Novel Germline ZCCHC8 Mutations Causes Dyskeratosis Congenita with Genetic Rescue By Uniparental Disomy of Chromosome 12q

Telomere-biology disorders (TBD) are caused by germline defects in telomere maintenance genes, most commonly in TERT, TERC, DKC1, TINF2 and RTEL1. Recently, ZCCHC8 was added to the list of genes that cause an adult-onset TBD as it is required for TERC maturation and telomerase function. The heterozygous ZCCHC8 p.P186L and p.E192K germline mutations are etiologic in familial pulmonary fibrosis with short telomeres. Here we expand the phenotypes associated with ZCCHC8 by describing two unrelated young patients with dyskeratosis congenita (DC) identified with distinct de novo pathogenic ZCCHC8 mutations in the p.G184 codon.

The first patient (UPN1) presented as a 4 year old boy with cerebellar hypoplasia, pancytopenia, and the clinical mucocutaneous triad. Bone marrow (BM) revealed signs of dyserythropoiesis and aplasia. Further workup showed he had very short telomere length (TL < 1^st percentile of age-matched controls) and normal karyotype. He was identified with a ZCCHC8 p.Gly184Arg mutation. UPN2 presented at age 25; she had typical mucocutaneous findings, mild anemia and thrombocytopenia, a hypocellular BM (<5%) without dysplasia, normal karyotype, and very short TL. Her sequence data revealed a ZCCHC8 p.Gly184Val mutation. Neither patient had signs of pulmonary or liver disease.

In both cases, the novel ZCCHC8 variants were in complete heterozygosity in patients’ fibroblasts but detected at somatic ranges in peripheral blood (PB) leukocytes by targeted sequencing (p.Gly184Arg/Val variant allele frequency [VAF] of 7.5% and 40-20%, respectively). The unusual VAFs in PB prompted the investigation of genomic imbalances by SNP array. A large copy-neutral loss of heterozygosity (uniparental disomy; UPD) of chromosome 12q was identified in both patients, confirming a somatic genetic rescue (SGR) of the germline p.Gly184Arg/Val mutation in 85% and >40% of PB cells, respectively. Direct SGR of these germline mutations is a proof-of-concept that they are pathogenic and disease-causing. In both cases, the parents and multiple siblings were wild-type.

Longitudinal screening of PB from UPN02 showed that the VAF of the p.Gly184Val mutation decreased from 41% to 22% in 7 years of follow-up, a consequence of the UPD(12q) clonal expansion. This coincided with an increased megakaryocyte frequency and preserved trilineage hematopoiesis in the BM. Despite the hypocellular BM and the typical DC phenotype, patients’ thrombocytopenia improved (from 33 to 74x10³cell/µL) and the absolute neutrophil and monocyte counts slightly increased without any specific treatment. Error-corrected sequencing (ECS) also detected four additional somatic ZCCHC8 mutations at VAFs <0.5-2.5% in her PB, mostly truncations before the p.G184 position. At last follow-up, SGR characterization in BM mononuclear cells from UPN2 by single-cell proteogenomic sequencing (Mission Bio) revealed that the UPD(12q) arose in hematopoietic stem cells and propagated downstream in all megakaryocyte–erythroid, myeloid and lymphoid progenitors cells; 84%, 77% and 64% of these progenitors were wild-type (as a consequence of UPD12q), respectively. Most myeloid and NK cells (>85%) harbored the UPD12q, while only less than 15% and 30% of T (both CD8+ and CD4+) and B cells, respectively, carried this SGR. These findings were validated in sorted populations from peripheral blood by ECS and Sanger sequencing.

In conclusion, we report novel de novo ZCCHC8 mutations that cause DC and very short telomeres; the disease onset of UPN2 was later than classically observed (>20 vs. <10 years old, respectively). Blood count recovery of UPN2 coincided with UPD(12q) expansion, suggesting that this natural gene therapy mechanism ameliorated marrow failure. The strong selective pressure for the p.G184 SGR by UPD(12q) in unrelated cases highlights the biological relevance of this protein, specifically this codon, for telomere maintenance. Further study is ongoing to precisely determine the functional impact of the p.G184 mutations on telomerase activity and telomere integrity. The somatic rescue of the germline mutations by UPD(12q) in myeloid and NK cells but not T/B lymphocytes changed the expected germline VAFs of this mutation to somatic ranges in PB. Therefore, SGR mechanisms should be considered when screening TBD patients, particularly those who have undergone extensive sequencing without a clear molecular etiology (about 20%).