PMC7506526_01
Family description.. a Pedigree. Both individuals II:1 and II:3 are affected by NPHP. Arrow indicates the proband. b Representative liver section of the proband showing periportal fibrosis (Sirius Red; x20 magnification). c All family members were subjected to genetic analysis. Zoomed in Integrative Genomics Viewer (IGV) screenshots showing the distribution of the two pathogenic mutations identified in NPHP3 by whole-genome sequencing (WGS) for each family member.
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Fig1
A total of 272 variants were reviewed, and only one heterozygous mutation affecting the NPHP3 gene (MIM:608002; RefSeq NM_153240.4) was categorized as "pathogenic" according to ACMG guidelines (Fig. 1). The c.520-1G>T variant was further validated using PCR and Sanger sequencing of the amplicons, confirming the diagnosis in all affected family members (II:1 and II:3) and the paternal origin of the mutation (Fig. 1).
PMC7506526_02
Family description.. a Pedigree. Both individuals II:1 and II:3 are affected by NPHP. Arrow indicates the proband. b Representative liver section of the proband showing periportal fibrosis (Sirius Red; x20 magnification). c All family members were subjected to genetic analysis. Zoomed in Integrative Genomics Viewer (IGV) screenshots showing the distribution of the two pathogenic mutations identified in NPHP3 by whole-genome sequencing (WGS) for each family member.
41525_2020_147_Fig1_HTML.jpg
PMC7506526_02_41525_2020_147_Fig1_HTML.jpg
Fig1
A total of 272 variants were reviewed, and only one heterozygous mutation affecting the NPHP3 gene (MIM:608002; RefSeq NM_153240.4) was categorized as "pathogenic" according to ACMG guidelines (Fig. 1). The c.520-1G>T variant was further validated using PCR and Sanger sequencing of the amplicons, confirming the diagnosis in all affected family members (II:1 and II:3) and the paternal origin of the mutation (Fig. 1).
Effects of the disease-causing genetic variants on NPHP3 function.. Both variants affect NPHP3 mRNA splicing and impact protein translation through distinct mechanisms. a-q Schematic representation of the effect of the splice acceptor site mutation c.520-1G>T and the c.671-996C>G deep intronic variant on NPHP3 mRNA splicing and its translation into protein. Wild-type sequences are indicated in panels (a-d) and (i-l), whereas sequences either bearing the c.520-1G>T mutation or the c.671-996C>G variant are shown in panels (e-h) and (m-q), respectively. a-d, i-l detail the normal splicing of NPHP3 exon 1 to 4. e-h show that the c.520-1G>T mutation activates a cryptic splice site within exon 3 of NPHP3, located 8 bp downstream from the authentic 3'-splice acceptor site and leading to a frameshift and premature stop codon, which terminates protein translation 6 codons downstream (p.Ile174Glyfs*6). Representative electropherograms of both normal and mutated cDNA sequences are shown in (c) and (k), respectively. m-q show that the c.671-996C>G mutation causes aberrant NPHP3 splicing with the insertion of a 163-nucleotide cryptic exon between exon 3 and 4 generating a frameshift and a premature stop codon at codon 240. o Agarose gel electrophoresis image showing aberrant NPHP3 splicing in affected family members harboring the c.671-996C>G variant. Samples were processed in parallel. Representative electropherograms of both normal and mutated cDNA sequences are shown in (k) and (p), respectively.
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Fig2
RT-PCR, using total RNAs from leukocytes obtained from affected and control family members. Results of this analysis indicated that the c.520-1G>T mutation activates a cryptic splice site within exon 3, located 8 bp downstream from the authentic 3'-splice acceptor site and leading to a frameshift and premature stop codon, which terminates protein translation 6 codons downstream (p.Ile174Glyfs*6) (Fig. 2a-h). This analysis revealed that the c.671-996C>G mutation was associated with the insertion of a 163-nucleotide cryptic exon within intron 3 (Genomic location: 3:132717955-132718117 (GRCh38)), located 48 bp downstream of the splice donor site and generating a frameshift as well as a premature stop codon at codon 240 (Fig. 2i-q).