Identification of single-gene causes of steroid-resistant nephrotic syndrome (SRNS) has furthered the understanding of the pathogenesis of this disease. Here, using a combination of homozygosity ...mapping and whole human exome resequencing, we identified mutations in the aarF domain containing kinase 4 (ADCK4) gene in 15 individuals with SRNS from 8 unrelated families. ADCK4 was highly similar to ADCK3, which has been shown to participate in coenzyme Q10 (CoQ10) biosynthesis. Mutations in ADCK4 resulted in reduced CoQ10 levels and reduced mitochondrial respiratory enzyme activity in cells isolated from individuals with SRNS and transformed lymphoblasts. Knockdown of adck4 in zebrafish and Drosophila recapitulated nephrotic syndrome-associated phenotypes. Furthermore, ADCK4 was expressed in glomerular podocytes and partially localized to podocyte mitochondria and foot processes in rat kidneys and cultured human podocytes. In human podocytes, ADCK4 interacted with members of the CoQ10 biosynthesis pathway, including COQ6, which has been linked with SRNS and COQ7. Knockdown of ADCK4 in podocytes resulted in decreased migration, which was reversed by CoQ10 addition. Interestingly, a patient with SRNS with a homozygous ADCK4 frameshift mutation had partial remission following CoQ10 treatment. These data indicate that individuals with SRNS with mutations in ADCK4 or other genes that participate in CoQ10 biosynthesis may be treatable with CoQ10.
Nephrotic syndrome (NS), the association of gross proteinuria, hypoalbuminaemia, edema, and hyperlipidemia, can be clinically divided into steroid-sensitive (SSNS) and steroid-resistant (SRNS) forms. ...SRNS regularly progresses to end-stage renal failure. By homozygosity mapping and whole exome sequencing, we here identify recessive mutations in Crumbs homolog 2 (CRB2) in four different families affected by SRNS. Previously, we established a requirement for zebrafish crb2b, a conserved regulator of epithelial polarity, in podocyte morphogenesis. By characterization of a loss-of-function mutation in zebrafish crb2b, we now show that zebrafish crb2b is required for podocyte foot process arborization, slit diaphragm formation, and proper nephrin trafficking. Furthermore, by complementation experiments in zebrafish, we demonstrate that CRB2 mutations result in loss of function and therefore constitute causative mutations leading to NS in humans. These results implicate defects in podocyte apico-basal polarity in the pathogenesis of NS.
Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease (CKD). Here we show that recessive mutations in FAT1 cause a distinct renal disease entity in four families with a ...combination of SRNS, tubular ectasia, haematuria and facultative neurological involvement. Loss of FAT1 results in decreased cell adhesion and migration in fibroblasts and podocytes and the decreased migration is partially reversed by a RAC1/CDC42 activator. Podocyte-specific deletion of Fat1 in mice induces abnormal glomerular filtration barrier development, leading to podocyte foot process effacement. Knockdown of Fat1 in renal tubular cells reduces migration, decreases active RAC1 and CDC42, and induces defects in lumen formation. Knockdown of fat1 in zebrafish causes pronephric cysts, which is partially rescued by RAC1/CDC42 activators, confirming a role of the two small GTPases in the pathogenesis. These findings provide new insights into the pathogenesis of SRNS and tubulopathy, linking FAT1 and RAC1/CDC42 to podocyte and tubular cell function.
Renal agenesis and hypodysplasia (RHD) are major causes of pediatric chronic kidney disease and are highly genetically heterogeneous. We conducted whole-exome sequencing in 202 case subjects with RHD ...and identified diagnostic mutations in genes known to be associated with RHD in 7/202 case subjects. In an additional affected individual with RHD and a congenital heart defect, we found a homozygous loss-of-function (LOF) variant in SLIT3, recapitulating phenotypes reported with Slit3 inactivation in the mouse. To identify genes associated with RHD, we performed an exome-wide association study with 195 unresolved case subjects and 6,905 control subjects. The top signal resided in GREB1L, a gene implicated previously in Hoxb1 and Shha signaling in zebrafish. The significance of the association, which was p = 2.0 × 10−5 for novel LOF, increased to p = 4.1 × 10−6 for LOF and deleterious missense variants combined, and augmented further after accounting for segregation and de novo inheritance of rare variants (joint p = 2.3 × 10−7). Finally, CRISPR/Cas9 disruption or knockdown of greb1l in zebrafish caused specific pronephric defects, which were rescued by wild-type human GREB1L mRNA, but not mRNA containing alleles identified in case subjects. Together, our study provides insight into the genetic landscape of kidney malformations in humans, presents multiple candidates, and identifies SLIT3 and GREB1L as genes implicated in the pathogenesis of RHD.
In steroid-resistant nephrotic syndrome (SRNS), >21 single-gene causes are known. However, mutation analysis of all known SRNS genes is time and cost intensive. This report describes a new ...high-throughput method of mutation analysis using a PCR-based microfluidic technology that allows rapid simultaneous mutation analysis of 21 single-gene causes of SRNS in a large number of individuals.
This study screened individuals with SRNS; samples were submitted for mutation analysis from international sources between 1996 and 2012. For proof of principle, a pilot cohort of 48 individuals who harbored known mutations in known SRNS genes was evaluated. After improvements to the method, 48 individuals with an unknown cause of SRNS were then examined in a subsequent diagnostic study. The analysis included 16 recessive SRNS genes and 5 dominant SRNS genes. A 10-fold primer multiplexing was applied, allowing PCR-based amplification of 474 amplicons in 21 genes for 48 DNA samples simultaneously. Forty-eight individuals were indexed in a barcode PCR, and high-throughput sequencing was performed. All disease-causing variants were confirmed via Sanger sequencing.
The pilot study identified the genetic cause of disease in 42 of 48 (87.5%) of the affected individuals. The diagnostic study detected the genetic cause of disease in 16 of 48 (33%) of the affected individuals with a previously unknown cause of SRNS. Seven novel disease-causing mutations in PLCE1 (n=5), NPHS1 (n=1), and LAMB2 (n=1) were identified in <3 weeks. Use of this method could reduce costs to 1/29th of the cost of Sanger sequencing.
This highly parallel approach allows rapid (<3 weeks) mutation analysis of 21 genes known to cause SRNS at a greatly reduced cost (1/29th) compared with traditional mutation analysis techniques. It detects mutations in about 33% of childhood-onset SRNS cases.
Hypophosphatemic rickets (HR) is a heterogeneous genetic phosphate wasting disorder. The disease is most commonly caused by mutations in the PHEX gene located on the X-chromosome or by mutations in ...CLCN5, DMP1, ENPP1, FGF23, and SLC34A3. The aims of this study were to perform molecular diagnostics for four patients with HR of Indian origin (two independent families) and to describe their clinical features.
We performed whole exome sequencing (WES) for the affected mother of two boys who also displayed the typical features of HR, including bone malformations and phosphate wasting. B-lymphoblast cell lines were established by EBV transformation and subsequent RT-PCR to investigate an uncommon splice site variant found by WES. An in silico analysis was done to obtain accurate nucleotide frequency occurrences of consensus splice positions other than the canonical sites of all human exons. Additionally, we applied direct Sanger sequencing for all exons and exon/intron boundaries of the PHEX gene for an affected girl from an independent second Indian family.
WES revealed a novel PHEX splice acceptor mutation in intron 9 (c.1080-3C>A) in a family with 3 affected individuals with HR. The effect on splicing of this mutation was further investigated by RT-PCR using RNA obtained from a patient's EBV-transformed lymphoblast cell line. RT-PCR revealed an aberrant splice transcript skipping exons 10-14 which was not observed in control samples, confirming the diagnosis of X-linked dominant hypophosphatemia (XLH). The in silico analysis of all human splice sites adjacent to all 327,293 exons across 81,814 transcripts among 20,345 human genes revealed that cytosine is, with 64.3%, the most frequent nucleobase at the minus 3 splice acceptor position, followed by thymidine with 28.7%, adenine with 6.3%, and guanine with 0.8%. We generated frequency tables and pictograms for the extended donor and acceptor splice consensus regions by analyzing all human exons. Direct Sanger sequencing of all PHEX exons in a sporadic case with HR from the Indian subcontinent revealed an additional novel PHEX mutation (c.1211_1215delACAAAinsTTTACAT, p.Asp404Valfs*5, de novo) located in exon 11.
Mutation analyses revealed two novel mutations and helped to confirm the clinical diagnoses of XLH in two families from India. WES helped to analyze all genes implicated in the underlying disease complex. Mutations at splice positions other than the canonical key sites need further functional investigation to support the assertion of pathogenicity.
Rare single-gene disorders cause chronic disease. However, half of the 6000 recessive single gene causes of disease are still unknown. Because recessive disease genes can illuminate, at least in ...part, disease pathomechanism, their identification offers direct opportunities for improved clinical management and potentially treatment. Rare diseases comprise the majority of chronic kidney disease (CKD) in children but are notoriously difficult to diagnose. Whole-exome resequencing facilitates identification of recessive disease genes. However, its utility is impeded by the large number of genetic variants detected. We here overcome this limitation by combining homozygosity mapping with whole-exome resequencing in 10 sib pairs with a nephronophthisis-related ciliopathy, which represents the most frequent genetic cause of CKD in the first three decades of life. In 7 of 10 sibships with a histologic or ultrasonographic diagnosis of nephronophthisis-related ciliopathy, we detect the causative gene. In six sibships, we identify mutations of known nephronophthisis-related ciliopathy genes, while in two additional sibships we found mutations in the known CKD-causing genes SLC4A1 and AGXT as phenocopies of nephronophthisis-related ciliopathy. Thus, whole-exome resequencing establishes an efficient, noninvasive approach towards early detection and causation-based diagnosis of rare kidney diseases. This approach can be extended to other rare recessive disorders, thereby providing accurate diagnosis and facilitating the study of disease mechanisms.
A rare syndrome was first described in 1997 in a 17-year-old male patient presenting with Retinitis pigmentosa, HYpopituitarism, Nephronophthisis and Skeletal dysplasia (RHYNS). In the single ...reported familial case, two brothers were affected, arguing for X-linked or recessive mode of inheritance. Up to now, the underlying genetic basis of RHYNS syndrome remains unknown. Here we applied whole-exome sequencing in the originally described family with RHYNS to identify compound heterozygous variants in the ciliary gene TMEM67. Sanger sequencing confirmed a paternally inherited nonsense c.622A > T, p.(Arg208*) and a maternally inherited missense variant c.1289A > G, p.(Asp430Gly), which perturbs the correct splicing of exon 13. Overall, TMEM67 showed one of the widest clinical continuum observed in ciliopathies ranging from early lethality to adults with liver fibrosis. Our findings extend the spectrum of phenotypes/syndromes resulting from biallelic TMEM67 variants to now eight distinguishable clinical conditions including RHYNS syndrome.
In African American (AA) children, focal segmental glomerulosclerosis (FSGS) is the leading cause of nephrotic syndrome (NS). It has been shown that AA children suffer from FSGS and steroid-resistant ...nephrotic syndrome (SRNS) at a higher frequency and with a more severe renal outcome in comparison with Caucasian children. Previous mutation analysis of large cohorts revealed that a high percentage of childhood SRNS is monogenic and that mutations in podocin (
NPHS2
) and Wilms’ tumor gene 1 (
WT1
) account for approximately 30% of SRNS in children. To test whether AA children with SRNS have a similar or a higher mutation rate, we performed mutation analysis of
NPHS2
and
WT1
in a cohort of AA children with SRNS. Direct sequencing was carried out for all exons of
NPHS2
and for exons 8 and 9 of
WT1
. We ascertained 18 children of AA descent in whom renal biopsy findings showed FSGS in 13 patients (72%) and minimal-change disease in five patients (28%). In both
NPHS2
and
WT1
, no disease-causing mutations were detected. Our data strongly suggest that in AA children with SRNS, the frequency of
NPHS2
mutations is much lower than in large cohorts of pediatric SRNS patients in the general population. Knowledge of mutation rate of
NPHS2
in different populations of SRNS patients facilitates the physician in planning a suitable genetic screening strategy for patients.
The risk of developing Wilms tumor (WT) can be present or absent in patients with nephrotic syndrome (NS) caused by WT1 mutations. Here, the genotype/phenotype correlation regarding the outcome and ...risk for WT in 52 patients from 51 families with NS due to WT1 mutations is described.
This study followed 19 patients with mutations in intron 9 splice donor site (KTS mutations), 27 patients with missense mutations, 4 patients with nonsense mutations, 1 patient with a splice site mutation in intron 8, and 1 patient with a deletion.
Twenty-four different WT1 mutations were detected. Sixteen of the 19 patients with KTS mutations were females. These patients had isolated NS if karyotype was 46,XX and Frasier syndrome if karyotype was 46,XY. Patients with KTS mutations presented at a significantly older age and with a slower progression toward chronic kidney disease (CKD) stage 5, compared with missense mutations. Patients with nonsense mutations presented initially with WT. Six patients with missense mutations developed WT after the diagnosis of NS (interval-range from NS onset to WT of 0.1 to 1.4 years).
(1) KTS mutations cause isolated NS with absence of WT in 46,XX females. (2) KTS mutations cause Frasier syndrome with gonadoblastoma risk in 46,XY phenotypic females. (3) KTS mutations cause NS with a slower progression when compared with missense mutations. (4) Missense mutations can occur with and without WT. (5) WT1 analysis is important in young patients with NS for early detection and tumor prophylaxis.
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