case_images: PMC8107470_01
case_images: An intron variant and a novel missense mutation in RNASEH2B gene are identified. (A) Pedigree of the family. Squares and circles represent male and female family members, respectively. (B) The two variants c.65-13G>A and c.253C>G are confirmed in the proband and parents by Sanger sequencing. (C) cDNAs from the proband and his parents were resolved on a 3% agarose-gel, showing the different splice variants. (D, E) Electropherograms of the two splice variants identified in the proband. The highest peak presents an intron retention due to the c.65-13G>A mutation whereas the lowest one the c.253C>G variant.
case_images: fimmu-12-672952-g001.jpg
case_images: PMC8107470_01_fimmu-12-672952-g001.jpg
case_images: f1
case_images: MRI showed white matter hyperintensity involving periventricular and deep regions and slight cerebral atrophy ( Figure S1 ). Gene segregation was studied by trio Sanger sequencing, and both parents were confirmed to be heterozygous carriers of each mutation ( Figures 1A, B  ). The cDNA derived from patient's and mother's RNA, both presenting the c.65-13G>A variant, showed two different transcripts, whereas the father presented only one transcript ( Figure 1C ). The upper band in the agarose gel represents a longer transcript that resulted from the retention of 11 nucleotides belonging to intron 1 ( Figure 1D ). The lower band, as well as the only band identified in the father, represents the wild-type transcript, and both father and proband presented the c.253C>G variant in exon 4 ( Figure 1E ).
case_images: Characterization of the two RNASEH2B mutations. (A) The L85 residue of RNASEH2B is evolutionarily conserved crossing all examined species. The conserved Leucine residue is shown in the rectangular box. Protein sequences were obtained from the National Center for Biotechnology (NCBI). (B) Expression levels of RNASEH2B transcripts in a healthy control, parents, and proband *P < 0.05, **P < 0.01, ***P < 0.001. (C) Western blot analysis of RNase H2B, RNase H2C, and RNase H2A protein levels in a healthy control, parents, and proband. GAPDH has been used as loading control. (D) The contacts involving amino acid L85 with V17, L19, W73, and F87 on the native (left panel, black broken lines) and L85V mutant (right panel, red broken lines) of RNASEH2B. (E) The crystal structure of RNASE H2 complex with RNASEH2A, RNASEH2B, RNASEH2C highlighted in blue, red and gray, respectively. On the same figure the first 30 residues of RNASEH2B (of which 1-12 are missing in the crystallographic structure) are highlighted in yellow.
case_images: fimmu-12-672952-g002.jpg
case_images: PMC8107470_02_fimmu-12-672952-g002.jpg
case_images: f2
case_images: We observed a significant decrease of RNASEH2B expression in all the members of the family by RT-qPCR ( Figure 2B ). This result was also confirmed by Western blot, which highlighted a reduction of RNase H2B protein levels in the family with a deeper decrease in the father ( Figure 2C ), as already observed for the transcript. Lower RNase H2A protein levels have been observed in all the members of the family, whereas a strong depletion of RNase H2C was identified only in the affected child ( Figure 2C ). The analysis of native and homology modeled mutant structures showed that replacement of Leucine by the shorter Valine sidechain eliminates many of the intramolecular contacts with hydrophobic moieties of V17, L19, W73, and F87, besides the intermolecular contact with V144 in RNase H2C ( Figure 2D ).
case_images: PMC8107470_02
case_images: Characterization of the two RNASEH2B mutations. (A) The L85 residue of RNASEH2B is evolutionarily conserved crossing all examined species. The conserved Leucine residue is shown in the rectangular box. Protein sequences were obtained from the National Center for Biotechnology (NCBI). (B) Expression levels of RNASEH2B transcripts in a healthy control, parents, and proband *P < 0.05, **P < 0.01, ***P < 0.001. (C) Western blot analysis of RNase H2B, RNase H2C, and RNase H2A protein levels in a healthy control, parents, and proband. GAPDH has been used as loading control. (D) The contacts involving amino acid L85 with V17, L19, W73, and F87 on the native (left panel, black broken lines) and L85V mutant (right panel, red broken lines) of RNASEH2B. (E) The crystal structure of RNASE H2 complex with RNASEH2A, RNASEH2B, RNASEH2C highlighted in blue, red and gray, respectively. On the same figure the first 30 residues of RNASEH2B (of which 1-12 are missing in the crystallographic structure) are highlighted in yellow.
case_images: fimmu-12-672952-g002.jpg
case_images: PMC8107470_02_fimmu-12-672952-g002.jpg
case_images: f2
case_images: We observed a significant decrease of RNASEH2B expression in all the members of the family by RT-qPCR ( Figure 2B ). This result was also confirmed by Western blot, which highlighted a reduction of RNase H2B protein levels in the family with a deeper decrease in the father ( Figure 2C ), as already observed for the transcript. Lower RNase H2A protein levels have been observed in all the members of the family, whereas a strong depletion of RNase H2C was identified only in the affected child ( Figure 2C ). The analysis of native and homology modeled mutant structures showed that replacement of Leucine by the shorter Valine sidechain eliminates many of the intramolecular contacts with hydrophobic moieties of V17, L19, W73, and F87, besides the intermolecular contact with V144 in RNase H2C ( Figure 2D ).