124 - KAT14 as a Novel Gene Associated with Diamond-Blackfan Anemia

Publication Number: 2119.124

Shiyu Luo, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United States; Anjali Pawar, University of California, Davis, School of Medicine, Sacramento, CA, United States; Haoyue sheng, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United States; Nicole Robinson, Goodman Cancer Institute- McGill University, Montreal, PQ, Canada; Lamrot Tulu, University of California Davis Children's Hospital, Sacramento, CA, United States; Casie A. Genetti, Boston Children's Hospital, Boston, MA, United States; Abdulrahman A.. Aldeeri, King Saud University and Boston Children’s Hospital, Boston, MA, United States; Timothy Yu, Harvard Medical School, Boston, MA, United States; Alan H.. Beggs, Boston Children's Hospital, Boston, MA, United States; Klaus Schmitz-Abe, University of Miami, Miami, FL, United States; Qifei Li, University of Miami Miller School of Medicine and Holtz Children's Hospital, Miami, FL, United States; Madesh Chinnathevar Ramesh, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United States; Victtoria O. Ardellini, Holtz Children's Hospital Jackson Memorial Hospital, Sunny Isles Beach, FL, United States; Ibrahim Ghemlas, King Faisal Specialist Hospital & Research Centre, Riyadh, Ar Riyad, Saudi Arabia; Firdous Abdulwahab, King faisal hospital, Riyadh, Ar Riyad, Saudi Arabia; Lama AlAbdi, King Faisal Specialist Hospital and Research Center, Riyadh, Ar Riyad, Saudi Arabia; Khadijah Bakur, Liferaomics, Riyadh, Ar Riyad, Saudi Arabia; Fowzan S. Alkuraya, Alfaisal University, Riyadh, Ar Riyad, Saudi Arabia; Xiang-Jiao Yang, McGill University Faculty of Medicine and Health Sciences, Montreal, PQ, Canada; Suma P. Shankar, University of California, Davis, School of Medicine, Sacramento, CA, United States; Pankaj Agrawal, Holtz Children's Hospital Jackson Memorial Hospital, Miami, FL, United States

Background: Diamond-Blackfan anemia (DBA) is a rare congenital bone marrow failure syndrome presenting in infancy with red cell aplasia of variable severity, and frequent growth or congenital anomalies. Pathogenic variants in ribosomal protein genes explain ~60-70% of cases, leaving up to 40% genetically unexplained. KAT14, encoding a histone acetyltransferase within the ATAC complex, is essential for chromatin regulation and development but has not previously been linked to a Mendelian disorder.

Objective: To define the genetic and functional basis of KAT14-associated anemia and establish its role in erythropoiesis.

Design/Methods: Patients identified through rare disease networks underwent deep phenotyping, pedigree analysis, and exome/genome sequencing with variant confirmation. Structural modeling used AlphaFold and PyMOL, and variant pathogenicity was evaluated in silico and in HEK293 cells. Transcriptomic profiling of patient blood was analyzed for differential gene expression. Functional studies in Kat14 hypomorph mice included hematologic profiling and bone marrow RT-qPCR for erythroid transcription factors.

Results: Five affected individuals from four unrelated families carried rare biallelic KAT14 variants: a homozygous frameshift (p.Ser67LeufsTer11) in one patient; compound heterozygous truncating and missense variants (p.R566Ter and p.R595W) in two siblings; and a homozygous p.R595W variant in two additional patients. Clinical presentations ranged from severe early-onset anemia to transfusion dependence, with variable steroid responses. Two females responded to steroids, while others were refractory, and one underwent bone marrow transplantation. Notably, two previously transfusion-dependent males showed spontaneous hematologic improvement in adolescence. Functional assays showed that the recurrent p.R595W variant destabilizes the KAT14 protein, while p.R566Ter produces a truncated, likely nonfunctional protein. Kat14 hypomorph mice exhibited embryonic lethality with incomplete penetrance, postnatal anemia and growth delay, although anemia resolved over time. Transcriptomic and RT-qPCR analyses revealed downregulation of key erythroid regulators (GATA1, GATA2, KLF1), consistent with impaired erythroid transcriptional control.

Conclusion(s): These findings identify KAT14 as a novel cause of DBA and a critical regulator of erythroid differentiation. Disruption of the ATAC complex emerges as a new mechanism in congenital red cell aplasia, expanding the genetic and epigenetic landscape of DBA beyond ribosomal protein dysfunction.

Characterization of KAT14 candidate variants identified in DBA patients.
(A) Schematic representation of the human KAT14 gene (NM_001392073.1) highlighting the locations of three variants identified in patients: a frameshift (p.Ser67LeufsTer11), a nonsense (p.R566Ter), and a missense (p.R595W) variant. Prosite scan of the KAT14 protein sequence reveals a Gcn5-related N-acetyltransferases (GNAT)-type histone acetyltransferase (HAT) domain at the C-terminal region (amino acids 637 - 781). An interaction domain spanning residues 542-662 mediates binding to SRF and CRP2. Additional annotated features include a PWAPA cassette, comprising a PHD-like finger and a helix-winged-helix (WH) domain, potentially involved in chromatin targeting through recognition of histone modifications and DNA. A serine-rich region is located between amino acids 311-350. (B) Structural modeling of the p.R595W variant using the AlphaFold predicted structure (AF‑Q9H8E8‑F1‑v4) shows residue R596 (UniProt Q9H8E8 numbering) located on a surface-exposed helix at the periphery of the C-terminal lobe, just outside the GNAT domain. The substitution of arginine with tryptophan eliminates a positive charge and introduces a bulky aromatic side chain, predicted to disrupt local electrostatics and packing, potentially destabilizing the protein. Residues 573-782 are shown in blue, remainder in green. (C) Immunoblot analysis of KAT14 protein expression in HEK293 cells transfected with wild-type or variant constructs. As predicted, the p.Arg566Ter variant produced truncated proteins, while the p.Arg595Trp variant showed reduced steady-state protein levels, consistent with impaired stability. The p.Ser67LeufsTer11 variant identified in P:1 was not tested, as the patient was enrolled after completion of these cellular validation experiments.

Growth and hematological defects in Kat14tm1a/tm1a mice from IMPC phenotyping pipeline.
Comprehensive phenotyping data from the International Mouse Phenotyping Consortium (IMPC), generated through the Sanger Institute's Mouse Genetics Project (MGP), revealed that (A-B) Homozygous mutants show significantly decreased body length (A) and body weight (B) compared to wild-type controls. (C-D) Female homozygous mice exhibit hematological abnormalities including reduced hemoglobin concentration (C) and hematocrit (D). Data represent means ± SEM. Statistical significance was determined using unpaired t-tests. ** p < 0.01, *** p < 0.001.

Transient anemia and reduced erythroid transcription factor expression in Kat14tm1a/tm1a mice.
(A-D) Kat14tm1a/tm1a mice exhibited reduced hemoglobin (Hb; A, B) and hematocrit (Hct; C, D) at ~3 weeks of age, with normalization by 5 weeks. Female mice (A, C): n = 3 at 3 weeks of age, n = 6 at 5 weeks; and male mice (B, D): n = 4 at 3-4 weeks of age. (E) RT-qPCR analysis of erythroid-specific transcription factors Gata1, Gata2, and Klf1 using total RNA extracted from bone marrow of Kat14tm1a/tm1a mice at ~3 weeks of age. Gene expression was normalized to housekeeping controls and analyzed using the ΔΔCt method. Data represent mean ± SEM. Statistical comparisons were performed using unpaired t-tests or ANOVA as appropriate. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.