Nineteen novel NPHS1 mutations in a worldwide cohort of patients with congenital nephrotic syndrome (CNS)

Background. Recessive mutations in the NPHS1 gene encoding nephrin account for ∼40% of infants with congenital nephrotic syndrome (CNS). CNS is defined as steroid-resistant nephrotic syndrome (SRNS) within the first 90 days of life. Currently, more than 119 different mutations of NPHS1 have been published affecting most exons. Methods. We here performed mutational analysis of NPHS1 in a worldwide cohort of 67 children from 62 different families with CNS. Results. We found bi-allelic mutations in 36 of the 62 families (58%) confirming in a worldwide cohort that about one-half of CNS is caused by NPHS1 mutations. In 26 families, mutations were homozygous, and in 10, they were compound heterozygous. In an additional nine patients from eight families, only one heterozygous mutation was detected. We detected 37 different mutations. Nineteen of the 37 were novel mutations (∼51.4%), including 11 missense mutations, 4 splice-site mutations, 3 nonsense mutations and 1 small deletion. In an additional patient with later manifestation, we discovered two further novel mutations, including the first one affecting a glycosylation site of nephrin. Conclusions. Our data hereby expand the spectrum of known mutations by 17.6%. Surprisingly, out of the two siblings with the homozygous novel mutation L587R in NPHS1, only one developed nephrotic syndrome before the age of 90 days, while the other one did not manifest until the age of 2 years. Both siblings also unexpectedly experienced an episode of partial remission upon steroid treatmen

A systematic approach to mapping recessive disease genes in individuals from outbred populations

The identification of recessive disease-causing genes by homozygosity mapping is often restricted by lack of suitable consanguineous families. To overcome these limitations, we apply homozygosity mapping to single affected individuals from outbred populations. In 72 individuals of 54 kindred ascertained worldwide with known homozygous mutations in 13 different recessive disease genes, we performed total genome homozygosity mapping using 250,000 SNP arrays. Likelihood ratio Z-scores (ZLR) were plotted across the genome to detect ZLR peaks that reflect segments of homozygosity by descent, which may harbor the mutated gene. In 93% of cases, the causative gene was positioned within a consistent ZLR peak of homozygosity. The number of peaks reflected the degree of inbreeding. We demonstrate that disease-causing homozygous mutations can be detected in single cases from outbred populations within a single ZLR peak of homozygosity as short as 2 Mb, containing an average of only 16 candidate genes. As many specialty clinics have access to cohorts of individuals from outbred populations, and as our approach will result in smaller genetic candidate regions, the new strategy of homozygosity mapping in single outbred individuals will strongly accelerate the discovery of novel recessive disease genes

The Jumonji-C oxygenase JMJD7 catalyzes (3S)-lysyl hydroxylation of TRAFAC GTPases

Biochemical, structural and cellular studies reveal Jumonji-C (JmjC) domain-containing 7 (JMJD7) to be a 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes (3S)-lysyl hydroxylation. Crystallographic analyses reveal JMJD7 to be more closely related to the JmjC hydroxylases than to the JmjC demethylases. Biophysical and mutation studies show that JMJD7 has a unique dimerization mode, with interactions between monomers involving both N- and C-terminal regions and disulfide bond formation. A proteomic approach identifies two related members of the translation factor (TRAFAC) family of GTPases, developmentally regulated GTP-binding proteins 1 and 2 (DRG1/2), as activity-dependent JMJD7 interactors. Mass spectrometric analyses demonstrate that JMJD7 catalyzes Fe(ii)- and 2OG-dependent hydroxylation of a highly conserved lysine residue in DRG1/2; amino-acid analyses reveal that JMJD7 catalyzes (3S)-lysyl hydroxylation. The functional assignment of JMJD7 will enable future studies to define the role of DRG hydroxylation in cell growth and disease.Fil: Markolovic, Suzana. University of Oxford; Reino UnidoFil: Zhuang, Qinqin. University Of Birmingham; Reino UnidoFil: Wilkins, Sarah E.. University of Oxford; Reino UnidoFil: Eaton, Charlotte D.. University Of Birmingham; Reino UnidoFil: Abboud, Martine I.. University of Oxford; Reino UnidoFil: Katz, Maximiliano Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: McNeil, Helen E.. University Of Birmingham; Reino UnidoFil: Leśniak, Robert K.. University of Oxford; Reino UnidoFil: Hall, Charlotte. University Of Birmingham; Reino UnidoFil: Struwe, Weston B.. University of Oxford; Reino UnidoFil: Konietzny, Rebecca. University of Oxford; Reino UnidoFil: Davis, Simon. University of Oxford; Reino UnidoFil: Yang, Ming. The Francis Crick Institute; Reino Unido. University of Oxford; Reino UnidoFil: Ge, Wei. University of Oxford; Reino UnidoFil: Benesch, Justin L. P.. University of Oxford; Reino UnidoFil: Kessler, Benedikt M.. University of Oxford; Reino UnidoFil: Ratcliffe, Peter J.. University of Oxford; Reino Unido. The Francis Crick Institute; Reino UnidoFil: Cockman, Matthew E.. The Francis Crick Institute; Reino Unido. University of Oxford; Reino UnidoFil: Fischer, Roman. University of Oxford; Reino UnidoFil: Wappner, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Chowdhury, Rasheduzzaman. University of Stanford; Estados Unidos. University of Oxford; Reino UnidoFil: Coleman, Mathew L.. University Of Birmingham; Reino UnidoFil: Schofield, Christopher J.. University of Oxford; Reino Unid

A novel TRPC6 mutation that causes childhood FSGS.

TRPC6, encoding a member of the transient receptor potential (TRP) superfamily of ion channels, is a calcium-permeable cation channel, which mediates capacitive calcium entry into the cell. Until today, seven different mutations in TRPC6 have been identified as a cause of autosomal-dominant focal segmental glomerulosclerosis (FSGS) in adults.Here we report a novel TRPC6 mutation that leads to early onset FSGS. We identified one family in whom disease segregated with a novel TRPC6 mutation (M132T), that also affected pediatric individuals as early as nine years of age. Twenty-one pedigrees compatible with an autosomal-dominant mode of inheritance and biopsy-proven FSGS were selected from a worldwide cohort of 550 families with steroid resistant nephrotic syndrome (SRNS). Whole cell current recordings of the mutant TRPC6 channel, compared to the wild-type channel, showed a 3 to 5-fold increase in the average out- and inward TRPC6 current amplitude. The mean inward calcium current of M132T was 10-fold larger than that of wild-type TRPC6. Interestingly, M132T mutants also lacked time-dependent inactivation. Generation of a novel double mutant M132T/N143S did not further augment TRPC6 channel activity.In summary, our data shows that TRPC6 mediated FSGS can also be found in children. The large increase in channel currents and impaired channel inactivation caused by the M132T mutant leads to an aggressive phenotype that underlines the importance of calcium dose channeled through TRPC6

Confirmation and mapping of the <i>Sd</i> mutation.

<p>A) Southern analysis and PCR showing the presence of a large DNA insertion at the <i>Sd</i> locus. B) Multiplex PCR from inbred mouse lines showing the mutation is only present in <i>Sd</i> mice, and is not a polymorphism. In this three primer PCR reaction the <i>Sd</i> amplimer is 406 bp, while the WT amplimer is 510 bp. C) Mapping of the <i>Sd</i> mutation which we identified as an ETn (early transposon) in relation to nearby gene/ESTs, figure not to scale.</p

Next-Generation Sequencing Identifies the Danforth's Short Tail Mouse Mutation as a Retrotransposon Insertion Affecting <em>Ptf1a</em> Expression

<div><p>The semidominant Danforth's short tail (<i>Sd</i>) mutation arose spontaneously in the 1920s. The homozygous <i>Sd</i> phenotype includes severe malformations of the axial skeleton with an absent tail, kidney agenesis, anal atresia, and persistent cloaca. The <i>Sd</i> mutant phenotype mirrors features seen in human caudal malformation syndromes including urorectal septum malformation, caudal regression, VACTERL association, and persistent cloaca. The <i>Sd</i> mutation was previously mapped to a 0.9 cM region on mouse chromosome 2qA3. We performed Sanger sequencing of exons and intron/exon boundaries mapping to the <i>Sd</i> critical region and did not identify any mutations. We then performed DNA enrichment/capture followed by next-generation sequencing (NGS) of the critical genomic region. Standard bioinformatic analysis of paired-end sequence data did not reveal any causative mutations. Interrogation of reads that had been discarded because only a single end mapped correctly to the <i>Sd</i> locus identified an early transposon (ETn) retroviral insertion at the <i>Sd</i> locus, located 12.5 kb upstream of the <i>Ptf1a</i> gene. We show that <i>Ptf1a</i> expression is significantly upregulated in <i>Sd</i> mutant embryos at E9.5. The identification of the <i>Sd</i> mutation will lead to improved understanding of the developmental pathways that are misregulated in human caudal malformation syndromes.</p> </div

Physical map of the <i>Sd</i> critical region as viewed on the UCSC genome browser (

<p><a href="http://www.genome.ucsc.edu" target="_blank">http://www.genome.ucsc.edu</a><b>).</b> The <i>Sd</i> region on chromosome 2 is defined by the flanking genetic markers D2Mit362 proximally and D2Mit364 distally (markers are highlighted in red) and represented by bases 18,686,882–20,915,358 on the July 2007 genome build (mm9). The <i>Sd</i> critical region contains 11 known genes. The mutation location is denoted by the red X.</p

Gene expression at E9.5 in <i>Sd</i> mutant embryos.

<p>Quantitative real time PCR plot of gene expression in RNA isolated from <i>Sd/Sd</i> (black bars), <i>Sd/</i>+ (hatched bars), and +/+ (grey bars) embryos of RefSeq genes mapping to the <i>Sd</i> interval. Y-axis shows fold change normalized to 1 for wildtype (+/+) expression. * denotes significance in expression levels where p<0.05 (For <i>Ptf1a</i>; <i>Sd</i>/+ versus +/+ p = 0.029, <i>Sd/Sd</i> versus +/+ p = 0.044).</p

Genes and ESTs mapping to the <i>Sd</i> critical region.

<p>Genes and ESTs mapping to the <i>Sd</i> critical region.</p