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1.  Mutation Analysis of 18 Nephronophthisis-associated Ciliopathy Disease Genes using a DNA Pooling and Next-Generation Sequencing Strategy 
Journal of medical genetics  2010;48(2):105-116.
Background
Nephronophthisis-associated ciliopathies (NPHP-AC) comprise a group of autosomal recessive cystic kidney diseases that includes nephronophthisis (NPHP), Senior-Loken syndrome (SLS), Joubert syndrome (JBTS), and Meckel-Gruber syndrome (MKS). To date, causative mutations in NPHP-AC have been described for 18 different genes, rendering mutation analysis tedious and expensive. To overcome the broad genetic locus heterogeneity we devised a strategy of DNA pooling with consecutive massively parallel resequencing (MPR).
Methods
In 120 patients with severe NPHP-AC phenotypes we prepared 5 pools of genomic DNA with 24 patients each which were used as templates in order to PCR-amplify all 376 exons of 18 NPHP-AC genes (NPHP1, INVS, NPHP3, NPHP4, IQCB1, CEP290, GLIS2, RPGRIP1L, NEK8, TMEM67, INPP5E, TMEM216, AHI1, ARL13B, CC2D2A, TTC21B, MKS1, and XPNPEP3). PCR products were then subjected to MPR on a Illumina Genome-Analyzer and mutations were subsequently assigned to their respective mutation carrier via CEL I endonuclease-based heteroduplex screening and confirmed by Sanger sequencing.
Results
For proof of principle we used DNA from patients with known mutations and demonstrated the detection of 22 out of 24 different alleles (92% sensitivity). MPR led to the molecular diagnosis in 30/120 patients (25%) and we identified 54 pathogenic mutations (27 novel) in 7 different NPHP-AC genes. Additionally, in 24 patients we only found single heterozygous variants of unknown significance.
Conclusions
The combined approach of DNA pooling followed by MPR strongly facilitates mutation analysis in broadly heterogeneous single-gene disorders. The lack of mutations in 75% of patients in our cohort indicates further extensive heterogeneity in NPHP-AC.
doi:10.1136/jmg.2010.082552
PMCID: PMC3913043  PMID: 21068128
Next-generation sequencing; Ciliopathy; Nephronophthisis
2.  ANKS6 is a central component of a nephronophthisis module linking NEK8 to INVS and NPHP3 
Nature genetics  2013;45(8):951-956.
Nephronophthisis (NPH) is an autosomal recessive cystic kidney disease that leads to renal failure in childhood or adolescence. Most NPHP gene products form molecular networks. We have identified ANKS6 as a new NPHP family member that connects NEK8 (NPHP9) to INVERSIN (INVS, NPHP2) and NPHP3 to form a distinct NPHP module. ANKS6 localizes to the proximal cilium and knockdown experiments in zebrafish and Xenopus confirmed a role in renal development. Genetic screening identified six families with ANKS6 mutations and NPH, including severe cardiovascular abnormalities, liver fibrosis and situs inversus. The oxygen sensor HIF1AN (FIH) hydroxylates ANKS6 and INVS, while knockdown of Hif1an in Xenopus resembled the loss of other NPHP proteins. HIF1AN altered the composition of the ANKS6/INVS/NPHP3 module. Network analyses, uncovering additional putative NPHP-associated genes, placed ANKS6 at the center of the NPHP module, explaining the overlapping disease manifestation caused by mutations of either ANKS6, NEK8, INVS or NPHP3.
doi:10.1038/ng.2681
PMCID: PMC3786259  PMID: 23793029
3.  Angiotensin‐converting enzyme inhibitor fetopathy: long‐term outcome 
Fetal exposure to angiotensin‐converting enzyme inhibitors (ACEIs) is associated with increased neonatal morbidity and mortality. Long‐term follow‐up of three patients with fetal ACEI exposure revealed impaired renal function in two, severe hypertension and proteinuria in one and isolated polycythaemia in all three. Careful long‐term follow‐up of children with ACEI fetopathy is recommended.
doi:10.1136/adc.2006.101717
PMCID: PMC2675369  PMID: 17284475
angiotensin‐converting enzyme inhibitor; fetopathy; neonatal renal failure; long‐term outcome; polycythaemia
4.  Nineteen novel NPHS1 mutations in a worldwide cohort of patients with congenital nephrotic syndrome (CNS) 
Nephrology Dialysis Transplantation  2010;25(9):2970-2976.
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 treatment.
doi:10.1093/ndt/gfq088
PMCID: PMC2948833  PMID: 20172850
mutation analysis; nephrotic syndrome; NPHS1
5.  TTC21B contributes both causal and modifying alleles across the ciliopathy spectrum 
Nature genetics  2011;43(3):189-196.
Ciliary dysfunction leads to a broad range of overlapping phenotypes, termed collectively as ciliopathies. This grouping is underscored by genetic overlap, where causal genes can also contribute modifying alleles to clinically distinct disorders. Here we show that mutations in TTC21B/IFT139, encoding a retrograde intraflagellar transport (IFT) protein, cause both isolated nephronophthisis (NPHP) and syndromic Jeune Asphyxiating Thoracic Dystrophy (JATD). Moreover, although systematic medical resequencing of a large, clinically diverse ciliopathy cohort and matched controls showed a similar frequency of rare changes, in vivo and in vitro evaluations unmasked a significant enrichment of pathogenic alleles in cases, suggesting that TTC21B contributes pathogenic alleles to ∼5% of ciliopathy patients. Our data illustrate how genetic lesions can be both causally associated with diverse ciliopathies, as well as interact in trans with other disease-causing genes, and highlight how saturated resequencing followed by functional analysis of all variants informs the genetic architecture of disorders.
doi:10.1038/ng.756
PMCID: PMC3071301  PMID: 21258341
6.  Metabolic and orthopedic management of X-linked vitamin D-resistant hypophosphatemic rickets 
Purpose
Therapy of vitamin D-resistant hypophosphatemic rickets (VDXLR) consists of oral phosphate and vitamin D supplements. Bone deformities, pain, and small stature can occur even in children with good compliance, requiring surgical correction and bone lengthening. However, only few surgical reports are available.
Methods
Twelve patients (three males) with VDXLR were followed at our institution. Median age at diagnosis was 3 9/12 years (range, birth to 11 10/12) with a follow-up period of 7 8/12 years (1 9/12–30) and age at last follow-up of 13 6/12 years (2–30). Eight patients underwent surgical correction, three of them in combination with bone lengthening. The corrections were performed at the end of growth in three patients. Clinical endpoints were height, leg axis, and pain.
Results
Single bilateral surgical correction was performed in six patients; one patient each had three and five corrections. Bone lengthening was performed in three patients. At last follow-up, the height of seven operated patients was within normal range. In addition, leg axis was normalized in six patients with mild genua vara in two. Only one patient complained of intermittent pain. Bone healing was excellent; surgical complications were rare. There was no radiological evidence of degenerative arthropathy.
Conclusions
Medical treatment remains the main pillar of therapy in children with VDXLR. In case of bone deformity, surgery can safely be performed, independent of age or bone maturation. All patients were satisfied with the results of axial corrective surgery and bone lengthening, and in the majority only one corrective intervention was needed.
doi:10.1007/s11832-008-0118-9
PMCID: PMC2656824  PMID: 19308556
Rickets; Correction; Axis; Lengthening; Growing children

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