Hiroyasu Tsukaguchi, Akulapalli Sudhakar, Tu Cam Le, Trang Nguyen, Jun Yao, Joshua A. Schwimmer, Asher D. Schachter, Esteban Poch, Patricia F. Abreu, Gerald B. Appel, Aparecido B. Pereira, Raghu Kalluri, Martin R. Pollak
NPHS2 missense mutations in family FS-W. (a) DNA sequence chromatograms. Left: DNA sequence analysis of exon 5 amplified from an affected individual in family FS-W. A G→A transition predicts an Arg→Gln substitution. Right: DNA sequence analysis of exon 7 shows a C→T transition resulting in an Arg→Trp substitution. (b) Cosegregation of mutations with disease. Affected individuals are showed by filled circles (female) and squares (male). Agarose gel electrophoresis demonstrates the presence or absence of mutations within family FS-W. Top: R229Q mutations detected by ClaI digestion. The point mutation G755A results in a loss of ClaI site, and mutant R229Q alleles are shown by the top bands (545 bp), representing PCR products missing a ClaI site. Bottom: R291W mutations identified by PflMI digestion. A C941T mutation creates a PflMI restriction site and is visualized as two bands of 155 bp and 128 bp. Affected individuals are compound heterozygotes for R229Q and R291W. (c) Alignment of species orthologs of podocin. Amino acid sequences encoded by human NPHS2 (positions 221–240, GenBank NP_055440), rat NPHS2 (GenBank AF309631), mouse EST (GenBank AI552534), Drosophila melanogaster EST (GenBank AA141235), and Globodera rostochiensis EST (GenBank AW505792) were aligned by the PILEUP program (Wisconsin package version 10.0 [Unix]; Genetic Computer Group, Accelrys, San Diego, California, USA). Identical amino acid residues are indicated by shading. The arginine residue at position 229 is highlighted by an arrow.