A long reads-based de-novo assembly of the genome of the Arlee homozygous line reveals structural genome variation in rainbow trout

Currently, there is still a need to improve the contiguity of the rainbow trout reference genome and to use multiple genetic backgrounds that will represent the genetic diversity of this species. The Arlee doubled haploid line was originated from a domesticated hatchery strain that was originally collected from the northern California coast. The Canu pipeline was used to generate the Arlee line genome de-novo assembly from high coverage PacBio long-reads sequence data. The assembly was further improved with Bionano optical maps and Hi-C proximity ligation sequence data to generate 32 major scaffolds corresponding to the karyotype of the Arlee line (2N=64). It is composed of 938 scaffolds with N50 of 39.16 Mb and a total length of 2.33 Gb, of which ∼95% was in 32 chromosome sequences with only 438 gaps between contigs and scaffolds. In rainbow trout the haploid chromosome number can vary from 29 to 32. In the Arlee karyotype the haploid chromosome number is 32 because chromosomes Omy04, 14 and 25 are divided into six acrocentric chromosomes. Additional structural variations that were identified in the Arlee genome included the major inversions on chromosomes Omy05 and Omy20 and additional 15 smaller inversions that will require further validation. This is also the first rainbow trout genome assembly that includes a scaffold with the sex-determination gene (sdY) in the chromosome Y sequence. The utility of this genome assembly is demonstrated through the improved annotation of the duplicated genome loci that harbor the IGH genes on chromosomes Omy12 and Omy13.Article SummaryA de-novo genome assembly was generated for the Arlee homozygous line of rainbow trout to enable identification and characterization of genome variants towards developing a rainbow trout pan-genome reference. The new assembly was generated using the PacBio sequencing technology and scaffolding with Hi-C contact maps and Bionano optical mapping. A contiguous genome assembly was obtained, with the contig and scaffold N50 over 15.6 Mb and 39 Mb, respectively, and 95% of the assembly in chromosome sequences. The utility of this genome assembly is demonstrated through the improved annotation of the duplicated genome loci that harbor the IGH genes.

Preferential Usage of Specific Immunoglobulin Heavy Chain Variable Region Genes with Unmutated Profile and Advanced Stage at Presentation Are Common Features in Patients with Chronic Lymphocytic Leukemia from Senegal

Introduction : Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in Western populations, being rarer in Asian and African people. It has been suggested that patients with CLL from Africa might have a more aggressive disease compared with Causasien patients. In this study, we aimed to identify genetic factors that may account for this difference Methods: We collected peripheral blood mononuclear cells (PBMCs) from a total of 75 patients with CLL, 25 from Senegal (Africa), and 50 from Siena. Since it is well known that there are differences in germline IGH repertoires between different populations, we also collected PBMCs from five healthy Senegalese individuals as control. We analyzed immunoglobulin heavy chain (IGH) genes mutational status by performing next-generation sequencing in these 2 groups of patients. Results: We found that Senegalese patients more frequently had adverse prognostic factors and an unmutated profile. Furthermore, we documented that IGHV1 (IGHV1-69), IGHD3, and IGHJ6 were significantly more frequent in Senegalese patients, whereas IGHV3-30 was common and limited to the Italian cohort. Stereotyped receptors commonly detected in the white population were not recorded in our Senegalese series. Conclusion: The different IGH repertoire we observed in the Senegalese cohort may reflect the diverse genetic and microenvironmental (ie, polymicrobial stimulation) background. Disclosures Gozzetti: Takeda: Honoraria; Amgen: Honoraria; Janssen: Honoraria, Research Funding.

The Immunoglobulins: New Insights, Implications, and Applications

Immunoglobulins (Igs), as one of the hallmarks of adaptive immunity, first arose approximately 500 million years ago with the emergence of jawed vertebrates. Two events stand out in the evolutionary history of Igs from cartilaginous fish to mammals: ( a) the diversification of Ig heavy chain (IgH) genes, resulting in Ig isotypes or subclasses associated with novel functions, and ( b) the diversification of genetic and structural strategies, leading to the creation of the antibody repertoire we know today. This review first gives an overview of the IgH isotypes identified in jawed vertebrates to date and then highlights the implications or applications of five new recent discoveries arising from comparative studies of Igs derived from different vertebrate species.

Lymphoma Heterogeneity: Three Different Histological Pictures and One Unique Clone

We report a patient who developed up to three different lymphomas with the same clonal IGH rearrangement. She was first diagnosed of splenic zone marginal lymphoma and relapsed for the first time with Hodgkin lymphoma histology and later with diffuse large B-cell lymphoma histology. Subsequent biopsies and analysis of clonally rearranged IGH genes helped to elucidate the clonal relationship between the three histologies and to confirm a common origin from the three tissue histologies. An integrated diagnosis should always be performed in order to achieve the most accurate diagnosis and be able to choose the best therapeutic options for our patients.

Clonotypic Analysis of Immunoglobulin Heavy Chain Sequences in Patients with Waldenström’s Macroglobulinemia: Correlation withMYD88L265P Somatic Mutation Status, Clinical Features, and Outcome

We performedIGHclonotypic sequence analysis in WM in order to determine whether a preferentialIGHgene rearrangement was observed and to assessIGHVmutational status in blood and/or bone marrow samples from 36 WM patients. In addition we investigated the presence ofMYD88L265P somatic mutation. AfterIGHVDJ locus amplification, monoclonal VDJ rearranged fragments were sequenced and analyzed.MYD88L265P mutation was detected by AS-PCR. The most frequent family usage wasIGHV3(74%);IGHV3-23andIGHV3-74segments were used in 26% and 17%, respectively. Somatic hypermutation was seen in 91% of cases.MYD88L265P mutation was found in 65,5% of patients and absent in the 3 unmutated. These findings did not correlate with clinical findings and outcome. Conclusion.IGH genes’repertoire differed in WM from those observed in other B-cell disorders with a recurrentIGHV3-23andIGHV3-74usage; monoclonalIGHVwas mutated in most cases, and a high but not omnipresent prevalence ofMYD88L265P mutation was observed. In addition, the identification of 3 patients with unmutatedIGHVgene segments, negative for theMYD88L265P mutation, could support the hypothesis that an extra-germinal B-cell may represent the originating malignant cell in this minority of WM patients.

Clonotypic Analysis Of Immunoglobulin Heavy Chain (IgH) Sequences In Patients With Waldenström’s Macroglobulinemia. Correlation With MYD88 L265P Somatic Mutation Status, Clinical Features and Outcome

Background and aim Waldenström’s Macroglobulinemia (WM) cells express a unique clonotypic rearrangement of heavy chain immunoglobulin (IgH) genes in each individual patient. Recent studies demonstrate high frequency of the MYD88 L265P somatic mutation in patients with WM, implying a new pathway for the pathogenesis of this disease. In this study we characterized clonal IgH genes rearrangements, IGHV gene use, somatic hypemutations (SHM) and presence of the ΜΥD88 L265P mutation in a cohort of WM patients and we investigated any eventual correlation with patients’ clinical features. Patients and methods 36 patients with WM were studied (median age 64y, 19 males and 17 females, symptomatic 72%, asymptomatic 28%) of whom 45%, 39% and 16% were stage IPSSWM 1, 2 and 3, respectively. Median time to treatment initiation was 13 months and median overall survival was 61 months. DNA was extracted from patients’ bone marrow/blood samples and clonal IgH VDJ rearrangements were amplified by PCR using the Biomed-2 Concerted Action protocol. Monoclonal VDJ rearranged PCR fragments were sequenced and analyzed using the IMGT database (www.imgt.org). Thirty-one patients’ DNA samples were also analyzed for the presence of the ΜΥD88 L265P mutation by using a mutation specific PCR assay. Results The most frequent clonal rearrangements observed in our patients were in the IGVH3 family (73% of cases); the IGVH3-23 gene and the IGVH3-74 gene were used in 24.4% and 18,9% of cases, respectively. Somatic hypermutation (>2%) was seen in all but three cases (92%). Mean percentage of mutations in all cases, IGVH3 family, IGVH3-23 and IGVH3-74 genes was 7,5%, 8%, 9,4% and 7,5%, respectively. Sixty-two percent of the patients were positive for the ΜΥD88 L265P mutation and notably all three unmutated WM cases were negative for this MYD88 point mutation. No correlation between VH or DH gene usage, CDR3 length, and mutational status with clinical parameters, time to treatment initiation, or survival was found. Conclusions WM IgH genes repertoire, as expected, differs from that observed in normal B-cells and other B-cell diseases such as MZL, MCL and B-CLL/SLL. In addition to the known hyper-representation of the IGVH3-23 gene, another member of the VH3 family, the IGVH3-74 gene is also over-represented in WM. The high IGVH mutation rate supports a derivation of WM cells from postgerminal center memory B cells in the majority (92%) of WM patients. However, the identification of a minority of WM patients (3 of 36) with unmutated IGHV gene segments, negative for the ΜΥD88 L265P mutation, supports the hypothesis that they represent a subgroup of WM not arising from post-germinal B cells with a different disease pathogenesis. Disclosures: No relevant conflicts of interest to declare.

Convergent differentiation: myeloid and lymphoid pathways to murine plasmacytoid dendritic cells

The developmental origin of IFN-producing plasmacytoid dendritic cells (pDCs) has been uncertain. In the present study, we tracked the development of pDCs in cultures of BM precursors stimulated with Flt3 ligand. Common myeloid precursors (CMPs) produced both conventional DCs (cDCs) and pDCs via the DC-restricted common DC precursor. Common lymphoid precursors (CLPs) produced only a few cDCs with variable efficiency, but produced pDCs via a transient intermediate precursor with B-cell potential. The pDCs of both origins produced IFN-α when stimulated with CpG oligonucleotides. The pDCs of CLP origin showed evidence of past RAG1 expression and had D-J rearrangements in IgH genes. Most pDCs and all cDCs of CMP origin lacked these signs of a lymphoid past. However, in these cultures, some pDCs of CMP origin showed evidence of past RAG1 expression and had D-J IgH gene rearrangements; most of these derived from a subset of CMPs already expressing RAG1.