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Hemolytic uremic syndrome (HUS) is a disease characterized by thrombotic microangiopathy with a triad of non-immune hemolytic anemia, thrombocytopenia, and renal impairment. Approximately 10% of cases of HUS are classified as atypical (aHUS). While today many genetically forms of aHUS pathology are known, only about 50% of carriers precipitate the disease. The reason remains unclear, and triggering events like intercurrent infections have been postulated. In rare cases, influenza A is the known trigger of aHUS; however, no cases of influenza B have been reported., Conclusion: We describe for the first time that influenza B strain as a trigger for aHUS in children with primary hereditary forms. We also showed in our three cases that immunization appears to be safe; however, this needs to be confirmed in a larger cohort. What is Known: • Known triggers of aHUS are infectious specimen. • Influenza A-associated aHUS cases are rarely published. What is New: • aHUS can be triggered by influenza B virus infection. • Influenza vaccination of patients with aHUS appears safe.

This review deals with podocyte proteins that play a significant role in the structure and function of the glomerular filter. Genetic linkage studies has identified several genes involved in the development of nephrotic syndrome and contributed to the understanding of the pathophysiology of glomerular proteinuria and/or focal segmental glomerulosclerosis. Here, we describe already well-characterized genetic diseases due to mutations in nephrin, podocin, CD2AP, alpha-actinin-4, WT1, and laminin beta2 chain, as well as more recently identified genetic abnormalities in TRPC6, phospholipase C epsilon, and the proteins encoded by the mitochondrial genome. In addition, the role of the proteins which have shown to be important for the structure and functions by gene knockout studies in mice, are also discussed. Furthermore, some rare syndromes with glomerular involvement, in which molecular defects have been recently identified, are briefly described. In summary, this review updates the current knowledge of genetic causes of congenital and childhood nephrotic syndrome and provides new insights into mechanisms of glomerular dysfunction.

Unlabelled: We describe two siblings with a Sengers-like syndrome, who presented with congenital hypertrophic cardiomyopathy, infantile cataract, mitochondrial myopathy, lactic acidosis and normal mental development. A mitochondrial adenine nucleotide translocator 1 (ANT1) defect was detected since the ANT1 protein was not detectable by immmunoblotting in muscle samples of the patients. Additionally to these features of classical Sengers syndrome (OMIM 212350), we found that the mitochondrial oxidative phosphorylation, measured by biochemical analysis, was severely compromised in skeletal muscle in both children. Biochemical and morphological analysis of the fibroblasts revealed normal results. The association of significantly decreased pyruvate oxidation rates, deficient energy production and decreased multiple mitochondrial enzyme-complex activities in the muscle samples of our patients is a new finding which differs from previous results in patients with Sengers syndrome., Conclusion: we recommend a muscle biopsy and the biochemical analysis of the oxidative phosphorylation system in patients with muscle hypotonia, cardiomyopathy and congenital or infantile cataract., (Copyright 2004 Springer-Verlag)

Unlabelled: A boy with recurrent episodes of hypoglycaemia and ataxia, microcephaly, mental retardation, permanent lactic acidaemia, intermittent 2-oxoglutaric aciduria as well as elevation of serum branched chain amino acids was diagnosed with dihydrolipoamide dehydrogenase (E3) deficiency. Analysis of genomic DNA revealed compound heterozygosity for two novel mutations: I393T in exon 11, located at the interface domain of the protein and possibly interfering with its dimerisation, and IVS9+1G>A located at a consensus splice site. A heterozygous polymorphism was also detected. In the patient's cDNA the I393T mutation and the polymorphism appeared to be homozygous, indicating that the mRNA coming from the IVS9+1G>A mutant allele is not stable., Conclusion: as opposed to the non-neurological phenotype of patients with a homozygous G229C mutation, this patient developed Leigh syndrome. Dihydrolipoamide dehydrogenase and pyruvate dehydrogenase complex activities in muscle were 29% and 14% of the lowest control values, respectively. Pyruvate dehydrogenase complex activity in fibroblasts was normal, however, indicating that the biochemical examination of defects in energy metabolism should be performed in a more energy demanding tissue.

Patients with primary renal glucosuria have normal blood glucose levels, normal oral glucose tolerance test results, and isolated persistant glucosuria. Congenital renal glucosuria is postulated to be attributable to defects in the SGLT2 gene. The Na(+)/glucose cotransporter gene SGLT2 (= SLC5A2) was analyzed in a Turkish patient with congenital isolated renal glucosuria. Genomic DNA was used as a template for amplification by the polymerase chain reaction of each of the 14 exons of the SGLT2 gene. The amplification products were sequenced. DNA sequence analysis revealed a homozygous nonsense mutation in exon 11 of the SGLT2 gene leading to the formation of a truncated cotransporter. Both parents and a younger brother, all three without renal glucosuria, are heterozygous for the nonsense mutation. Our data provide the first direct evidence of an etiologic role for the sodium/glucose cotransporter type 2 in the pathogenesis of renal glucosuria.

The human mitochondrial NADH:ubiquinone oxidoreductase (complex I), the first complex of the oxidative phosphorylation system, is composed of at least 42 subunits. Little is known about the assembly process of these subunits into the mature complex. Recently, two proteins in Neurospora crassa have been found to be involved in the assembly of complex I. These proteins are not constituent parts of the mature complex but are associated with smaller intermediate complexes of the assembly process and have a chaperone-like function. We have characterized the human homologue of one of these two complex I intermediate associated proteins, named CIA30, and show that expression of the human CIA30 protein is ubiquitous with a slightly higher expression in various heart tissues, kidney, lung and liver. As deletion of the Neurospora crassa CIA genes results in severe disruption of the assembly process, human CIA30 can be considered as a candidate gene related to complex I deficiency. Thirteen patients with an isolated complex I deficiency, but who were ruled out for mutations in the 35 nuclear genes of the complex and mtDNA, were subjected to mutational analysis of the gene coding for the human CIA30 protein. Four new single nucleotide polymorphisms (SNPs) were detected but no functional mutation was found.

Cerebrovascular deposition of amyloid beta protein (A beta) is a characteristic lesion of Alzheimer's disease (AD) and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D). Besides A beta, several other proteins and proteoglycans accumulate in cerebral amyloid angiopathy (CAA). We have now analyzed the expression of the heparan sulfate proteoglycan (HSPG) subtypes agrin, perlecan, glypican-1, syndecans 1-3 and HS glycosaminoglycan (GAG) side chains in CAA in brains of patients with AD and HCHWA-D. Hereto, specific well-characterized antibodies directed against the core protein of these HSPGs and against the GAG side chains were used for immunostaining. Glypican-1 was abundantly expressed in CAA both in AD and HCHWA-D brains, whereas perlecan and syndecans-1 and -3 were absent in both. Colocalization of agrin with vascular A beta was clearly observed in CAA in HCHWA-D brains, but only in a minority of the AD cases. Conversely, syndecan-2 was frequently associated with vascular A beta in AD, but did not colocalize with vascular A beta deposits in HCHWA-D. The three different syndecans, agrin, glypican-1 and HS GAG, but not perlecan, were associated with the majority of senile plaques (SPs) in all brains. Our results suggest a role for agrin in the formation of SPs and of CAA in HCHWA-D, but not in the pathogenesis of CAA in AD. Both syndecan-2 and glypican, but not perlecan, may be involved in the formation of CAA. We conclude that specific HSPG species may be involved in the pathogenesis of CAA in both AD and HCHWA-D, and that the pathogenesis of CAA and SPs may differ with regard to the involvement of HSPG species.

Unlabelled: Knowledge concerning the approximately 70 human nuclear genes creating the essential building-blocks of the five multi-protein subunit complexes of the oxidative phosphorylation (OXPHOS) system has been expanded greatly in the past few years. However, knowledge concerning the numerous human genes involved in the regulation of transcription, translation, post-translational modification, mitochondrial signalling, import, quality control, folding and assembly of the OXPHOS system is still rather scanty. It may be expected that this scenario, by the application of direct (candidate gene identification by comparison between known genes in lower species and the human expressed sequence tag database) and indirect genetic strategies (the chromosome transfer technique, linkage analysis and positional cloning) will rapidly change. By now, a limited number of structural and non-structural nuclear gene defects have been found., Conclusion: This review summarises the state of our current knowledge of nuclear gene mutations in oxidative phosphorylation disorders.

Deficiency of NADH:ubiquinone oxidoreductase, the first enzyme complex of the mitochondrial respiratory chain, is one of the most frequent causes of human mitochondrial encephalomyopathies. A relatively small percentage of human complex I deficiency is associated with mitochondrial DNA mutations. cDNA characterization and mutational analysis of the structural complex I genes in 19 complex I-deficient patients, in whom common mtDNA mutations have been excluded, has so far revealed five patients with alterations in evolutionary conserved nuclear-encoded proteins. In order to complete our knowledge about the expected 36 structural nuclear complex I genes, we characterized the NDUFB7 and the 17.2-kDa cDNA sequences of the hydrophobic (HP) fraction of the complex. Subsequently, we screened all subunits of this fraction for the presence of mutations in those 14 patients of our initial patient cohort in whom the underlying genetic cause had not been elucidated. Strikingly, no pathogenic mutations were found in the HP subunits that would explain the complex I deficiency in our patients. Other strategies are needed to unravel proteins involved in the pathogenesis of the complicated cellular network of transcription until correct assemblage of complex I.

We report the cloning of the cDNA sequence of the nuclear-encoded NDUFA8 subunit of NADH: ubiquinone oxidoreductase, the first mitochondrial respiratory chain complex. The NDUFA8 open reading frame (ORF) includes 519 bp and encodes 172 amino acids (Mr=20.1 kDa). The human cDNA sequence shows 86.2% identity with the bovine sequence, whereas the human NDUFA8 amino acid sequence is 87.8% similar to its bovine PGIV protein counterpart. Both human and bovine NDUFA8 contain a conserved cysteine motif. Polymerase chain reaction analysis of rodent/human somatic cell hybrids maps the human NDUFA8 gene to chromosome 9. A multiple tissue blot has revealed the highest NDUFA8 mRNA expression in human heart, skeletal muscle, and fetal heart. Mutation analysis of the NDUFA8 fibroblast cDNA in 20 patients with an isolated enzymatic complex I deficiency in cultured skin fibroblasts has revealed two polymorphisms, one within the ORF and the other in the 3' untranslated region of the NDUFA8 cDNA sequence. The allelic frequency of both polymorphisms was similar in controls and complex-I-deficient patients.

Bovine NADH:ubiquinone oxidoreductase (complex 1) of the mitochondrial respiratory chain consists of about 36 nuclear-encoded subunits. We review the current knowledge of the 15 human complex I subunits cloned so far, and report the 598-bp cDNA sequence, the chromosomal localization and the tissue expression of an additional subunit, the B17 subunit. The cDNA open reading frame of B17 comprises 387 bp and encodes a protein of 128 amino acids (calculated Mr 15.5 kDa). There is 82.7% and 78.1% homology, respectively, at the cDNA and amino acid level with the bovine counterpart. The gene of the B17 subunit has been mapped to chromosome 2. Multiple-tissue dot-blots showed ubiquitous expression of the mRNA with relatively higher expression in tissues known for their high energy demand. Of these, kidney showed the highest expression. Mutational analysis of the subunit revealed no mutations or polymorphisms in 20 patients with isolated enzymatic complex I deficiency in cultured skin fibroblasts.

This report concerns one new mutation in the tyrosine hydroxylase (TH) gene in three patients originating from three unrelated Dutch families with autosomal recessive L-DOPA-responsive dystonia (DRD). In this study, all exons of the TH gene were amplified by the polymerase chain reaction and subjected to analyses by single-strand conformation polymorphism. An aberrant migration pattern was observed for exon 6 of the TH gene in all patients. Direct sequencing of the coding region of exon 6 revealed the presence of one novel missense mutation. An a698g transition resulted in the substitution of the evolutionary conserved arginine 233 by a histidine (R233H). All patients were homozygous for the mutation. This new mutation in the TH gene was confirmed by restriction enzyme analysis with the restriction enzyme HhaI. Thus, a high proportion of defective TH alleles may be R233H in The Netherlands.

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by cerebellar ataxia, telangiectasia, immunodeficiency, elevated alpha-fetoprotein levels, chromosomal instability, predisposition to cancer, and radiation sensitivity. We report the identification of a new, double missense mutation in the ataxia telangiectasia gene (ATM) of a Dutch family. This homozygous mutation consists of two consecutive base substitutions in exon 55: a T-->G transversion at position 7875 of the ATM cDNA and a G-->C transversion at position 7876. These transversions were confirmed by polymerase chain reaction/primer-induced restriction analysis with CelII. The double base substitution results in an amino acid change of an aspartic acid to a glutamic acid at codon 2625 and of an alanine to a proline at codon 2626 of the ATM protein. Both amino acids are conserved between the ATM protein and its functional homolog, the Atm gene product in the mouse. Furthermore, the Chou-Fasman and Robson predictions both demonstrate a change in the secondary structure of the ATM protein carrying the D2625E/A2626P mutation. These findings suggest that the double base substitution in the ATM gene is a disease-causing mutation.

We report a new mutation in the sterol 27-hydroxylase (CYP 27) gene in a Dutch family with cerebrotendinous xanthomatosis: a G-->A transition in the splice donor site in intron 4. This mutation leads to skipping of exon 4, resulting in a loss of 66 amino acids in the CYP 27 enzyme molecule.

This report concerns two new mutations in the sterol 27-hydroxylase gene in two patients with cerebrotendinous xanthomatosis (CTX). In a Surinam-Creole patient (patient A), a G deletion on position cDNA 546/547 in exon 3 led to a frameshift and the introduction of a premature termination codon. In a Dutch patient (patient B), a C-->T transition at position 496 in exon 3 also led to a premature termination codon. Patient A was homozygous for the mutation, whereas patient B was compound heterozygous, a C-->T transition also being found in exon 6 at position 1204. The two new mutations were confirmed by restriction analysis with the restriction enzymes FokI and MaeI, respectively.

Sequencing of the arylsulfatase A gene in a late infantile metachromatic leukodystrophy patient showed the presence of a 12-bp deletion in exon 2. This deletion was found in a compound heterozygous state with the previously described 287 C-->T transition.

Direct sequencing of the coding region of the cystathionine beta-synthase (CBS) gene in two homocystinuric patients revealed the presence of two novel missense mutations. The first mutation, a 1111G-->A transition, resulted in the substitution of the evolutionary conserved valine-371 by a methionine residue (V371M) and created a new NlaIII restriction site. The second mutation, a G-->A transition at base-pair 494, resulted in an amino acid change from cysteine to tyrosine (C165Y) and abolished a BsoFI restriction site. Both mutations were found in a compound heterozygous state with the previously described 833T-->C transition.

In the present study we investigated whether glomerular hyperfiltration and albuminuria in streptozotocin-induced diabetic nephropathy in male Wistar-Münich rats are associated with changes in the heparan sulphate content of the glomerular basement membrane. Rats with a diabetes mellitus duration of 8 months, treated with low doses of insulin, showed a significant increase in glomerular filtration rate (p < 0.01) and effective renal plasma flow (p < 0.05), without alterations in filtration fraction or mean arterial blood pressure. Diabetic rats developed progressive albuminuria (at 7 months, diabetic rats (D): 42 +/- 13 vs control rats (C): 0.5 +/- 0.2 mg/24 h, p < 0.002) and a decrease of the selectivity index (clearance IgG/clearance albumin) of the proteinuria (at 7 months, D: 0.20 +/- 0.04 vs C: 0.39 +/- 0.17, p < 0.05), suggesting loss of glomerular basement membrane charge. Light- and electron microscopy demonstrated a moderate increase of mesangial matrix and thickening of the glomerular basement membrane in the diabetic rats. Immunohistochemically an increase of laminin, collagen III and IV staining was observed in the mesangium and in the glomerular basement membrane, without alterations in glomerular basement membrane staining of heparan sulphate proteoglycan core protein or heparan sulphate. Glomerular basement membrane heparan sulphate content, quantitated in individual glomerular extracts by a new inhibition ELISA using a specific anti-glomerular basement membrane heparan sulphate monoclonal antibody (JM403), was not altered (median (range) D: 314 (152-941) vs C: 262 (244-467) ng heparan sulphate/mg glomerulus). However, the amount of glomerular 4-hydroxyproline, as a measure for collagen content, was significantly increased (D: 1665 (712-2014) vs C: 672 (515-1208) ng/mg glomerulus, p < 0.01). Consequently, a significant decrease of the heparan sulphate/4-hydroxyproline ratio (D: 0.21 (0.14-1.16) vs C: 0.39 (0.30-0.47), p < 0.05) was found. In summary, we demonstrate that in streptozotocin-diabetic rats glomerular hyperfiltration and a progressive, selective proteinuria are associated with a relative decrease of glomerular basement membrane heparan sulphate. Functionally, a diminished heparan sulphate-associated charge density within the glomerular basement membrane might explain the selective proteinuria in the diabetic rats.