PMC9086540_01
PMC9086540_02
Clinical and radiology manifestation in the current proband. (A) The patient exhibited abnormal bilirubin in plasma, while the total bilirubin remained high during his illness. At the beginning, DBIL elevated rapidly to a high level, while IDIL remained normal. However, after 3 months follow-up, DBIL dropped to normal level, and IDIL increased. (B) Hepatic ultrasound demonstrated a normal density of liver. (C) Gallbladder demonstrated a normal morphology. (D,E) The cerebral MRI identified hematoma near right cerebral ventricle.
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F1
IDIL dropped to approximately 50-80 mumol/L from the 3rd postnatal day (Figure 1A), but DBIL had increased to 193.4 mumol/L by the 15th postnatal day (Figure 1A). In tests for specific pathogens, quantitative polymerase chain reaction (qPCR) to detect pathogenic bacteria, viruses, and fungi, TORCH IgM antibody screening, group Hepatic ultrasound indicated normal liver density (Figure 1B) and normal gallbladder morphology (Figure 1C), thus liver fibrosis and biliary atresia were ruled out as causes of the elevated DBIL. Cerebral magnetic resonance imaging revealed hematoma signs in the right cerebral ventricle (T2 image in Figure 1D and T1 image in Figure 1E).
PMC9086540_03
The PKLR mutations in this family. (A) Family pedigree in the current proband. (B) Sanger sequencing validation of the current proband and his parents, revealing the maternal carrier of PKLR c.707T > C (p.L236R) and his paternal carrier of PKLR c.826_827insAGGAGCATGGG. The current proband exhibited PPHN and metabolic disorder in neonatal period and congenital non-spherocytic hemolytic anemia with a novel compound heterozygous mutation of PKLR. (C) The crystal structure of full length of AA sequence built by AlphaFold 2.0.
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F2
PKD, and no other relatives in his family exhibited any potential symptoms (Figure 2A). PPHN was the primary consideration in this patient, as well as substantial elevations in total bilirubin, DBIL, and IDIL, consistent with metabolic disorder. These data were confirmed by Sanger sequencing, and the patient's mother carried the heterozygous variant of c.707T > G, and his father carried the c.826_827insAGGAGCATGGGG frame insertion as a heterozygous variant (Figure 2B). The crystal structure of the full amino acid sequence is shown in Figure 2C.
The effects of PKLR c.707T > C and c.826_827insAGGAGCATGGG mutations on the molecular structure of the protein. (A) The reported cases of the identified mutations in database 1000G and ExAC. And the PROVEAN protein batch and MutationTaster scores revealed the two newly identified mutations would be protein damaging and diseasing causing variants. (B) Individual crystal structures of wild type, p.L236R and p.G275_V276insEEHG according to the 36eck.1.A model template. (C) Ramachandran plots of AA with wild type, p.L236R and p.G275_V276insEEHG. (D) The detailed space structure between wild type and mutant proteins. (E) The comparisons of free energy on crystal structure of wild type sequence and p.L236R and p.G275_V276insEEHG variants, respectively.
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F3
According to updated data in ExAC and 1000G neither of the mutations had been reported, indicating that they were novel variants (Figure 3A). PolyPhen_2, and MutationTaster indicated that both the c.707T > G (probability 0.999) and c.826_827insAGGAGCATGGGG (probability 0.998) mutations were disease causing (Figure 3A). PROVEAN protein batch indicated that the p.L236R protein was deleterious (Provean score -4.71) and damaging (SIFT prediction 0.00), as was the p.G275_V276insEEHG protein (deleterious Provean score -12.00, damaging SIFT prediction 0.00) (Figure 3A). The altered crystal structures of PLKR complexes are shown in Figure 3B. Generally there were no significant changes in crystal structure in the PKLR p.L236R complex, and an additional loop was identified in the PKLR p.G275_V276insEEHG complex (Figure 3B). Notably, however, differences in Ramachandran plots were observed in both PKLR p.L236R and PKLR p.G275_V276insEEHG compared to wild-type (Figure 3C). Detailed structures including changes in positions in p.L276R and p.G275_V276insEEHG are shown in Figure 3D. Structural comparisons were completed for both mutations, and are shown in Figure 3E.
PMC9086540_04
PMC9086540_05
PMC9086540_06