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1.  Identification of two novel CAKUT-causing genes by massively parallel exon resequencing of candidate genes in patients with unilateral renal agenesis 
Kidney international  2011;81(2):10.1038/ki.2011.315.
Congenital abnormalities of the kidney and urinary tract (CAKUT) constitute the most frequent cause of chronic kidney disease in children, accounting for ~50% of all cases. Although many forms of CAKUT are likely caused by single-gene defects, only few causative genes have been identified. To identify new causative genes many candidate genes need to be analyzed due to the broad genetic locus heterogeneity of CAKUT. We therefore applied our newly developed approach of DNA pooling with consecutive massively parallel exon resequencing to overcome this problem. We pooled DNA of 20 individuals and amplified by PCR all 313 exons of 30 CAKUT candidate genes. PCR products were then subjected to massively parallel exon resequencing. Mutation carriers were identified using Sanger sequencing. We repeated the experiment to cover 40 patients in total (29 with unilateral renal agenesis and 11 with other CAKUT phenotypes). We detected 5 heterozygous missense mutations in 2 candidate genes that were not previously implicated in non-syndromic CAKUT in humans, 4 mutations in the FRAS1 gene and 1 in FREM2. All mutations were absent from 96 healthy control individuals and had a PolyPhen score of >1.4 (“possibly damaging”). Recessive truncating mutations in FRAS1 and FREM2 were known to cause Fraser syndrome in humans and mice, whereas a phenotype in heterozygous carriers has not been described. We hereby identify heterozygous missense mutations in FRAS1 and FREM2 as a new cause of non-syndromic CAKUT in human.
doi:10.1038/ki.2011.315
PMCID: PMC3836012  PMID: 21900877
2.  Arrayed primer extension technology simplifies mutation detection in Bardet–Biedl and Alström syndrome 
Bardet–Biedl syndrome (BBS; OMIM no. 209 900) and Alström syndrome (ALMS; OMIM no. 203 800) are rare, multisystem genetic disorders showing both a highly variable phenotype and considerable phenotypic overlap; they are included in the emerging group of diseases called ciliopathies. The genetic heterogeneity of BBS with 14 causal genes described to date, serves to further complicate mutational analysis. The development of the BBS–ALMS array which detects known mutations in these genes has allowed us to detect at least one mutation in 40.5% of BBS families and in 26.7% of ALMS families validating this as an efficient and cost-effective first pass screening modality. Furthermore, using this method, we found two BBS families segregating three BBS alleles further supporting oligogenicity or modifier roles for additional mutations. We did not observe more than two mutations in any ALMS family.
doi:10.1038/ejhg.2010.207
PMCID: PMC3060323  PMID: 21157496
Bardet–Biedl syndrome; BBS; Alström syndrome; ALMS1; arrayed primer extension; mutation analysis
3.  Mapping of a new locus for congenital anomalies of the kidney and urinary tract on chromosome 8q24 
Nephrology Dialysis Transplantation  2009;25(5):1496-1501.
Background. Congenital anomalies of the kidney and urinary tract (CAKUT) account for the majority of end-stage renal disease in children (50%). Previous studies have mapped autosomal dominant loci for CAKUT. We here report a genome-wide search for linkage in a large pedigree of Somalian descent containing eight affected individuals with a non-syndromic form of CAKUT.
Methods. Clinical data and blood samples were obtained from a Somalian family with eight individuals with CAKUT including high-grade vesicoureteral reflux and unilateral renal agenesis. Total genome search for linkage was performed using a 50K SNP Affymetric DNA microarray. As neither parent is affected, the results of the SNP array were analysed under recessive models of inheritance, with and without the assumption of consanguinity.
Results. Using the non-consanguineous recessive model, a new gene locus (CAKUT1) for CAKUT was mapped to chromosome 8q24 with a significant maximum parametric Logarithm of the ODDs (LOD) score (LODmax) of 4.2. Recombinations were observed in two patients defining a critical genetic interval of 2.5 Mb physical distance flanked by markers SNP_A-1740062 and SNP_A-1653225.
Conclusion. We have thus identified a new non-syndromic recessive gene locus for CAKUT (CAKUT1) on chromosome 8q24. The identification of the disease-causing gene will provide further insights into the pathogenesis of urinary tract malformations and mechanisms of renal development.
doi:10.1093/ndt/gfp650
PMCID: PMC2910330  PMID: 20007758
congenital anomalies of the kidney and urinary tract (CAKUT); kidney development; total genome search for linkage; ureteropelvic junction obstruction; vesicoureteral reflux
4.  Thirteen novel NPHS1 mutations in a large cohort of children with congenital nephrotic syndrome 
Nephrology Dialysis Transplantation  2008;23(11):3527-3533.
Background. Congenital nephrotic syndrome (CNS) is de- fined as nephrotic syndrome that manifests at birth or within the first 3 months of life. Most patients develop end-stage renal disease (ESRD) within 2 to 3 years of life. CNS of the Finnish-type (CNF) features a rather specific renal histology and is caused by recessive mutations in the NPHS1 gene encoding nephrin, a major structural protein of the glomerular slit-diaphragm. So far, more than 80 different mutations of NPHS1 causing CNF have been published.
Methods. Here, we performed mutation analysis of NPHS1 by exon sequencing in a worldwide cohort of 32 children with CNS from 29 different families.
Results. Sixteen of the 29 families (55%) were found to have two disease-causing alleles in NPHS1. Two additional patients had a single heterozygous mutation in NPHS1. Thirteen of a total of 20 different mutations detected were novel (65%). These were five missense mutations, one nonsense mutation, three deletions, one insertion and three splice-site mutations.
Conclusion. Our data expand the spectrum of known NPHS1 mutations by >15% in a worldwide cohort. Surprisingly, two patients with disease-causing mutations showed a relatively mild phenotype, as one patient had a partial remission with steroid treatment and one patient had normal renal function 1 year after the onset of disease. The increased number of known mutations will facilitate future studies into genotype/phenotype correlations.
doi:10.1093/ndt/gfn271
PMCID: PMC2720813  PMID: 18503012
CNS; novel mutations; NPHS1
6.  Individuals with mutations in XPNPEP3, which encodes a mitochondrial protein, develop a nephronophthisis-like nephropathy  
The autosomal recessive kidney disease nephronophthisis (NPHP) constitutes the most frequent genetic cause of terminal renal failure in the first 3 decades of life. Ten causative genes (NPHP1–NPHP9 and NPHP11), whose products localize to the primary cilia-centrosome complex, support the unifying concept that cystic kidney diseases are “ciliopathies”. Using genome-wide homozygosity mapping, we report here what we believe to be a new locus (NPHP-like 1 [NPHPL1]) for an NPHP-like nephropathy. In 2 families with an NPHP-like phenotype, we detected homozygous frameshift and splice-site mutations, respectively, in the X-prolyl aminopeptidase 3 (XPNPEP3) gene. In contrast to all known NPHP proteins, XPNPEP3 localizes to mitochondria of renal cells. However, in vivo analyses also revealed a likely cilia-related function; suppression of zebrafish xpnpep3 phenocopied the developmental phenotypes of ciliopathy morphants, and this effect was rescued by human XPNPEP3 that was devoid of a mitochondrial localization signal. Consistent with a role for XPNPEP3 in ciliary function, several ciliary cystogenic proteins were found to be XPNPEP3 substrates, for which resistance to N-terminal proline cleavage resulted in attenuated protein function in vivo in zebrafish. Our data highlight an emerging link between mitochondria and ciliary dysfunction, and suggest that further understanding the enzymatic activity and substrates of XPNPEP3 will illuminate novel cystogenic pathways.
doi:10.1172/JCI40076
PMCID: PMC2827951  PMID: 20179356
7.  A Systematic Approach to Mapping Recessive Disease Genes in Individuals from Outbred Populations 
PLoS Genetics  2009;5(1):e1000353.
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.
Author Summary
Many childhood diseases are caused by single-gene mutations of recessive genes, in which a child has inherited one mutated gene copy from each parent causing disease in the child, but not in the parents who are healthy heterozygous carriers. As the two mutations represent the disease cause, gene mapping helped understand disease mechanisms. “Homozygosity mapping” has been particularly useful. It assumes that the parents are related and that a disease-causing mutation together with a chromosomal segment of identical markers (i.e., homozygous markers) is transmitted to the affected child through the paternal and the maternal line from an ancestor common to both parents. Homozygosity mapping seeks out those homozygous regions to map the disease-causing gene. Homozygosity mapping requires families, in which the parents are knowingly related, and have multiple affected children. To overcome these limitations, we applied homozygosity mapping to single affected individuals from outbred populations. In 72 individuals with known homozygous mutations in 13 different recessive disease genes, we performed homozygosity mapping. In 93% we detected the causative gene in a segment of homozygosity. We demonstrate that disease-causing homozygous mutations can be detected in single cases from outbred populations. This will strongly accelerate the discovery of novel recessive disease genes.
doi:10.1371/journal.pgen.1000353
PMCID: PMC2621355  PMID: 19165332

Results 1-7 (7)