Panel A: Pedigree chart showing two families with W/M, W/W, M/M, W/M1, W/M2, and M1/M2 genotypes. Panel B: Microscopy images showing blood-cell morphology for Patient 1 and a second cellular sample. Panel C: DNA sequencing chromatograms showing variants c.1331C greater than A and p.P444H in family members and wild-type sequence. Panel D: DNA sequencing chromatograms showing variant c.785G>A and p.R262Q compared with wild-type sequence. Panel E: DNA sequencing chromatograms showing c.2270_2272dupCCG and p.P757_E758insA variants compared with wild-type sequence. Panel F: Western blot showing HYOU1 and GAPDH protein bands for Patient 1 and four controls. Panel G: Bar graph showing HYOU1 signal intensity for Patient 1 and controls. Panel H: Bar graph showing LFQ signal intensity for Patient 1 and controls. Panel I: Bar graph showing relative quantification for Patient 1 and controls, with no significant difference. Panel J: Protein structure diagram showing HYOU1 domains and locations of P757, P444, and R262. Panel K: Molecular structure model showing the P444H variant and surrounding P444 residues. Panel L: Molecular structure model showing R262Q with R262, A370, and E371 and indicated distances. Panel M: Molecular structure model showing P757, E758, and K373 with indicated distances.
Genetics, cytology, transcription, translation, and structure of HYOU1 variants in two patients. (A) Pedigrees of patients. Roman numerals and Arabic numerals indicate generations and subjects, respectively. Double lines connecting the parents connote consanguinity. Squares, male subjects; circles, female subjects; filled (black) symbols represent patients; white symbols represent unaffected family members; arrows indicate the probands in each family; stars show family members subjected to whole-exome sequencing (W: WT allele, M: mutant allele). (B) The peripheral blood (upper panel) and bone marrow smears (lower panel) of patient 1 before G-CSF treatment. (C) Sanger traces of the HYOU1 c.1331C>A, p.Pro444His variant in family 1 confirm the homozygous status of the proband and heterozygous status of the parents for the mutation. The electropherograms denote single base pair substitutions of Pro444His in HYOU1. (D) Sanger traces of HYOU1 in patient 2 confirm the compound heterozygous status of the patient for the maternally inherited HYOU1 c.785G>A, p.Arg262Gln variant and (E) paternally inherited HYOU1 c.2270_2272dupCCG>A, p.Pro757_Glu758insAla variant. (F) Western blot analyses of HYOU1 protein expression in dermal fibroblasts of patient 1 and four controls. GAPDH was used as the internal control. (G) Signal intensity of HYOU1 protein relative to the expression of GAPDH (*P < 0.05). (H) Label-free quantification (LFQ) signal intensity of HYOU1 in the proteomics study of the dermal fibroblasts of patient 1 and controls; ****P < 0.0001. (I) Quantitative real-time PCR of dermal fibroblasts of patient 1 and controls, normalized to β-actin revealed no difference in HYOU1 mRNA expression (P = 0.57); ns, not significant. A t test was used for statistical comparison. (J) Full-length structural modelling of HYOU1 superimposing the 7A4U PDB model (green) and AlphaFold3-predicted structures (blue). Mutated amino acids are indicated in red. (K–M) Predicted structural effects of the variants. Wild-type residues are shown in red and mutant residues in gray. Distances between the residues are shown in Å. Source data are available for this figure: SourceData F1.