Frequency Of Gene Usage and Copy Number Variation Within The Rearranged Immunoglobin Heavy-Chain Variable Locus Based On Immune Repertoire Sequencing

Abstract The human adaptive immune system is composed of both B and T cells that undergo somatic recombination at specific loci to create rearrangements of Variable (V), Diversity (D) and Joining (J) gene segments. For the B-cell immunoglobin receptor heavy-chain (IGH), the CDR3 regions are defined by the VDJ gene segments and nucleotide insertions/deletions at these junctions that create the vast sequence diversity of the IGH repertoire. Characterizing the germline DNA in these regions is impeded by the high sequence similarity between gene segments, mutation and copy-number variation (i.e. large insertions/deletions). Currently, there is a fundamental lack of information about the baseline IGH immune repertoire V gene usage and diversity within healthy human controls. To provide an estimate of this, we sequenced functionally recombined gene segments to infer the underlying gene structure. From a set of 132 healthy controls we sorted C19+/CD27+ B-cells from whole blood and amplified genomic DNA using a highly multiplexed PCR assay that targeted the rearranged IGH receptor locus. Following DNA sequencing and data processing to assign V, D and J gene families and names, we examined the usage frequency of IGHV gene segments across all individuals. We found that of the 98 V gene segments only 56 (57%) were used at a frequency > 0.1%, and ∼10 showed little to no usage (present in<1% of individuals). This data also allowed us to identify two IGHV genes currently annotated as orphons (pseudogenes assigned to an alternate chromosomal location) that had unambiguous functional usage (IGHV4/OR15-8; IGHV3/OR16-09) and therefore must reside at the IGH locus on chromosome 14. Finally, by taking this functional approach we were able to screen all V gene segments for germline copy-number variation (e.g. large insertion/deletion events encompassing individual genes) by looking for an excess of deletion events or modal changes in gene usage. We confirmed that existence of 12 of 15 previously identified deleted IGHV gene segments. Strong deletion evidence was observed for an additional six IGHV genes (IGHV3-NL1, IGHV3-33, IGHV1-24, IGHV4-04, IGHV3-41, IGHV3-35) and ten with highly likely germline deletion events. These data suggest that functional immune profiling of rearranged immune receptors provides a more robust method of identifying individual structural variation and provides insight into the immune repertoire of healthy controls. Disclosures: Rieder: Adaptive Biotechnologies: Employment, Equity Ownership. Williamson:Adaptive Biotechnologies: Employment, Equity Ownership. Sherwood:Adaptive Biotechnologies: Employment, Equity Ownership. Emerson:Adaptive Biotechnologies: Employment, Equity Ownership. Desmarais:Adaptive Biotechnologies: Employment, Equity Ownership. Chung:Adaptive Biotechnologies: Employment, Equity Ownership. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Carlson:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties.

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Optimization of Droplet Digital Polymerase Chain Reaction (Ddpcr) For Dna Copy Number Variation Analysis of Cnv Esv27061 in Young Adults with Higher Blood Pressure

IIUM Medical Journal Malaysia ◽

10.31436/imjm.v16i1.1158 ◽

2017 ◽

Vol 16 (1) ◽

Author(s):

Siti Radziah Shaik Alaudeen ◽

Aszrin Abdullah ◽

Azarisman Shah Mohd Shah ◽

Norlelawati Abdul Talib

Keyword(s):

Blood Pressure ◽

Young Adults ◽

Copy Number Variation ◽

High Blood Pressure ◽

Dna Sequences ◽

Copy Number ◽

Healthy Controls ◽

Cross Sectional ◽

Dna Concentration ◽

Number Variation

Introduction: Copy number variation (CNV) caused by changes in DNA sequences of 1000 or more bases is implicated with susceptibility to common diseases. A study on CNV esv27061 among hypertensive Australian adults reported association with high blood pressure (BP). In Malaysia, no study on CNV among hypertensive young adults is available. Thus, this investigation aimed to assess the CNV esv27061 of young Malaysian adults with high blood pressure using optimized ddPCR. Materials and method: Ten samples each from hypertensive and healthy controls were randomly selected from samples collected for an on-going comparative cross-sectional research project among young adults living in Kuantan. The DNAs were purified using Maxwell RSC Buffy Coat DNA Kit and the concentration was measured using SimpliNano spectrophotometer. Subsequently, restriction digestion of DNAs by EcoRV was performed prior to ddPCR. The products were later subjected to droplet generation (QX100 Droplet Generator), PCR amplification and finally CNV was read by QX100 Droplet reader. Unfortunately, the above method did not yield any result. Thus, an alternative method in which purified DNA concentration was determined by QuantiFluor ONE dsDNA System (Quantus fluorometer). The DNAs (60 ng) and Alu1 were added in master mix during ddPCR and CNV esv27061 analysis was performed as stated above. Results: Optimization of method in this study showed that the detection of CNV esv27061 was possible by the use of more sensitive measurement of DNA concentration, Alu1 restriction enzyme instead of EcoRV and digestion in ddPCR reaction method rather than prior digestion. The finalized protocol run on selected hypertensive and healthy controls has shown to be reproducible and easily interpretable discrimination of gene's copy numbers. Conclusion: This optimized protocol for CNV esv27061 analysis proved useful in identifying CNV and will allow a reproducible assay evaluation and the application of this method to a bigger sample size.

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Two Novel Distinct Subtypes of Myeloid Neoplasms Molecularly Associated with Histone H3K36 Methylations

Blood ◽

10.1182/blood.v126.23.2841.2841 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2841-2841 ◽

Cited By ~ 1

Author(s):

Yosaku Watatani ◽

Yasunobu Nagata ◽

Vera Grossmann ◽

Yusuke Okuno ◽

Tetsuichi Yoshizato ◽

...

Keyword(s):

Gene Expression ◽

Copy Number ◽

Copy Number Variations ◽

Healthy Controls ◽

Employment Equity ◽

Gene Deletions ◽

Myeloid Neoplasms ◽

Frequent Mutations ◽

Equity Ownership

Abstract Myelodysplastic syndromes (MDS) and related disorders are a heterogeneous group of chronic myeloid neoplasms with a high propensity to acute myeloid leukemia. A cardinal feature of MDS, as revealed by the recent genetic studies, is a high frequency of mutations and copy number variations (CNVs) affecting epigenetic regulators, such as TET2, IDH1/2, DNMT3A, ASXL1, EZH2, and other genes, underscoring a major role of deregulated epigenetic regulation in MDS pathogenesis. Meanwhile, these mutations/deletions have different impacts on the phenotype and the clinical outcome of MDS, suggesting that it should be important to understand the underlying mechanism for abnormal epigenetic regulation for better classification and management of MDS. SETD2 and ASH1L are structurally related proteins that belong to the histone methyltransferase family of proteins commonly engaged in methylation of histone H3K36. Both genes have been reported to undergo frequent somatic mutations and copy number alterations, and also show abnormal gene expression in a variety of non-hematological cancers. Moreover, germline mutation of SETD2 has been implicated in overgrowth syndromes susceptible to various cancers. However, the role of alterations in these genes has not been examined in hematological malignancies including myelodysplasia. In this study, we interrogated somatic mutations and copy number variations, among a total of 1116 cases with MDS and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), who had been analyzed by target deep sequencing (n=944), and single nucleotide polymorphism-array karyotyping (SNP-A) (n=222). Gene expression was analyzed in MDS cases and healthy controls, using publically available gene expression datasets. SETD2 mutations were found in 6 cases, including 2 with nonsense and 4 with missense mutations, and an additional 10 cases had gene deletions spanning 1.8-176 Mb regions commonly affecting the SETD2 locus in chromosome 3p21.31, where SETD2 represented the most frequently deleted gene within the commonly deleted region. SETD2 deletion significantly correlated with reduced SETD2 expression. Moreover, MDS cases showed a significantly higher SETD2 expression than healthy controls. In total, 16 cases had either mutations or deletions of the SETD2 gene, of which 70% (7 out of 10 cases with detailed diagnostic information) were RAEB-1/2 cases. SETD2 -mutated/deleted cases had frequent mutations in TP53 (n=4), SRSF2 (n=3), and ASXL1 (n=3) and showed a significantly poor prognosis compared to those without mutations/deletions (HR=3.82, 95%CI; 1.42-10.32, P=0.004). ASH1L, on the other hand, was mutated and amplified in 7 and 13 cases, respectively, of which a single case carried both mutation and amplification with the mutated allele being selectively amplified. All the mutations were missense variants, of which 3 were clustered between S1201 and S1209. MDS cases showed significantly higher expression of ASH1L compared to healthy controls, suggesting the role of ASH1L overexpression in MDS development. Frequent mutations in TET2 (n=8) and SF3B1 (n=6) were noted among the 19 cases with ASH1L lesions. RAEB-1/2 cases were less frequent (n=11) compared to SETD2-mutated/deleted cases. ASH1L mutations did not significantly affect overall survival compared to ASH1L-intact cases. Gene Set Expression Analysis (Broad Institute) on suppressed SETD2 and accelerated ASH1L demonstrated 2 distinct expression signatures most likely due to the differentially methylated H3K36. We described recurrent mutations and CNVs affecting two histone methyltransferase genes, which are thought to represent novel driver genes in MDS involved in epigenetic regulations. Given that SETD2 overexpression and reduced ASH1L expression are found in as many as 89% of MDS cases, deregulation of both genes might play a more role than expected from the incidence of mutations and CNVs alone. Although commonly involved in histone H3K36 methylation, both methyltransferases have distinct impacts on the pathogenesis and clinical outcome of MDS in terms of the mode of genetic alterations and their functional consequences: SETD2 was frequently affected by truncating mutations and gene deletions, whereas ASH1L underwent gene amplification without no truncating mutations, suggesting different gene targets for both methyltransferases, which should be further clarified through functional studies. Disclosures Alpermann: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Shih:Novartis: Research Funding.

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