A gene for familial juvenile nephronophthisis (recessive medullary cystic kidney disease) maps to chromosome 2p.

Familial juvenile nephronophthisis (NPH) is a chronic autosomal recessive kidney disease responsible for 15% of end stage renal failure in children. NPH is frequently (16% of cases) associated with Leber amaurosis (termed Senior-Løken syndrome, SLS). Linkage analyses, performed in 22 multiplex NPH families (18 without and 4 with ocular abnormalities), have localized the gene to a region between D2S48 and D2S51 on chromosome 2p. This was confirmed using adjacent microsatellite markers, one of which (AFM220ze3 at the D2S160 locus) gave a lod score of 4.78 at theta = 0.05 in the 18 families with isolated NPH, whereas the same marker excluded linkage with SLS. These results demonstrate linkage of the purely renal form of NPH to chromosome 2p, and suggest that there may be genetic heterogeneity between NPH and SLS.

NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome.

Familial idiopathic nephrotic syndromes represent a heterogeneous group of kidney disorders, and include autosomal recessive steroid-resistant nephrotic syndrome, which is characterized by early childhood onset of proteinuria, rapid progression to end-stage renal disease and focal segmental glomerulosclerosis. A causative gene for this disease, NPHS2, was mapped to 1q25-31 and we report here its identification by positional cloning. NPHS2 is almost exclusively expressed in the podocytes of fetal and mature kidney glomeruli, and encodes a new integral membrane protein, podocin, belonging to the stomatin protein family. We found ten different NPHS2 mutations, comprising nonsense, frameshift and missense mutations, to segregate with the disease, demonstrating a crucial role for podocin in the function of the glomerular filtration barrier.

Identification of mutations in the alpha 3(IV) and alpha 4(IV) collagen genes in autosomal recessive Alport syndrome.

Alport syndrome (AS) is an hereditary disease of basement membranes characterized by progressive renal failure and deafness. Changes in the glomerular basement membrane (GBM) in AS suggest that the type IV collagen matrix, the major structural component of GBM, is disrupted. We recently isolated the genes for two type IV collagens, alpha 3(IV) and alpha 4(IV), that are encoded head-to-head on human chromosome 2. These chains are abundant in normal GBM but are sometimes absent in AS. We screened for mutations in families in which consanguinity suggested autosomal recessive inheritance. Homozygous mutations were found in alpha 3(IV) in two families and in alpha 4(IV) in two others, demonstrating that these chains are important in the structural integrity of the GBM and that there is an autosomal form of AS in addition to the previously-defined X-linked form.

A novel gene encoding an integral membrane protein is mutated in nephropathic cystinosis.

Nephropathic cystinosis, an autosomal recessive disorder resulting from defective lysosomal transport of cystine, is the most common inherited cause of renal Fanconi syndrome. The cystinosis gene has been mapped to chromosome 17p13. We found that the locus D17S829 was homozygously deleted in 23 out of 70 patients, and identified a novel gene, CTNS, which mapped to the deletion interval. CTNS encodes an integral membrane protein, cystinosin, with features of a lysosomal membrane protein. Eleven different mutations, all predicted to cause loss of function of the protein, were found to segregate with the disorder.

Mutation of BSND causes Bartter syndrome with sensorineural deafness and kidney failure.

Antenatal Bartter syndrome (aBS) comprises a heterogeneous group of autosomal recessive salt-losing nephropathies. Identification of three genes that code for renal transporters and channels as responsible for aBS has resulted in new insights into renal salt handling, diuretic action and blood-pressure regulation. A gene locus of a fourth variant of aBS called BSND, which in contrast to the other forms is associated with sensorineural deafness (SND) and renal failure, has been mapped to chromosome 1p. We report here the identification by positional cloning, in a region not covered by the human genome sequencing projects, of a new gene, BSND, as the cause of BSND. We examined ten families with BSND and detected seven different mutations in BSND that probably result in loss of function. In accordance with the phenotype, BSND is expressed in the thin limb and the thick ascending limb of the loop of Henle in the kidney and in the dark cells of the inner ear. The gene encodes a hitherto unknown protein with two putative transmembrane alpha-helices and thus might function as a regulator for ion-transport proteins involved in aBS, or else as a new transporter or channel itself.

Dietary supplementation of cystinotic mice by lysine inhibits the megalin pathway and decreases kidney cystine content.

Megalin/LRP2 is a major receptor supporting apical endocytosis in kidney proximal tubular cells. We have previously reported that kidney-specific perinatal ablation of the megalin gene in cystinotic mice, a model of nephropathic cystinosis, essentially blocks renal cystine accumulation and partially preserves kidney tissue integrity. Here, we examined whether inhibition of the megalin pathway in adult cystinotic mice by dietary supplementation (5x-fold vs control regular diet) with the dibasic amino-acids (dAAs), lysine or arginine, both of which are used to treat patients with other rare metabolic disorders, could also decrease renal cystine accumulation and protect cystinotic kidneys. Using surface plasmon resonance, we first showed that both dAAs compete for protein ligand binding to immobilized megalin in a concentration-dependent manner, with identical inhibition curves by L- and D-stereoisomers. In cystinotic mice, 2-month diets with 5x-L-lysine and 5x-L-arginine were overall well tolerated, while 5x-D-lysine induced strong polyuria but no weight loss. All diets induced a marked increase of dAA urinary excretion, most prominent under 5x-D-lysine, without sign of kidney insufficiency. Renal cystine accumulation was slowed down approx. twofold by L-dAAs, and totally suppressed by D-lysine. We conclude that prolonged dietary manipulation of the megalin pathway in kidneys is feasible, tolerable and can be effective in vivo.

Disruption of pathways regulated by Integrator complex in Galloway-Mowat syndrome due to WDR73 mutations.

Several studies have reported WDR73 mutations to be causative of Galloway-Mowat syndrome, a rare disorder characterised by the association of neurological defects and renal-glomerular disease. In this study, we demonstrate interaction of WDR73 with the INTS9 and INTS11 components of Integrator, a large multiprotein complex with various roles in RNA metabolism and transcriptional control. We implicate WDR73 in two Integrator-regulated cellular pathways; namely, the processing of uridylate-rich small nuclear RNAs (UsnRNA), and mediating the transcriptional response to epidermal growth factor stimulation. We also show that WDR73 suppression leads to altered expression of genes encoding cell cycle regulatory proteins. Altogether, our results suggest that a range of cellular pathways are perturbed by WDR73 loss-of-function, and support the consensus that proper regulation of UsnRNA maturation, transcription initiation and cell cycle control are all critical in maintaining the health of post-mitotic cells such as glomerular podocytes and neurons, and preventing degenerative disease.

Hypomorphic mutations in meckelin (MKS3/TMEM67) cause nephronophthisis with liver fibrosis (NPHP11).

BACKGROUND: Nephronophthisis (NPHP), a rare recessive cystic kidney disease, is the most frequent genetic cause of chronic renal failure in children and young adults. Mutations in nine genes (NPHP1-9) have been identified. NPHP can be associated with retinal degeneration (Senior-Løken syndrome), brainstem and cerebellar anomalies (Joubert syndrome), or liver fibrosis. METHODS: To identify a causative gene for the subset of patients with associated liver fibrosis, the authors performed a genome wide linkage search in a consanguineous family with three affected patients using 50K SNP microarrays and homozygosity mapping. RESULTS: The authors obtained a significant maximum parametric LOD (logarithm of odds) score of Z(max) = 3.72 on chromosome 8q22 and identified a homozygous missense mutation in the gene MKS3/TMEM67. When examining a worldwide cohort of 62 independent patients with NPHP and associated liver fibrosis we identified altogether four novel mutations (p.W290L, p.C615R, p.G821S, and p.G821R) in five of them. Mutations of MKS3/TMEM67, found recently in Meckel-Gruber syndrome (MKS) type 3 and Joubert syndrome (JBTS) type 6, are predominantly truncating mutations. In contrast, the mutations detected here in patients with NPHP and associated liver fibrosis are exclusively missense mutations. This suggests that they may represent hypomorphic alleles, leading to a milder phenotype compared with the more severe MKS or JBTS phenotype. Additionally, mutation analysis for MKS3/TMEM67 in 120 patients with JBTS yielded seven different (four novel) mutations in five patients, four of whom also presented with congenital liver fibrosis. CONCLUSIONS: Hypomorphic MKS3/TMEM67 mutations cause NPHP with liver fibrosis (NPHP11). This is the first report of MKS3 mutations in patients with no vermian agenesis and without neurological signs. Thus NPHP, JBTS, and MKS represent allelic disorders.

Deletion of the paired alpha 5(IV) and alpha 6(IV) collagen genes in inherited smooth muscle tumors.

The gene encoding alpha 6(IV) collagen, COL4A6, was identified on the human X chromosome in a head-to-head arrangement and within 452 base pairs of the alpha 5(IV) collagen gene, COL4A5. In earlier studies, intragenic deletions of COL4A5 were detected in a subset of patients with Alport syndrome (AS), a hereditary defect of basement membranes. In some families, AS cosegregates with diffuse leiomyomatosis (DL), a benign smooth muscle tumor diathesis. Here it is shown that patients with AS-DL harbor deletions that disrupt both COL4A5 and COL4A6. Thus, type IV collagen may regulate smooth muscle differentiation and morphogenesis.

A missense mutation in podocin leads to early and severe renal disease in mice.

Mutations in the NPHS2 gene, encoding podocin, are responsible for familial autosomal recessive and sporadic cases of steroid-resistant nephrotic syndrome. We have successfully generated a mouse model in which the common p.R138Q mutation found in nephrotic patients is expressed in the kidney. Homozygous mice express the mutant protein, which is mislocated to the cytoplasm, along with a portion of the nephrin pool. These mice die within the first month of life, but their survival depends on the genetic background. Albuminuria manifests early and leads to progressive renal insufficiency, characterized histologically by diffuse mesangiolysis and mesangial sclerosis, endothelial lesions along with podocyte abnormalities such as widespread foot process effacement. Gene expression profiling revealed marked differences between these and the podocin-null mice, including significant perturbations of podocyte-expressed genes such as Cd2ap, Vegfa and the transcription factors Lmx1b and Zhx2. Upregulation of Serpine1 and Tgfb1 implicates these as potential mediators of disease progression in these mice. This mouse model of nephrotic syndrome may serve as a valuable tool in studies of in vivo intracellular protein trafficking of podocyte proteins, as well as testing therapeutic modalities aimed at correcting the targeting of mutant proteins.

A novel renal carbonic anhydrase type III plays a role in proximal tubule dysfunction.

Dysfunction of the proximal tubule (PT) is associated with variable degrees of solute wasting and low-molecular-weight proteinuria. We measured metabolic consequences and adaptation mechanisms in a model of inherited PT disorders using PT cells of ClC-5-deficient (Clcn5Y/-) mice, a well-established model of Dent's disease. Compared to cells taken from control mice, those from the mutant mice had increased expression of markers of proliferation (Ki67, proliferative cell nuclear antigen (PCNA), and cyclin E) and oxidative scavengers (superoxide dismutase I and thioredoxin). Transcriptome and protein analyses showed fourfold induction of type III carbonic anhydrase in a kidney-specific manner in the knockout mice located in scattered PT cells. Kidney-specific carbonic anhydrase type III (CAIII) upregulation was confirmed in other mice lacking the multiligand receptor megalin and in a patient with Dent's disease due to an inactivating CLCN5 mutation. The type III enzyme was specifically detected in the urine of mice lacking ClC-5 or megalin, patients with Dent's disease, and in PT cell lines exposed to oxidative stress. Our study shows that lack of PT ClC-5 in mice and men is associated with CAIII induction, increased cell proliferation, and oxidative stress.

Bilateral macular detachment caused by bilateral optic nerve malformation in a papillorenal syndrome due to a new PAX2 mutation.

PURPOSE: Papillorenal syndrome is an autosomal dominant entity due to PAX2 gene mutation, involving optic nerve and renal malformations. METHODS: The authors report a 19-year-old man with bilateral macular detachment associated with optic nerve pit in one eye and morning glory syndrome in the other eye. The patient underwent three-port pars plana vitrectomy, endolaser photocoagulation, and C3F8 gas tamponade in his best eye. A medical history of vesicoureteric reflux and kidney hypoplasia led to genetic analysis. RESULTS: Molecular genetic PAX2 analysis revealed a novel nondescribed mutation in exon 3. One year postoperatively, ophthalmologic outcomes were satisfactory with complete flattening of the retina and improvement of the best-corrected visual acuity to 11/10. CONCLUSIONS: PAX2 is involved in the optic vesicles, genital tracts, kidney, and central nervous system embryogenic development. The association of optic nerve and renal malformations should lead to the suspicion of papillorenal syndrome with PAX2 mutation.

Deletion of the donor splice site of intron 4 in the glucokinase gene causes maturity-onset diabetes of the young.

Missense and nonsense mutations in the glucokinase gene have recently been shown to result in maturity-onset diabetes of the young (MODY), a subtype of non-insulin-dependent diabetes mellitus with early age of onset. Glucokinase catalyzes the formation of glucose-6-phosphate and is involved in the regulation of insulin secretion and integration of hepatic intermediary metabolism. Nucleotide sequence analysis of exon 4 and its flanking intronic regions of the glucokinase gene, in four hyperglycemic individuals of a MODY family, revealed a deletion of 15 base pairs, which removed the t of the gt in the donor splice site of intron 4, and the following 14 base pairs. This deletion resulted in two aberrant transcripts, which were analyzed by reverse transcription of RNA from lymphoblastoid cells obtained from a diabetic patient. In one of the abnormal transcripts, exon 5 is missing, while in the other, the activation of a cryptic splice site leads to the removal of the last eight codons of exon 4. This intronic deletion in a donor splice site seems to cause a more severe form of glucose intolerance, compared with point mutations described in glucokinase. This might be due to a more pronounced effect on insulin secretion.

Deletions in the COL4A5 collagen gene in X-linked Alport syndrome. Characterization of the pathological transcripts in nonrenal cells and correlation with disease expression.

The type IV collagen alpha 5 chain (COL4A5) gene of 88 unrelated male patients with X-linked Alport syndrome was tested for major gene rearrangements by Southern blot analysis, using COL4A5 cDNA probes. 14 different deletions were detected, providing a 16% deletion rate in the COL4A5 gene in the patient population. The deletions are dispersed all over the gene with different sizes, ranging from 1 kb to the complete absence of the gene (> 250 kb) in one patient. In four patients with intragenic deletions, absence of the alpha 3 (IV) chain in the glomerular basement membrane was demonstrated by immunohistochemical studies. This finding supports the hypothesis that abnormalities in the alpha 5 (IV) chain may prevent normal incorporation of the alpha 3 (IV) chain into the glomerular basement membrane. Direct sequencing of cDNA amplified from lymphoblast mRNA of four patients with internal gene deletions, using appropriate combinations of primers amplifying across the predicted boundaries of the deletions, allowed us to determine the effect of the genomic rearrangements on the transcripts and, by inference, on the alpha 5 (IV) chain. Regardless of the extent of deletion and of the putative protein product, the 14 deletions occur in patients with juvenile-type Alport syndrome.

Isolation of kidney complementary DNAs down-expressed in Wilms' tumor by a subtractive hybridization approach.

We applied a subtractive hybridization approach to isolate genes differentially expressed between mature kidney and Wilms' tumor. We constructed a complementary DNA library from a total mature kidney complementary DNA subtracted by an excess of mRNA from a Wilms' tumor, WAGR4, with a germline deletion of 11p13 and a somatic loss of alleles at 11p15. Six clones presenting a differential pattern of expression, positive with mRNA from the mature kidney and negative with mRNA from the Wilms' tumor WAGR4, were characterized. Among these clones were two as yet unknown expressed sequences (D11S877E and D15S109E) and four sequences from known genes: renal dipeptidase (DPEP1), alpha B-crystallin (CRYA2), uromodulin (UMOD), and plasma glutathione peroxidase (GPX2). The different patterns of expression of these genes in 11 Wilms' tumors, whether or not they are hereditary, reflect the well-documented pathogenetic heterogeneity for Wilms' tumors. We propose that these clones could be helpful for an improved histological characterization of Wilms' tumors.

Familial juvenile nephronophthisis.

Familial juvenile nephronophthisis (NPH) is an autosomal recessive interstitial nephritis leading to terminal renal failure around puberty. Associations with extrarenal symptoms have been reported, mainly with Leber amaurosis (termed Senior-Løken syndrome). By means of linkage analysis a gene NPH1 for the purely renal form of NPH has been localized to chromosome 2. Genetic heterogeneity has been shown between NPH and Senior-Løken syndrome and also within the group of isolated NPH cases. Further characterization of the NPH1 region led to the isolation of large homozygous deletions in approximately 70% of patients with NPH. The detection of these deletions by PCR represents a simple noninvasive method for precise diagnosis in the majority of patients suspected of having NPH.

Molecular basis of steroid-resistant nephrotic syndrome.

The identification of the underlying gene defect in some cases of steroid resistant nephrotic syndrome (SRNS) has recently led to a critical breakthrough in the understanding of the pathogenesis of nephrotic syndromes. The more severe form of hereditary nephrotic syndromes is the congenital nephrotic syndrome of the Finnish type (CNF). The causative gene, NPHS1, encodes a novel protein, nephrin which is a transmembrane protein belonging to the immunoglobulin superfamily specifically expressed in the podocyte at the slit diaphragm. Using a positional cloning approach, our group identified a gene, NPHS2, involved in a specific entity of familial SRNS characterized by early onset, complete steroid-resistance, rapid progression to ESRD and no recurrence after renal transplantation. NPHS2 encodes a novel membrane protein named podocin localized at the cytoplasmic part of the slit diaphragm. Familial autosomal dominant cases of primary FSGS have been described in adulthood. Two corresponding genes have been mapped to date, one to 19q13 and the second to 11q21-22. The former has been identified as ACTN4, the gene encoding the actin-binding protein, a-actinin 4. Other genes involved in the slit-diaphragm or the nephrotic syndrome are CD2-associated protein (CD2AP), FAT1, WT1, LMX1B, SMARCAL1. Altogether, these data demonstrate the pivotal role of the podocyte in the development and the maintenance of the glomerular filtration barrier and the crucial role of the genetic factors in the development of SRNS.

[Immunohistochemistry contribution in Alport syndrome diagnosis]. / Apport de l'immunohistochimie dans le diagnostic du syndrome d'Alport.

UNLABELLED: Alport syndrome (AS) is an hereditary disease characterised by the association of progressive hematuria nephritis. The diagnosis is based on clinical genetic and ultrastructural findings. Nowadays, immunohistochemical technique is of great interest. It enables us to analyze the distribution of the different chains of the type IV collagen in renal basement membrane (RBM) and epidermal basement membrane (EBM) which appeared to be abnormal in 70% of cases. METHODS: We report a prospective study of five families affected with AS. Six patients were investigated by immunohistochemical studies of kidney (3 cases) and skin (6 cases) frozen specimens. Monoclonal antibodies recognizing the collagenous domain of alpha1 (MAB1), alpha3 (MAB3) and alpha5 (MAB5) chains of type IV collagen were used. Two methods were performed: direct immunofluorescence and immunohistochemical (ultravision) analysis. RESULTS: The different chains distribution of type IV collagen in the EBM and RBM was normal in four cases (4 men), abnormal in two patients (1 man and woman). Based on the clinical, genetical and immunohistochemical findings we established three transmission modes: autosomal recessive in two families, dominant X linked in two other familiales, and autosomal dominant in one family. CONCLUSION: Immunohistochemical studies is a simple technique of an easy interpretation accomplished on kidney frozen specimen, or even on a simple cutaneous biopsy. It could be very useful for the diagnosis and enables us in addition to determine the mode of transmission of AS.

A mouse model suggests two mechanisms for thyroid alterations in infantile cystinosis: decreased thyroglobulin synthesis due to endoplasmic reticulum stress/unfolded protein response and impaired lysosomal processing.

Thyroid hormones are released from thyroglobulin (Tg) in lysosomes, which are impaired in infantile/nephropathic cystinosis. Cystinosis is a lysosomal cystine storage disease due to defective cystine exporter, cystinosin. Cystinotic children develop subclinical and then overt hypothyroidism. Why hypothyroidism is the most frequent and earliest endocrine complication of cystinosis is unknown. We here defined early alterations in Ctns(-/-) mice thyroid and identified subcellular and molecular mechanisms. At 9 months, T4 and T3 plasma levels were normal and TSH was moderately increased (∼4-fold). By histology, hyperplasia and hypertrophy of most follicles preceded colloid exhaustion. Increased immunolabeling for thyrocyte proliferation and apoptotic shedding indicated accelerated cell turnover. Electron microscopy revealed endoplasmic reticulum (ER) dilation, apical lamellipodia indicating macropinocytic colloid uptake, and lysosomal cystine crystals. Tg accumulation in dilated ER contrasted with mRNA down-regulation. Increased expression of ER chaperones, glucose-regulated protein of 78 kDa and protein disulfide isomerase, associated with alternative X-box binding protein-1 splicing, revealed unfolded protein response (UPR) activation by ER stress. Decreased Tg mRNA and ER stress suggested reduced Tg synthesis. Coordinated increase of UPR markers, activating transcription factor-4 and C/EBP homologous protein, linked ER stress to apoptosis. Hormonogenic cathepsins were not altered, but lysosome-associated membrane protein-1 immunolabeling disclosed enlarged vesicles containing iodo-Tg and impaired lysosomal fusion. Isopycnic fractionation showed iodo-Tg accumulation in denser lysosomes, suggesting defective lysosomal processing and hormone release. In conclusion, Ctns(-/-) mice showed the following alterations: 1) compensated primary hypothyroidism and accelerated thyrocyte turnover; 2) impaired Tg production linked to ER stress/UPR response; and 3) altered endolysosomal trafficking and iodo-Tg processing. The Ctns(-/-) thyroid is useful to study disease progression and evaluate novel therapies.