Defective nephrin trafficking caused by missense mutations in the NPHS1 gene: insight into the mechanisms of congenital nephrotic syndrome.

Congenital nephrotic syndrome of the Finnish type (CNF or NPHS1) is an autosomal recessive kidney disorder resulting in severe proteinurea and renal dysfunction. Although the disease occurs predominantly in the Finnish population, many cases in other populations have also been reported. The disease gene (NPHS1) encodes nephrin, a podocyte transmembrane protein that is an essential component of the podocyte slit diaphragm, the renal ultrafilter. Since the discovery of the gene, many mutations have been reported in the NPHS1 gene in patients with diverse ethnic background. A surprisingly large number of these mutations are missense mutations resulting in single amino acid substitutions. In order to study the pathomechanism of these missense mutations, we have investigated the fate of 21 such mutations hitherto identified in NPHS1 patients. Immunostaining of stable transfected cells expressing the nephrin mutants demonstrated that most of the mutants showed only endoplasmic reticulum (ER) staining and no detectable cell surface localization. Immunoelectron microscopy of cells expressing the wild-type and a mutant nephrin further confirmed that the mutant nephrin could be abundantly found in the ER but not on the plasma membrane. Subcellular fractionation of wild-type and a mutant cell line clearly showed an altered subcellular distribution and molecular mobility of the mutant nephrin. In summary, our data indicate that a defective intracellular nephrin transport, most likely due to misfolding, is the most common consequence of missense mutations in NPHS1.     

Mutation spectrum in the nephrin gene (NPHS1) in congenital nephrotic syndrome

Congenital nephrotic syndrome, Finnish type (CNF or NPHS1), is an autosomal recessive disease characterized by massive proteinuria and development of nephrotic syndrome shortly after birth. The disease is most common in Finland, but many patients have been identified in other populations. The disease is caused by mutations in the gene for nephrin which is a key component of the glomerual ultrafilter, the podocyte slit diaphragm. A total of 30 mutations have been reported in the nephrin gene in patients with congenital nephrotic syndrome worldwide. In the Finnish population, two main mutations have been found. These two nonsense mutations account for over 94% of all mutations in Finland. Most mutations found in non-Finnish patients are missense mutations, but they include also nonsense and splice site mutations, as well as deletions and insertions. This mutation update summarizes the nature of all previously reported nephrin mutations and, additionally, describes 20 novel mutations recently identified in our laboratory.     

Genotype/phenotype correlations of NPHS1 and NPHS2 mutations in nephrotic syndrome advocate a functional inter-relationship in glomerular filtration

Mutations of the novel renal glomerular genes NPHS1 and NPHS2 encoding nephrin and podocin cause two types of severe nephrotic syndrome presenting in early life, Finnish type congenital nephrotic syndrome (CNF) and a form of autosomal recessive familial focal segmental glomerulosclerosis (SRN1), respectively. To investigate the mechanisms by which mutations might cause glomerular protein leak, we analysed NPHS1/NPHS2 genotype/phenotype relationships in 41 non-Finnish CNF patients, four patients with congenital (onset 0 to 3 months) focal segmental glomerulosclerosis and five patients with possible SRN1 (onset 6 months to 2 years). We clarify the range of NPHS1 mutations in CNF, detecting mutation 'hot-spots' within the NPHS1 coding sequence. In addition, we describe a novel discordant CNF phenotype characterized by variable clinical severity, apparently influenced by gender. Moreover, we provide evidence that CNF may be genetically heterogeneous by detection of NPHS2 mutations in some CNF patients in whom NPHS1 mutations were not found. We confirm an overlap in the NPHS1/NPHS2 mutation spectrum with the characterization of a unique di-genic inheritance of NPHS1 and NPHS2 mutations, which results in a 'tri-allelic' hit and appears to modify the phenotype from CNF to one of congenital focal segmental glomerulosclerosis (FSGS). This may result from an epistatic gene interaction, and provides a rare example of multiple allelic hits being able to modify an autosomal recessive disease phenotype in humans. Our findings provide the first evidence for a functional inter-relationship between NPHS1 and NPHS2 in human nephrotic disease, thus underscoring their critical role in the regulation of glomerular filtration.     

Molecular basis of intermittent maple syrup urine disease: novel mutations in the E2 gene of the branched-chain α-keto acid dehydrogenase complex

The E2 gene of the branched-chain α-keto acid dehydrogenase (BCKDH) complex was studied at the molecular level in three patients with intermittent maple syrup urine disease (MSUD). All three patients had higher BCKDH activity than did those with the classical phenotype. In the first patient, a single base substitution from A to G in intron 8 created a new 5′ splice site and caused an insertion of 126 nucleotides between exons 8 and 9 by activating an upstream cryptic 3′ splice site in the same intron. The predicted mRNA encoded a truncated protein with 282 amino acids including 4 novel ones at the carboxyl terminus, compared with the normal protein with 421 amino acids. In vitro, the region from the patient but not from a normal control was recognized and was recovered as a novel exon, indicating that the single substitution was responsible for incorporation of the region into mRNA. This mutation probably supports an exon definition model in which the spliceosome recognizes a 3′ splice site and then scans downstream for an acceptable 5′ splice site, thereby defining an exon. The second patient was homozygous for a G to T transversion at nucleotide 1463 in exon 11, which predicted a substitution of the termination codon by a leucine residue and the addition of 7 extra amino acids at the carboxyl terminus. For each mutation, these two patients were homozygous and their parents were heterozygous. The third patient was a compound heterozygote for a C to G transversion at nucleotide 309 in exon 4 and a G to A transition at nucleotide 1165 in exon 9, causing an Ile-to-Met substitution at amino acid 37 and a Gly-to-Ser substitution at amino acid 323, respectively. Taken together, these results indicate that the molecular basis of intermittent phenotype MSUD in some patients can be due to mutations in the E2 gene, giving rise to a low but significant residual activity of the BCKDH complex. Received: October 29, 1997 / Accepted: November 27, 1997     

Clustering of MHC–peptide complexes prior to their engagement in the immunological synapse: lipid raft and tetraspan microdomains

Summary: Protein reorganization at the interface of a T cell and an antigen‐presenting cell (APC) plays an important role in T cell activation. Imaging techniques reveal that reorganization of particular receptor–ligand pairs gives rise to an intercellular junction, termed the immunological synapse. In this synapse antigenic peptides associated with major histocompatibility complex (MHC) molecules form multimolecular arrays on the APC side, engaging an equivalent number of clustered T cell receptors (TCRs) on the T cell. The accumulation of MHC molecules carrying cognate peptide in the APC–T cell interface was thought to depend on the specificity and presence of TCRs. Recent evidence, however, suggests that the APC is equipped to preorganize MHC–peptide complexes in the absence of T cells. To this end, MHC molecules become incorporated into two types of membrane microdomains: (i) cholesterol‐ and glycosphingolipid‐enriched domains, denoted lipid rafts, that preconcentrate MHC class II molecules; and (ii) microdomains made up of tetraspan proteins, such as CD9, CD63, CD81 or CD82, that mediate enrichment of MHC class II molecules loaded with a select set of peptides. It follows that the integrity, composition and dynamics of these microdomains are candidate determinants favoring activation or silencing of T cells.     

Molecular basis of familial hypercholesterolemia in Brazil: Identification of seven novel LDLR gene mutations

Low-density lipoprotein receptor (LDLR) gene mutations cause familial hypercholesterol-emia (FH), one of the most common single gene disorders. The spectrum of LDLR mutations in Brazil is not known. The aim of this study was the characterization of LDLR mutations in 35 unrelated Brazilian patients with heterozygous FH. The promoter region, the 18 exons and the flanking intron sequences of the LDLR gene were screened by PCR-SSCP analysis and by DNA sequencing. In addition, we have screened the apolipoprotein B gene (APOB) for known mutations (R3500Q and R3531C) that cause Familial defective apo B-100 (FDB) by PCR-RFLP procedure. We found two nonsense (E92X and C371X) and six missense LDLR mutations (R236W, G322S, G352D, A370T, C675W and C677Y), that were previously described in FH patients from other populations. We also found five novel missense [G(-20)R, T476P, V503G, D580H and S652R] and two novel frame shift LDLR mutations (FsR757 and FsS828). Four patients were found to carry two different mutations in the LDLR gene: G352D and A370T (one patient), S652R and C675W (one patient) and T476P and V503G (two patients). APOB mutations were not found. These findings demonstrate that there is a broad spectrum of mutations in the LDLR gene in FH individuals from Brazil.     

Misleading findings of homozygosity mapping resulting from three novel mutations in NPHS1 encoding nephrin in a highly inbred community.

PURPOSE: Congenital nephrotic syndrome of the Finnish type (CNF, NPHS1) is a rare autosomal recessive disease caused by mutations in the NPHS1 gene encoding nephrin. We diagnosed congenital nephrotic syndrome in 12 children living in a village near Jerusalem. Most of the inhabitants are descendants of one Muslim family and have maintained their isolation by preference of consanguineous marriages. The aim of this study was to confirm that the NPHS1 gene is responsible for congenital nephrotic syndrome in our population, applying homozygosity mapping. METHODS: DNA samples were genotyped by four microsatellite markers that were in linkage disequilibrium with the NPHS1 gene on chromosome 19q13.1. Immunoperoxidase staining was used to study the expression of nephrin, and mutations were subsequently identified by direct sequencing of the entire coding region of the NPHS1 gene. RESULTS: Haplotype analysis revealed several different haplotypes, leading us to assume erroneously that there was genetic heterogeneity of congenital nephrotic syndrome. Because nephrin was completely absent in kidney tissue of one patient, direct sequencing of all DNA samples was performed, yielding three novel mutations: c.1138C>T (p.Gln380X), c.2160_ 2161insC (p.Cys721fs), and c.1707C>G (p.Ser569Arg). Patients were either homozygous for one of these mutations or compound heterozygotes, and they differed in their phenotype. CONCLUSION: We report the potential pitfalls of performing homozygosity mapping in a highly consanguineous population and discuss the phenomenon of multiple mutations in a given gene within an isolate.     

Molecular basis of multiple exostoses: mutations in the EXT1 and EXT2 genes

Hereditary multiple exostoses (EXT) is an autosomal dominant disorder characterized by the formation of exostoses, which are cartilage-capped bony protuberances mainly located on long bones. Two genes, EXT1 and EXT2, and at least one other unidentified gene, are known to be involved in the formation of exostoses. To date, 49 different EXT1 and 25 different EXT2 mutations have been found in EXT patients, and there is evidence that mutations in these two genes are responsible for over 70% of the EXT cases. Among the 49 EXT1 mutations there are 9 nonsense, 21 frameshift, and 5 splice site mutations; 2 in-frame deletions of 1 and 5 amino acids respectively; and 12 missense mutations. For EXT2, 8 nonsense, 11 frameshift, 3 splice site and 3 missense mutations are described. The majority of these mutations are mutations causing loss of function, which is consistent with the presumed tumor suppressor function of the EXT genes.     

GH-1 gene splicing mutations: Molecular basis of hereditary isolated growth hormone deficiency in children

Children, residents of the Russian Federation, with congenital isolated growth hormone deficiency, were screened for mutations of GH-1 gene, the main gene of this deficiency. Twenty-eight children from 26 families with total congenital isolated growth hormone deficiency were examined. Direct sequencing of GH-1 detected five splicing mutations in intron 2, intron 3, and exon 4, two of them were never described previously. Three dominant negative mutations of GH-1 splicing, the basis for autosomal dominant isolated growth hormone deficiency (type II), are presented: IVS2 −2A>T, IVS3 +2T>C, and IVS3 +1G<A. GH-1 is the main gene of type II isolated growth hormone deficiency in patients living in the Russian Federation. All detected mutations of GH-1 impair splicing processes, which distinguishes them from mutations in other forms of isolated growth hormone deficiency. The detected variety of GH-1 splicing mutations attests to allele genetic heterogeneity of this pathology. The “hot spot” of mutations is 5′-donor splicing site of GH-1 intron 3, while IVS3 +1G>A mutation can be regarded as the most incident in type II isolated growth hormone deficiency in the Russian population. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 141, No. 3, pp. 324–329, March, 2006     

Molecular basis of phenylketonuria and related hyperphenylalaninemias: mutations and polymorphisms in the human phenylalanine hydroxylase gene.

Mutations in the human phenylalanine hydroxylase gene producing phenylketonuria or hyperphenylalaninemia have now been identified in many patients from various ethnic groups. These mutations all exhibit a high degree of association with specific restriction fragment-length polymorphism haplotypes at the PAH locus. About 50 of these mutations are single-base substitutions, including six nonsense mutations and eight splicing mutations, with the remainder being missense mutations. One splicing mutation results in a 3 amino acid in-frame insertion. Two or 3 large deletions, 2 single codon deletions, and 2 single base deletions have been found. Twelve of the missense mutations apparently result from the methylation and subsequent deamination of highly mutagenic CpG dinucleotides. Recurrent mutation has been observed at several of these sites, producing associations with different haplotypes in different populations. About half of all missense mutations have been examined by in vitro expression analysis, and a significant correlation has been observed between residual PAH activity and disease phenotype. Since continuing advances in molecular methodologies have dramatically accelerated the rate in which new mutations are being identified and characterized, this register of mutations will be updated periodically.     

Molecular basis of mucopolysaccharidosis type II: Mutations in the iduronate-2-sulphatase gene

A number of mutations in the X-chromosomal human iduronate-2-sulphatase gene have now been identified as the primary genetic defect leading to the clinical condition known as Hunter syndrome or mucopolysaccharidosis type II. The mutations that are tabulated include different deletions, splice-site and point mutations. From the group of 319 patients thus far studied by Southern analysis, 14 have a full deletion of the gene and 48 have a partial deletion or other gross rearrangements. All patients with full deletions or gross rearrangements have severe clinical presentations. Twenty-nine different “small” mutations have so far been characterised in a total of 32 patients. These include 4 nonsense and 13 missense mutations, 7 different small deletions from 1 to 3 bp, with most leading to a frameshift and premature chain termination, and 5 different splice-site mutations also leading to small insertions or deletions in the mRNA. A 60 bp deletion, that results from a new donor splice-site, has been observed in five unrelated patients with relatively mild clinical phenotypes. This information will not only be useful for MPS II patient and carrier diagnosis, but also will aid in the understanding of the structure and function of iduronate-2-sulphatase, and possibly in correlating genotype with phenotype. © 1993 Wiley-Liss, Inc.     

Interferon-gamma and tumor necrosis factor-alpha disrupt epithelial barrier function by altering lipid composition in membrane microdomains of tight junction

Tight junctions (TJs) are specialized membrane microdomains of plasma membrane and play an important role in barrier function. IFN-gamma and TNF-alpha have been implicated in intestinal barrier dysfunction. In the present study, we analyzed the effect of IFN-gamma and TNF-alpha on epithelial barrier function and determined the contribution of apoptosis to this process using T84 cells, a model intestinal epithelial cell line. We found that TNF-alpha and IFN-gamma synergistically affected the epithelial barrier and disrupted the structure of TJs. We demonstrated for the first time that treatment with TNF-alpha and IFN-gamma changed lipid composition and fatty acyl substitutions of phospholipids in membrane microdomains of TJs. Alterations of lipid environment affected TJs barrier function and partly removed flotillin-1 and displaced occludin from membrane microdomains of TJs to detergent-soluble fractions. The distribution of claudin isoforms was unaffected by TNF-alpha and IFN-gamma treatment. These findings indicated the interaction between inflammatory cytokines and alterations of lipid composition in membrane microdomains of TJs in the inflammatory processes. The apoptosis inhibitor did not prevent cytokine-induced decrease in TER and increase in permeability to FITC-dextran. Our results suggest that the cytokines directly influence TJ function and modulate both the membrane microdomain localization of TJ proteins and lipid composition of TJs. The effects of proinflammatory cytokines on TJ structure and function provide a mechanism in the pathophysiology of Crohn's disease (CD). Understanding the intracellular mechanisms involved could be important in devising future therapeutic strategies to induce retightening of the leaky TJ barrier.     

Molecular basis of hereditary fructose intolerance in Italy: identification of two novel mutations in the aldolase B gene.

We screened the aldolase B gene in 14 unrelated Italian patients with hereditary fructose intolerance (HFI), and found two novel disease related mutations: a single nucleotide deletion in exon 2 (delta A20) that leads to an early stop codon, and a C-->T transition in exon 8 that substitutes an Arg with a Trp residue at codon 303 (R303W).     

Molecular basis for clinical heterogeneity in inherited cardiomyopathies due to myopalladin mutations

Abnormalities in Z-disc proteins cause hypertrophic (HCM), dilated (DCM) and/or restrictive cardiomyopathy (RCM), but disease-causing mechanisms are not fully understood. Myopalladin (MYPN) is a Z-disc protein expressed in striated muscle and functions as a structural, signaling and gene expression regulating molecule in response to muscle stress. MYPN was genetically screened in 900 patients with HCM, DCM and RCM, and disease-causing mechanisms were investigated using comparative immunohistochemical analysis of the patient myocardium and neonatal rat cardiomyocytes expressing mutant MYPN. Cardiac-restricted transgenic (Tg) mice were generated and protein-protein interactions were evaluated. Two nonsense and 13 missense MYPN variants were identified in subjects with DCM, HCM and RCM with the average cardiomyopathy prevalence of 1.66%. Functional studies were performed on two variants (Q529X and Y20C) associated with variable clinical phenotypes. Humans carrying the Y20C-MYPN variant developed HCM or DCM, whereas Q529X-MYPN was found in familial RCM. Disturbed myofibrillogenesis with disruption of α-actinin2, desmin and cardiac ankyrin repeat protein (CARP) was evident in rat cardiomyocytes expressing MYPN(Q529X). Cardiac-restricted MYPN(Y20C) Tg mice developed HCM and disrupted intercalated discs, with disturbed expression of desmin, desmoplakin, connexin43 and vinculin being evident. Failed nuclear translocation and reduced binding of Y20C-MYPN to CARP were demonstrated using in vitro and in vivo systems. MYPN mutations cause various forms of cardiomyopathy via different protein-protein interactions. Q529X-MYPN causes RCM via disturbed myofibrillogenesis, whereas Y20C-MYPN perturbs MYPN nuclear shuttling and leads to abnormal assembly of terminal Z-disc within the cardiac transitional junction and intercalated disc.     

Molecular basis of phenylketonuria in Venezuela: Presence of two novel null mutations

This report describes the mutational spectrum and linked haplotypes of the phenylalanine hydroxylase gene in Venezuela. In this study, we have detected European mutations such as IVS10nt-11, R243Q, and R408W on the same haplotype background (6.7, 1.8, and 2.3, respectively) as in Europe. In this sample, we have found two novel mutations: S349L detected in two homozygous siblings on the background of haplotype 6.7, and a small deletion, P314fsdelC, that results in a frameshift and a premature stop codon detected on the background of haplotype 4.3. The definite demonstration that mutation S349L results in a nonfunctional protein was shown by expression analysis in prokaryotic and eukaryotic systems. This mutation results in an unstable phenylalanine hydroxylase (PAH) protein completely devoid of enzymatic activity well correlated with the severe form of the disease exhibited by the homozygous patients. Hum Mutat 11:354–359, 1998. © 1998 Wiley-Liss, Inc.     

Molecular bases of antithrombin deficiency: twenty-two novel mutations in the antithrombin gene

Antithrombin (AT) is a major physiological inhibitor of hemostasis. We report 22 novel antithrombin gene (SERPINC1) mutations associated with antithrombin deficiency in 17 French and five German families. They were all present at the heterozygous state. Nine missense mutations accounted for type I deficiency, defined by equally low antithrombin activity and antigen level. Most of them (7/9) affected highly conserved serpin residues and were associated with venous thrombosis occurring at a young age (before age 32). One splice site, one nonsense mutation, three small deletions and one insertion were also identified as a cause for type I antithrombin deficiency. Seven other missense mutations were identified in type II or unclassified AT deficiency; g.5270C>T (p.T147I, T115I) and g.5281A>T (p.I151F, I119F) change residues in the heparin binding region, g.13267C>G (p.P439A, P407A) and g.13271T>C (p.F440S, F408S) affect amino acids in the pleiotropic region, g.2372G>A (p.G25D, G-8D) changes a signal peptide amino acid, g.2456G>C (p.C53S, C21S) affects one of the three disulfide bonds of the protein, and g.7585A>T (p.M347K, M315K) changes a nonconserved residue on strand 2C.     

Molecular and functional characterization of polymorphisms in the secreted phospholipase A2 group X gene: relevance to coronary artery disease

Among secreted phospholipases A2 (sPLA2s), human group X sPLA2 (hGX sPLA2) is emerging as a novel attractive therapeutic target due to its implication in inflammatory diseases. To elucidate whether hGX sPLA2 plays a causative role in coronary artery disease (CAD), we screened the human PLA2G10 gene to identify polymorphisms and possible associations with CAD end-points in a prospective study, AtheroGene. We identified eight polymorphisms, among which, one non-synonymous polymorphism R38C in the propeptide region of the sPLA2. The T-512C polymorphism located in the 5′ untranslated region was associated with a decreased risk of recurrent cardiovascular events during follow-up. The functional analysis of the R38C polymorphism showed that it leads to a profound change in expression and activity of hGX sPLA2, although there was no detectable impact on CAD risk. Due to the potential role of hGX sPLA2 in inflammatory processes, these polymorphisms should be investigated in other inflammatory diseases. Claire Perret and Ikram Jemel contributed equally to this paper.