters and differentially expressed genes were identified in each cluster relative to other clusters, which were considered potential marker genes in each cluster. The top differentially expressed genes in HSPCs (S100A8, RETN, and TNFAIP3), monocytes (CXCL8, JUN, and IL1B), and neutrophils and granulocytes (CXCL8, NFKBIA, and MT-CYB) were related to immune and inflammatory injury. Then, the B-cell receptor (BCR) diversities and pairing frequencies of V and J genes were analyzed. The highest pairing frequencies in AA patients were IGHV3-20-IGKJ2, IGHV3-20-IGKJ4, and IGHV3-20-IGHLJ2. Meanwhile, there were 3 V genes, including IGHV3-7, IGHV3-33, and IGLV2-11, with elevated expression in B cells from AA patients. Cell type–specific ligand–receptor was further identified in B-cell interaction with hematopoietic cells in the bone marrow. The changed ligand–receptor pairs involved antigen presentation, inflammation, apoptosis, and proliferation of B cells. These data showed the transcriptomic landscape of hematopoiesis in AA at single-cell resolution, providing new insights into hematopoiesis failure related with aberrance of B cells, and provide available targets of treatment for AA.

Coronavirus 19 (COVID-19) infection and vaccination in patients with Shwachman-Diamond syndrome

Because they can experience neutropenia due to bone marrow failure, patients with Shwachman-Diamond syndrome (SDS) carry increased risk for serious infections compared to the general population; however, it has been unknown whether this predisposes them to COVID-19 infection or more significant complications. We compiled results from a survey distributed to participants in the Shwachman-Diamond Syndrome Registry between May and June 2021. In this report we describe the characteristics and outcomes of patients with SDS who had COVID-19. Patients reported a short clinical course without significant complications or severe cytopenias. Additionally, COVID-19 vaccines were well tolerated with only minor side effects.

Fanconi Anemia Patients from an Indigenous Community in Mexico Carry a New Founder Pathogenic Variant in FANCG

Fanconi anemia (FA) is a rare genetic disorder caused by pathogenic variants (PV) in at least 22 genes, which cooperate in the FA/BRCA pathway to maintain genome stability. PV in FANCA, FANCC, and FANCG account for most cases (~90%). This study evaluated the chromosomal, molecular, and phenotypic findings of a novel founder FANCG PV, identified in three patients with FA from the Mixe community of Oaxaca, Mexico. All patients presented chromosomal instability and a homozygous PV, FANCG: c.511-3_511-2delCA, identified by next-generation sequencing analysis. Bioinformatics predictions suggest that this deletion disrupts a splice acceptor site promoting the exon 5 skipping. Analysis of Cytoscan 750K arrays for haplotyping and global ancestry supported the Mexican origin and founder effect of the variant, reaffirming the high frequency of founder PV in FANCG. The degree of bone marrow failure and physical findings (described through the acronyms VACTERL-H and PHENOS) were used to depict the phenotype of the patients. Despite having a similar frequency of chromosomal aberrations and genetic constitution, the phenotype showed a wide spectrum of severity. The identification of a founder PV could help for a systematic and accurate genetic screening of patients with FA suspicion in this population.

Early Onset Colorectal Cancer: An Emerging Cancer Risk in Patients with Diamond Blackfan Anemia

Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome, the founding member of a class of disorders known as ribosomopathies. Most cases result from loss of function mutations or deletions in 1 of 23 genes encoding either a small or large subunit-associated ribosomal protein (RP), resulting in RP haploinsufficiency. DBA is characterized by red cell hypoplasia or aplasia, poor linear growth and congenital anomalies. Small case series and case reports demonstrate DBA to be a cancer predisposition syndrome. Recent analyses from the Diamond Blackfan Anemia Registry of North America (DBAR) have quantified the cancer risk in DBA. These studies reveal the most prevalent solid tumor, presenting in young adults and in children and adolescents, to be colorectal cancer (CRC) and osteogenic sarcoma, respectively. Of concern is that these cancers are typically detected at an advanced stage in patients who, because of their constitutional bone marrow failure, may not tolerate full-dose chemotherapy. Thus, the inability to provide optimal therapy contributes to poor outcomes. CRC screening in individuals over the age of 50 years, and now 45 years, has led to early detection and significant improvements in outcomes for non-DBA patients with CRC. These screening and surveillance strategies have been adapted to detect familial early onset CRC. With the recognition of DBA as a moderately penetrant cancer risk syndrome a rational screening and surveillance strategy will be implemented. The downstream molecular events, resulting from RP haploinsufficiency and leading to cancer, are the subject of significant scientific inquiry.