Figure 4.
Natural evolution of disease and somatic genetic rescue in P1. (A) White blood cells (WBC, triangles), hemoglobin (Hgb, circles), and platelets (diamonds) plotted over 23 years for P1 (pedigree 1). Red arrow indicates time of clinical presentation. (B) Illustration of germline RPA1 variant in exon 9 and somatic mutation in exon 16 with respective DNA Sanger electropherograms from BM. Bottom table depicts variant allelic frequencies from exome sequencing performed in BM and skin fibroblast DNA and RNA sequencing from BM when patient was aged 20 years. (C) Copy number neutral UPD encompassing RPA1 locus at 17p13.3 (red arrow) identified by using an SNP array. Serial SNP array analysis in BM granulocytes shows UPD expansion over time (denoted by purple brackets). (D) Schematic of RPA1 locus (gray bar) with germline (c.718) and somatic (c.1735) mutational spots 17 kb apart. Three haplotype orientations between c.718 and c.1735 identified in marrow DNA of P1 at age 19 years from 2 independent experiments using digital droplet polymerase chain reaction: left haplotype, wt/wt (c.718G wild type/c.1735G wild type) denoted by black boxes; middle haplotype, mut/wt (c.718A mutant/c.1735G wild type) denoted by green and black boxes; and right haplotype, mut/mut (c.718A mutant/c.1735T mutant) denoted by green and red boxes. (E) Ultra-deep amplicon sequencing of bone marrow DNA and RNA targeting position of RPA1 somatic mutation (c.1735) confirms near total loss of mutant RNA. (F) Longitudinal deep sequencing in BM samples from diagnosis to age 25 years showing decrease in allele frequency of the germline c.G718G>A variant (red line) and increase of the somatic c.1735G>T mutation (blue line). (G) Single cells from P1 BM at ages 13 and 17 years were sequenced for germline (RPA1:chr17:1782314:G>A) and somatic (RPA1:chr17:1798378:G>T) mutational positions using single-cell DNA (scDNA) sequencing Tapestri Platform. Violin plot shows 3 clonal populations, including homozygous wild type (blue, RPA1WT/WT; rescue clone 1 = UPD17p), heterozygous RPA1E240K/WT (gold, native state hematopoiesis), and heterozygous c.718G>A with concurrent c.1735G>T stop-gain (red, RPA1E240K/WT + K579*= rescue clone 2). (H) Tapestri single-cell multi-omic analysis combining DNA mutation data and surface protein expression performed in P1 BM at age 17 years. Panels depict 3 clones (color coding identical to that in panel G) constructed from 2110 high-quality cells with normalized protein expression of markers for hematopoietic stem and progenitor cells (CD34), stem cells (CD90), progenitors (CD38), and terminally differentiated cells, including myeloid (CD11b), B-lymphoid (CD19), and T-lymphoid (CD3) cells.

Natural evolution of disease and somatic genetic rescue in P1. (A) White blood cells (WBC, triangles), hemoglobin (Hgb, circles), and platelets (diamonds) plotted over 23 years for P1 (pedigree 1). Red arrow indicates time of clinical presentation. (B) Illustration of germline RPA1 variant in exon 9 and somatic mutation in exon 16 with respective DNA Sanger electropherograms from BM. Bottom table depicts variant allelic frequencies from exome sequencing performed in BM and skin fibroblast DNA and RNA sequencing from BM when patient was aged 20 years. (C) Copy number neutral UPD encompassing RPA1 locus at 17p13.3 (red arrow) identified by using an SNP array. Serial SNP array analysis in BM granulocytes shows UPD expansion over time (denoted by purple brackets). (D) Schematic of RPA1 locus (gray bar) with germline (c.718) and somatic (c.1735) mutational spots 17 kb apart. Three haplotype orientations between c.718 and c.1735 identified in marrow DNA of P1 at age 19 years from 2 independent experiments using digital droplet polymerase chain reaction: left haplotype, wt/wt (c.718G wild type/c.1735G wild type) denoted by black boxes; middle haplotype, mut/wt (c.718A mutant/c.1735G wild type) denoted by green and black boxes; and right haplotype, mut/mut (c.718A mutant/c.1735T mutant) denoted by green and red boxes. (E) Ultra-deep amplicon sequencing of bone marrow DNA and RNA targeting position of RPA1 somatic mutation (c.1735) confirms near total loss of mutant RNA. (F) Longitudinal deep sequencing in BM samples from diagnosis to age 25 years showing decrease in allele frequency of the germline c.G718G>A variant (red line) and increase of the somatic c.1735G>T mutation (blue line). (G) Single cells from P1 BM at ages 13 and 17 years were sequenced for germline (RPA1:chr17:1782314:G>A) and somatic (RPA1:chr17:1798378:G>T) mutational positions using single-cell DNA (scDNA) sequencing Tapestri Platform. Violin plot shows 3 clonal populations, including homozygous wild type (blue, RPA1WT/WT; rescue clone 1 = UPD17p), heterozygous RPA1E240K/WT (gold, native state hematopoiesis), and heterozygous c.718G>A with concurrent c.1735G>T stop-gain (red, RPA1E240K/WT + K579*= rescue clone 2). (H) Tapestri single-cell multi-omic analysis combining DNA mutation data and surface protein expression performed in P1 BM at age 17 years. Panels depict 3 clones (color coding identical to that in panel G) constructed from 2110 high-quality cells with normalized protein expression of markers for hematopoietic stem and progenitor cells (CD34), stem cells (CD90), progenitors (CD38), and terminally differentiated cells, including myeloid (CD11b), B-lymphoid (CD19), and T-lymphoid (CD3) cells.

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