PMC9557171_01
Graphical representation of the clinical history of P1 and P2. Stars and triangles represent strokes and T-LGL onset, respectively.
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Both patients developed hematological manifestations ( Figure 1  ).
Plasmatic IgM levels (A), absolute counts of CD4+ T cells (B) and CD8+ T cells (C), and percentage of T-LGL (D) in the peripheral blood of P1 (black line) and P2 (grey line) after diagnosis. Dashed lines represent reference ranges in healthy controls.
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P2's BM analyzed at 23 and 27 years of age revealed an infiltrate of lymphocytes gradually increasing from 30% to 40% of total BM cells, with a consistent component of small CD8+ T lymphocytes classifiable as T-LGL. Phenotypically, the CD8+CD57+ T-cell subset expanded from 11.8% to 19% of total BM lymphocytes, mirroring the expansion observed in the PB ( Figure 2D ). ADA2 activity ( Figure 2A ). At present, she is completely independent of any immunosuppression and anti-infectious prophylaxis.
(A) ADA2 plasmatic activity in P1 (before and after HSCT), P2, parents, carriers (n= 46), and healthy controls (n=27). (B) Visualization of mapped reads (as pairs) with Integrative Genomics Viewer (IGV) around the ADA2 exon 7. The tracks related to P1, P2, and the mother are depicted from top to bottom. On the top of each track, pairs (in red) spot the deletion event. Reads colored in grey represent properly mapped paired-end reads. (C) Graphical representation of PCR amplification used for validating the deletion. (D) Short amplicons of 300 bp visualized by agarose gel electrophoresis in P1, P2, and the mother. (E) Pedigree of the affected twin sisters, compound heterozygous for the missense mutation L188P and IVS6_IVS7 deletion in the ADA2 gene.
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No second pathogenic variant could be identified in the coding sequence even though plasma ADA2 activity was completely absent in patients and reduced in parents, compatible with a carrier status for both of them ( Figure 3A ). Thus, on the same WGS data, we explored the possible presence of large structural variants using Delly, which revealed a heterozygous deletion (chr22: 17187840 and chr22:17188716) causing the exon 7 excision both in the mother and daughters ( Figures 3B, C ). This structural variant was confirmed by amplifying the interested region using flanked primers, which revealed the presence of a short amplicon of 300 bp in the patients and the mother, but not in the father or a healthy control ( Figure 3D ). ADA2 putative receptor binding domain) and exon 7 deletion (disrupting the catalytic domain of the protein) ( Figure 3E ). AmpFlSTR  Identifiler  Plus, PCR Amplification Kit short tandem repeat (STR) multiplex assay, amplifying 15 tetranucleotide repeat loci and the Amelogenin in single PCR amplification showed complete overlap of all loci of P1 and P2, confirming that the patients were genetically identical twins.