Journal: Frontiers in pediatrics

Article Title: A Sphingosine-1-Phosphate Lyase Mutation Associated With Congenital Nephrotic Syndrome and Multiple Endocrinopathy.

doi: 10.3389/fped.2020.00151

Figure Lengend Snippet: FIGURE 1 | Our patient harbors a homozygous mutation in SGPL1, c.511A>G, p.N171D. (A) Partial sequence chromatograms of genomic DNA from the parents who are heterozygote carriers and the homozygote patient, showing the base change from A to G in exon 7. (B) Pedigree of affected patient. Black-filled symbols indicate individual homozygous, half-filled indicate individuals heterozygous for the mutation. White-filled symbols indicate wild-type individuals. (C) Partial alignment of SGPL1 protein sequences, showing conservation of asparagine (N) at position 171 across several species, highlighted in red, numbering relative to human sequence. Sequence conservation is beneath the alignment, *total conservation, : partial conservation.

Article Snippet: Site directed mutagenesis of SGPL1 Human Tagged ORF Clone (Origene) and oligonucleotide primer design was performed using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent) and web based QuikChange program in keeping with the manufacturer’s guidelines.

Techniques: Mutagenesis, Sequencing

Journal: Frontiers in pediatrics

Article Title: A Sphingosine-1-Phosphate Lyase Mutation Associated With Congenital Nephrotic Syndrome and Multiple Endocrinopathy.

doi: 10.3389/fped.2020.00151

Figure Lengend Snippet: FIGURE 2 | A conformational change in SGPL1 occurs with the p.N171D mutation, resulting in reduced protein expression. (A) Protein modeling showed a minor conformational change in SGPL1 due to amino acid substitution at position 171, predicted to result in reduced protein stability. (B) An in vitro splicing assay showed effective splicing of both wild type and mutant SGPL1 exon 7 constructs providing no evidence of aberrant splicing. (C) Expression of p.N171D mutant in an SGPL1 KO cell line is reduced when compared to wild type suggesting increased susceptibility to degradation.

Article Snippet: Site directed mutagenesis of SGPL1 Human Tagged ORF Clone (Origene) and oligonucleotide primer design was performed using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent) and web based QuikChange program in keeping with the manufacturer’s guidelines.

Techniques: Mutagenesis, Expressing, In Vitro, Splicing Assay, Construct