Investigation of Clonal Hematopoiesis in New Japanese Cohort
Authors: Makishima Kenichi , Yukie Kashima , Kuze Yuta , Suma Sakurako , Abe Yoshiaki , Suehara Yasuhito , Sakamoto Tatsuhiro , Hattori Keiichiro , Kito Sakura , Nguyen Tran Bich , Fujisawa Manabu , Chiba Shigeru , Sakata-Yanagimoto Mamiko , Suzuki Yutaka 
Published: December 2023

Introduction: Clonal hematopoiesis (CH) is an age-related change in which blood cells with somatic mutations are clonally expanded. CH is known to be a predisposing factor for various age-related diseases. However, the characteristics of blood cells with somatic mutations derived from CH at the single-cell level is not fully understood.

Objective: We performed this study to explore the comprehensive properties of mutant cells derived from CH.

Methods: We enrolled 51 healthy elderly individuals (male, 13; female, 38) from the Kashiwanoha cohort for this study.

To investigate somatic mutations frequently mutated in CH, we designed a custom panel targeting 49 genes. Targeted deep sequencing (TDS) was performed on mononuclear cells, and T, B, and monocyte fractions of peripheral blood (PB). Error-correction process was performed on TDS data. The error-correction process to the TDS data was as follows: Firstly, read families which have more than 5 identical unique molecular identifier (UMI) amplicon were included. Second, at each position, nucleotides were compared and a consensus nucleotide was called if at least 90% the nucleotides were identical. If the agreement was below 90%, the nucleotides were changed to “N ”at that position. Third, if the “N” constituted less than 10% of a read family, it was recognized as a consensus read.

Single-cell multiome analysis (sc Multiome) was performed on PB samples of 43 individuals by 10xGenomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression. Sc Multiome analysis was conducted using Seurat and Signac for quality control, integration, and clustering. Additionally, we employed long-read sequencing with the PromethION platform to identify CH mutations in the sc Multiome libraries.

Results: The median age of this cohort was 72 years old (range, 50 - 85).

After error correction was applied to the TDS data, a total of 56 mutations were detected in 34 individuals ( DNMT3A R882, 1; DNMT3A nonR882, 16; TET2, 10; GNAS, 4; STAT3, 3; KRAS, 1; MYD88, 1; others, 20). The median VAF was 0.0091 (range, 0.003 to 0.164). The number of mutated genes in each individual increased with age, and individuals of under the age of 69 had significantly fewer mutations compared to those over 70 years old.

Regarding the TDS data of each fraction, DNMT3A mutations were detected in both monocyte and B-cell fractions in 5 cases, while they were restricted to T-cell fraction in one case. In 3 cases, mutations were detected in all fractions. TET2 mutations were present in both monocyte and B-cell fractions in all 5 cases. On the other hand, KRAS, STAT3 and MYD88 mutations were restricted to T-, T-, and B-cell fractions, respectively.

In sc Multiome data, the median number of cells included was 7931 (range, 2998 to 13413). By the sc Multiome long read sequencing, 39 mutations were detected in 22 individuals ( DNMT3A R882, 1; DNMT3A nonR882, 5; TET2, 6; GNAS, 1; STAT3, 3; KRAS, 1; others, 22). The median rate of DNMT3A mutated cells was 0.007(range, 0.003 to 0.014). Similarly, the median TET2 mutated cell population rate was 0.007(range, 0.004 to 0.044). The average cell number of B cells, T cells, monocytes, and NK cells in sc Muliome data were 955 (range, 305 to 2275), 3875 (range, 955 to 7726), 1812 (range, 352 to 4750), and 868 (range, 289 to 6022) respectively. Among B cells, 16 mutations were detected in 13 individuals ( DNMT3A nonR882, 2; TET2, 2; STAT3, 3; others, 9). For T cells, 27 mutations were detected in 20 individuals ( DNMT3A R882, 1; DNMT3A nonR882, 4; TET2, 4; GNAS, 1; STAT3, 3;others, 12). In Monocytes cells 24, mutations were detected in 16 individuals ( DNMT3A R882, 1; DNMT3A nonR882, 4; TET2, 5; GNAS, 1; STAT3, 3;others, 10). Lastly, among NK cells, 23 mutations were detected in 17 individuals ( DNMT3A nonR882, 5; TET2, 4; GNAS, 1; STAT3, 3;others, 10). The mutated cell populations in each cell fraction were 0.037(range, 0.014 to 0.208), 0.013 (range, 0.002 to 0.180), 0.013(range, 0.003 to 0.178), and 0.043(range, 0.009 to 0.284).

Conclusion: This study clarified the detailed distribution of CH-derived mutant cells in PB of elderly individuals. We identified 17 mutations that expanded in myeloid fraction and 12 mutations expanded in lymphoid fraction. Sc Multiome analysis combined with long-read sequencing allowed us to gain a deeper understanding of CH mutated cell populations. These findings provide the valuable insights into properties of CH mutant cells.