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1.  Mutations in 12 known dominant disease-causing genes clarify many congenital anomalies of the kidney and urinary tract 
Kidney international  2014;85(6):1429-1433.
Congenital anomalies of the kidney and urinary tract (CAKUT) account for approximately half of children with chronic kidney disease. CAKUT can be caused by monogenic mutations, however, data are lacking on their frequency. Genetic diagnosis has been hampered by genetic heterogeneity and lack of genotype-phenotype correlation. To determine the percentage of cases with CAKUT that can be explained by mutations in known CAKUT genes, we analyzed the coding exons of the 17 known dominant CAKUT-causing genes in a cohort of 749 individuals from 650 families with CAKUT. The most common phenotypes in this CAKUT cohort were 288 with vesicoureteral reflux, 120 with renal hypodysplasia and 90 with unilateral renal agenesis. We identified 37 different heterozygous mutations (33 novel) in 12 of the 17 known genes in 47 patients from 41 of the 650 families (6.3%). These mutations include (number of families): BMP7 (1), CDC5L (1), CHD1L (5), EYA1 (3), GATA3 (2), HNF1B (6), PAX2 (5), RET (3), ROBO2 (4), SALL1 (9), SIX2 (1), and SIX5 (1). Furthermore, several mutations previously reported to be disease-causing are most likely benign variants. Thus, in a large cohort over 6% of families with isolated CAKUT are caused by a mutation in 12 of 17 dominant CAKUT genes. Our report represents one of the most in-depth diagnostic studies of monogenic causes of isolated CAKUT in children.
doi:10.1038/ki.2013.508
PMCID: PMC4040148  PMID: 24429398
renal agenesis; renal development; genetic renal disease
2.  Clinical utility of chitotriosidase enzyme activity in nephropathic cystinosis 
Background
Nephropathic cystinosis is an inherited autosomal recessive lysosomal storage disorder characterized by the pathological accumulation and crystallization of cystine inside different cell types. WBC cystine determination forms the basis for the diagnosis and therapeutic monitoring with the cystine depleting drug (cysteamine). The chitotriosidase enzyme is a human chitinase, produced by activated macrophages. Its elevation is documented in several lysosomal storage disorders. Although, about 6% of Caucasians have enzyme deficiency due to homozygosity of 24-bp duplication mutation in the chitotriosidase gene, it is currently established as a screening marker and therapeutic monitor for Gaucher’s disease.
Methods
Plasma chitotriosidase activity was measured in 45 cystinotic patients, and compared with 87 healthy controls and 54 renal disease patients with different degrees of renal failure (CKD1-5). Chitotriosidase levels were also correlated with WBC cystine in 32 treated patients. Furthermore, we incubated control human macrophages in-vitro with different concentrations of cystine crystals and monitored the response of tumor necrosis factor-alpha (TNF-α) and chitotriosidase activity. We also compared plasma chitotriosidase activity in cystinotic knocked-out (n = 10) versus wild-type mice (n = 10).
Results
Plasma chitotriosidase activity in cystinotic patients (0–3880, median 163 nmol/ml/h) was significantly elevated compared to healthy controls (0–90, median 18 nmol/ml/h) and to CKD patients (0–321, median 52 nmol/ml/h), P < 0.001 for both groups. Controls with decreased renal function had mild to moderate chitotriosidase elevations; however, their levels were significantly lower than in cystinotic patients with comparable degree of renal insufficiency. Chitotriosidase activity positively correlated with WBC cystine content for patients on cysteamine therapy (r = 0.8), P < 0.001. In culture, human control macrophages engulfed cystine crystals and released TNF-α into culture supernatant in a crystal concentration dependent manner. Chitotriosidase activity was also significantly increased in macrophage supernatant and cell-lysate. Furthermore, chitotriosidase activity was significantly higher in cystinotic knocked-out than in the wild-type mice, P = 0.003.
Conclusions
This study indicates that cystine crystals are potent activators of human macrophages and that chitotriosidase activity is a useful marker for this activation and a promising clinical biomarker and therapeutic monitor for nephropathic cystinosis.
doi:10.1186/s13023-014-0155-z
PMCID: PMC4269071  PMID: 25407738
Lysosomal storage disorders; Nephropathic cystinosis; Cystine crystals; Macrophage activation; Chitotriosidase enzyme; Clinical screening; Cysteamine; Therapeutic monitoring
4.  Clinical Characterization and NPHP1 Mutations in Nephronophthisis and Associated Ciliopathies: A Single Center Experience 
Nephronophthisis (NPHP) is a recessive disorder of the kidney that is the leading genetic cause of end-stage renal failure in children. Egypt is a country with a high rate of consanguineous marriages; yet, only a few studies have investigated the clinical and molecular characteristics of NPHP and related ciliopathies in the Egyptian population. We studied 20 children, from 17 independent families, fulfilling the clinical and the ultrasonographic criteria of NPHP. Analysis for a homozygous deletion of the NPHP1 gene was performed by polymerase chain reaction on the genomic DNA of all patients. Patients were best categorized as 75% juvenile NPHP, 5% infantile NPHP, and 20% Joubert syndrome-related disorders (JSRD). The mean age at diagnosis was 87.5 + 45.4 months, which was significantly late as compared with the age at onset of symptoms, 43.8 ± 29.7 months (P <0.01). Homozygous NPHP1 deletions were detected in six patients from five of 17 (29.4%) studied families. Our study demonstrates the clinical phenotype of NPHP and related disorders in Egyptian children. Also, we report that homozygous NPHP1 deletions account for 29.4% of NPHP in the studied families in this cohort, thereby confir-ming the diagnosis of type-1 NPHP. Moreover, our findings confirm that NPHP1 deletions can indeed be responsible for JSRD.
doi:10.4103/1319-2442.100968
PMCID: PMC4154542  PMID: 22982934
5.  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
6.  Mutational Spectrum of the CTNS Gene in Egyptian Patients with Nephropathic Cystinosis 
JIMD Reports  2014;14:87-97.
Background: Nephropathic cystinosis is a rare autosomal recessive disorder caused by mutations in the CTNS gene, encoding for cystinosin, a carrier protein transporting cystine out of lysosomes. Its deficiency leads to cystine accumulation and cell damage in multiple organs, especially in the kidney. In this study, we aimed to provide the first report describing the mutational spectrum of Egyptian patients with nephropathic cystinosis and their genotype–phenotype correlation.
Methods: Fifteen Egyptian patients from 13 unrelated families with infantile nephropathic cystinosis were evaluated clinically, biochemically, and genetically. Screening for the common 57-kb deletion was performed by standard multiplex PCR, followed by direct sequencing of the ten coding exons, exon-intron interfaces, and promoter region.
Results: None of the 15 Egyptian patients had the 57-kb deletion. Twenty-seven mutant alleles and 12 pathogenic mutations were detected including six novel mutations: two frameshift (c.260_261delTT; p.F87SfsX36, c.1032delCinsTG; p.F345CfsX19), one nonsense (c.734G>A; p.W245fsX), two missense (c.1084G>A; pG362R, c.560A>G; p.K187R), and one intronic splicing mutation (IVS3+5g>t). A novel promoter region mutation (1-593-41C>T) seemed to be detected but was excluded as a pathogenic mutation by quantitative real-time PCR analysis.
Conclusions: This study could be the basis for future genetic counseling and prenatal diagnosis of patients with nephropathic cystinosis in Egyptian and surrounding populations. The screening for the 57-kb deletion is not recommended anymore outside its geographical distribution, especially in the region of the Middle East. A common Middle Eastern mutation (c.681G>A; E227E) was pointed out and discussed.
doi:10.1007/8904_2013_288
PMCID: PMC4213330  PMID: 24464559
7.  ARHGDIA mutations cause nephrotic syndrome via defective RHO GTPase signaling  
The Journal of Clinical Investigation  2013;123(8):3243-3253.
Nephrotic syndrome (NS) is divided into steroid-sensitive (SSNS) and -resistant (SRNS) variants. SRNS causes end-stage kidney disease, which cannot be cured. While the disease mechanisms of NS are not well understood, genetic mapping studies suggest a multitude of unknown single-gene causes. We combined homozygosity mapping with whole-exome resequencing and identified an ARHGDIA mutation that causes SRNS. We demonstrated that ARHGDIA is in a complex with RHO GTPases and is prominently expressed in podocytes of rat glomeruli. ARHGDIA mutations (R120X and G173V) from individuals with SRNS abrogated interaction with RHO GTPases and increased active GTP-bound RAC1 and CDC42, but not RHOA, indicating that RAC1 and CDC42 are more relevant to the pathogenesis of this SRNS variant than RHOA. Moreover, the mutations enhanced migration of cultured human podocytes; however, enhanced migration was reversed by treatment with RAC1 inhibitors. The nephrotic phenotype was recapitulated in arhgdia-deficient zebrafish. RAC1 inhibitors were partially effective in ameliorating arhgdia-associated defects. These findings identify a single-gene cause of NS and reveal that RHO GTPase signaling is a pathogenic mediator of SRNS.
doi:10.1172/JCI69134
PMCID: PMC3726174  PMID: 23867502
8.  Tumor necrosis factor-α and interleukin-6 in cirrhotic patients with spontaneous bacterial peritonitis 
AIM: To evaluate the role of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in cirrhotic patients who have hepatic and renal impairment with spontaneous bacterial peritonitis (SBP).
METHODS: We prospectively studied 120 cirrhotic patients with SBP and 80 cirrhotic patients with sterile ascitic fluid. They included 144 males and 56 females with ages ranging between 34 and 62 years. The diagnosis of cirrhosis was established by clinical and laboratory criteria that did not require histological confirmation. The severity of underlying liver disease was evaluated using Pugh’s modification of Child’s criteria (Child-Pugh scores). Ascitic fluid was sent to the laboratory for cell count, culture, sensitivity testing, and measurement of chemical elements (i.e., albumin, glucose). Specimens were inoculated into aerobic and anaerobic blood culture bottles. Serum and ascitic fluid were also collected in sterile tubes at study entry (before the initiation of antibiotic treatment) and 48 h later. Assays for TNF-α and IL-6 in the serum and ascitic fluid were performed with an immunoenzymometric assay using manufacture’s instructions.
RESULTS: Cytokine levels in serum and ascitic fluid were significantly higher in the patients with SBP. (plasma TNF-α: 135.35 ng/mL ± 11.21 ng/mL vs 92.86 ng/mL ± 17.56 ng/mL, P < 0.001; plasma IL-6: 32.30 pg/mL ± 7.07 pg/mL vs 12.11 pg/mL ± 6.53 pg/mL, P < 0.001; ascitic fluid TNF-α: 647.54 ± 107.11 ng/mL vs 238.43 ng/mL ± 65.42 ng/mL, P < 0.001); ascitic fluid IL-6: 132.84 ng/mL ± 34.13 vs 40.41 ± 12.85 pg/mL, P < 0.001). About 48 (40%) cirrhotic patients with SBP developed renal and hepatic impairment and showed significantly higher plasma and ascitic fluid cytokine levels at diagnosis of infection. [(plasma TNF-α: 176.58 ± 17.84 vs 135.35 ± 11.21 ng/mL) (P < 0.001) and (IL-6: 57.83 ± 7.85 vs 32.30 ± 7.07 pg/mL) (P < 0.001); ascitic fluid TNF-α: 958.39 ± 135.72 vs 647.54 ± 107.11 ng/mL, (P < 0.001), ascitic fluid IL-6: 654.74 ± 97.43 vs 132.84 ± 34.13 pg/mL, (P < 0.001)]. Twenty nine patients (60.4%) with SBP and renal impairment died whereas, only four patients (5.55%) with SBP but without renal impairment died from gastrointestinal hemorrhage (P < 0.0005).
CONCLUSION: It appears that TNF-α production may enhance liver cell injury and lead to renal impairment. This correlated well with the poor prognosis and significantly increased mortality associated with SBP in cirrhotic patients.
doi:10.4291/wjgp.v3.i5.92
PMCID: PMC3602440  PMID: 23515243
Tumor necrosis factor; Interleukin-6; Spontaneous bacterial peritonitis; Cirrhosis; Tumor necrosis factor
9.  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
10.  COQ6 mutations in human patients produce nephrotic syndrome with sensorineural deafness  
The Journal of Clinical Investigation  2011;121(5):2013-2024.
Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of end-stage renal failure. Identification of single-gene causes of SRNS has generated some insights into its pathogenesis; however, additional genes and disease mechanisms remain obscure, and SRNS continues to be treatment refractory. Here we have identified 6 different mutations in coenzyme Q10 biosynthesis monooxygenase 6 (COQ6) in 13 individuals from 7 families by homozygosity mapping. Each mutation was linked to early-onset SRNS with sensorineural deafness. The deleterious effects of these human COQ6 mutations were validated by their lack of complementation in coq6-deficient yeast. Furthermore, knockdown of Coq6 in podocyte cell lines and coq6 in zebrafish embryos caused apoptosis that was partially reversed by coenzyme Q10 treatment. In rats, COQ6 was located within cell processes and the Golgi apparatus of renal glomerular podocytes and in stria vascularis cells of the inner ear, consistent with an oto-renal disease phenotype. These data suggest that coenzyme Q10–related forms of SRNS and hearing loss can be molecularly identified and potentially treated.
doi:10.1172/JCI45693
PMCID: PMC3083770  PMID: 21540551

Results 1-10 (10)