Zebrafish Models Provide Novel Insights into the Disease Biology of Parn-Mutant Dyskeratosis Congenita and DNAJC21-Mutant Shwachman-Diamond Syndrome

Inherited bone marrow failure syndromes (IBMFS) are a group of rare inherited genetic disorders defined by impaired hematopoiesis and multi- or unilineage cytopenias. Dyskeratosis Congenita (DC) is characterized by pancytopenia, abnormal skin pigmentation and leukoplakia and is caused by mutations in genes involved in telomere biogenesis and/or RNA processing such as TERT, TERC, DKC1 and PARN. Shwachman-Diamond syndrome (SDS) is characterized by neutropenia and exocrine pancreatic insufficiency and occurs as a result of mutations in genes required for ribosome subunit maturation such as SBDS, DNAJC21 and EFL1. How the disruption of such ubiquitous cellular processes leads to distinct cytopenic phenotypes is not fully understood. Further, IBMFS patients have a high risk of developing myelodysplastic syndrome and/or acute myeloid leukemia with a cumulative incidence of 36% by 30 years of age for SDS patients and 13% for DC patients who do not undergo a stem cell transplant.

As IBMFS are rare and large numbers of primary human samples are not readily available for mechanistic studies, we employed zebrafish (Danio rerio) given their highly conserved hematopoietic program and ease of genetic manipulation. Using CRISPR-Cas9 genomic editing, we generated deletion mutations predicted to introduce premature stop codons in the zebrafish orthologs, parn and dnajc21, two more recently identified causes of DC and SDS, respectively. Homozygous zygotic parn mutants had normal morphology and were viable to adulthood. However, fertile homozygous adult females were not recoverable in subsequent generations, implicating parn as an essential factor in oocyte specification. In contrast, homozygous dnajc21 mutants showed normal development to adulthood. Using whole mount in situ hybridization (WISH), we found increased expression of hematopoietic precursor markers (erythroid - gata1 and myeloid - lcp1) and concurrently decreased expression of mature hematopoietic markers (erythroid - hbbe3, myeloid - mpx and thrombocyte - CD41) in parn mutants at 24- and 48-hours post-fertilization (hpf). Further, o-dianisidine staining showed reduced hemoglobinized erythrocytes at 48 hpf. These data indicate that multilineage embryonic hematopoiesis is compromised in parn mutants, recapitulating the pancytopenia observed in patients with DC. WISH for mpx in dnajc21 mutants revealed reduced expression at 24 and 48 hpf, recapitulating the neutropenia seen in SDS.

Activation of the TP53 tumor suppressor pathway has been suggested to mediate marrow failure and leukemic progression in some types of IBMFS. Using quantitative PCR to measure the expression of tp53 and its downstream effector p21 at 48 hpf, we observed no significant changes in parn mutants but significantly upregulated tp53 expression in dnajc21 mutants. To further study the role of tp53 in dnajc21-mutant SDS, we crossed dnajc21 mutants with a zebrafish line carrying a tp53 ^R217H point mutation that confers anti-apoptotic phenotypes. Heterozygous tp53 loss in a dnajc21-/- or +/- background resulted in reduced overall larval growth and abnormal yolk sac development, suggesting defective lipid metabolism. Using the lipophilic dye, Oil Red O, we observed reduced lipid distribution in the vasculature and caudal hematopoietic tissue region (equivalent to mammalian fetal liver) of dnajc21 mutants at 48 hpf.

In summary, these findings support a role for PARN in hematopoietic lineage specification through mechanisms that are predominantly TP53-independent. By contrast, DNAJC21 is required for neutrophil specification and normal lipid metabolism and may function in a TP53-dependent manner. These zebrafish models provide new insights into the unique biology underlying these IBMFS and can serve as an in vivo platform for identifying therapeutic compounds that restore normal hematopoiesis and prevent leukemic transformation.