Mutation databases for inherited renal disease: are they complete, accurate, clinically relevant, and freely available?

This study examined whether gene-specific DNA variant databases for inherited diseases of the kidney fulfilled the Human Variome Project recommendations of being complete, accurate, clinically relevant and freely available. A recent review identified 60 inherited renal diseases caused by mutations in 132 genes. The disease name, MIM number, gene name, together with "mutation" or "database," were used to identify web-based databases. Fifty-nine diseases (98%) due to mutations in 128 genes had a variant database. Altogether there were 349 databases (a median of 3 per gene, range 0-6), but no gene had two databases with the same number of variants, and 165 (50%) databases included fewer than 10 variants. About half the databases (180, 54%) had been updated in the previous year. Few (77, 23%) were curated by "experts" but these included nine of the 11 with the most variants. Even fewer databases (41, 12%) included clinical features apart from the name of the associated disease. Most (223, 67%) could be accessed without charge, including those for 50 genes (40%) with the maximum number of variants. Future efforts should focus on encouraging experts to collaborate on a single database for each gene affected in inherited renal disease, including both unpublished variants, and clinical phenotypes.

Clinical and genetic features in autosomal recessive and X-linked Alport syndrome.

BACKGROUND: This study determined the family history and clinical features that suggested autosomal recessive rather than X-linked Alport syndrome. METHODS: All patients had the diagnosis of Alport syndrome and the mode of inheritance confirmed by genetic testing, and underwent examination at a single centre. RESULTS: Patients comprised 9 males and 6 females with autosomal recessive Alport syndrome, and 18 males and 22 females with X-linked disease. Fourteen (93 %) individuals with autosomal recessive Alport syndrome developed early end-stage renal failure, all 15 had hearing loss, and most had lenticonus (12, 80 %), and a central (13, 87 %) or peripheral (13, 87 %) retinopathy. These features occurred as often as in males with X-linked disease. Females with autosomal recessive inheritance were less likely to have an affected family member in another generation (p = 0.01) than females with X-linked disease. They were more likely to have renal failure (p = 0.003), hearing loss (p = 0.02) and lenticonus (p < 0.001). Fifty percent had a central retinopathy compared with 18 % with X-linked disease (p = 0.14), but peripheral retinopathy prevalence was not different (p = 0.64). Nonsense mutations accounted for 67 % (8/12) of these disease-causing mutations. CONCLUSIONS: Autosomal recessive inheritance is increased in females with Alport syndrome and early onset renal failure, hearing loss, lenticonus, and, possibly, central retinopathy.

DNA variant databases improve test accuracy and phenotype prediction in Alport syndrome.

X-linked Alport syndrome is a form of progressive renal failure caused by pathogenic variants in the COL4A5 gene. More than 700 variants have been described and a further 400 are estimated to be known to individual laboratories but are unpublished. The major genetic testing laboratories for X-linked Alport syndrome worldwide have established a Web-based database for published and unpublished COL4A5 variants ( https://grenada.lumc.nl/LOVD2/COL4A/home.php?select_db=COL4A5 ). This conforms with the recommendations of the Human Variome Project: it uses the Leiden Open Variation Database (LOVD) format, describes variants according to the human reference sequence with standardized nomenclature, indicates likely pathogenicity and associated clinical features, and credits the submitting laboratory. The database includes non-pathogenic and recurrent variants, and is linked to another COL4A5 mutation database and relevant bioinformatics sites. Access is free. Increasing the number of COL4A5 variants in the public domain helps patients, diagnostic laboratories, clinicians, and researchers. The database improves the accuracy and efficiency of genetic testing because its variants are already categorized for pathogenicity. The description of further COL4A5 variants and clinical associations will improve our ability to predict phenotype and our understanding of collagen IV biochemistry. The database for X-linked Alport syndrome represents a model for databases in other inherited renal diseases.

Rhinovirus 3C protease can localize in the nucleus and alter active and passive nucleocytoplasmic transport.

The degradation of nuclear pore components and disruption of nucleocytoplasmic trafficking during rhinovirus infection have been attributed to viral 2A protease. Here we show for the first time that rhinovirus 3C protease may also have a role. Specifically, we show that 3C and its precursor, 3CD, can target green fluorescent protein to the nucleus of living cells, leading to degradation of nuclear pore components, and that incubation with recombinant 3C disrupts active and passive nucleocytoplasmic transport in a semi-intact cell nuclear transport system dependent on 3C protease activity. 3C may thus contribute to host cell shutoff in infected cells by localizing in the nucleus and facilitating nuclear pore breakdown.

Effects of PPAR gamma ligands on TGF-beta1-induced epithelial-mesenchymal transition in alveolar epithelial cells.

BACKGROUND: Transforming growth factor beta1 (TGF-beta1)-mediated epithelial mesenchymal transition (EMT) of alveolar epithelial cells (AEC) may contribute to lung fibrosis. Since PPAR gamma ligands have been shown to inhibit fibroblast activation by TGF-beta1, we assessed the ability of the thiazolidinediones rosiglitazone (RGZ) and ciglitazone (CGZ) to regulate TGF-beta1-mediated EMT of A549 cells, assessing changes in cell morphology, and expression of cell adhesion molecules E-cadherin (epithelial cell marker) and N-cadherin (mesenchymal cell marker), and collagen 1 alpha 1 (COL1A1), CTGF and MMP-2 mRNA. METHODS: Serum-deprived A549 cells (human AEC cell line) were pre-incubated with RGZ and CGZ (1 - 30 microM) in the absence or presence of the PPAR gamma antagonist GW9662 (10 microM) before TGFbeta-1 (0.075-7.5 ng/ml) treatment for up to 72 hrs. Changes in E-cadherin, N-cadherin and phosphorylated Smad2 and Smad3 levels were analysed by Western blot, and changes in mRNA levels including COL1A1 assessed by RT-PCR. RESULTS: TGFbeta-1 (2.5 ng/ml)-induced reductions in E-cadherin expression were associated with a loss of epithelial morphology and cell-cell contact. Concomitant increases in N-cadherin, MMP-2, CTGF and COL1A1 were evident in predominantly elongated fibroblast-like cells. Neither RGZ nor CGZ prevented TGF beta 1-induced changes in cell morphology, and PPAR gamma-dependent inhibitory effects of both ligands on changes in E-cadherin were only evident at submaximal TGF-beta1 (0.25 ng/ml). However, both RGZ and CGZ inhibited the marked elevation of N-cadherin and COL1A1 induced by TGF-beta1 (2.5 ng/ml), with effects on COL1A1 prevented by GW9662. Phosphorylation of Smad2 and Smad3 by TGF-beta1 was not inhibited by RGZ or CGZ. CONCLUSIONS: RGZ and CGZ inhibited profibrotic changes in TGF-beta1-stimulated A549 cells independently of inhibition of Smad phosphorylation. Their inhibitory effects on changes in collagen I and E-cadherin, but not N-cadherin or CTGF, appeared to be PPAR gamma-dependent. Further studies are required to unravel additional mechanisms of inhibition of TGF-beta1 signalling by thiazolidinediones and their implications for the contribution of EMT to lung fibrosis.

Transforming growth factor-beta enhances rhinovirus infection by diminishing early innate responses.

Individuals with asthma are prone to viral and bacterial infections, and most asthma exacerbations have been linked to viruses, particularly rhinovirus. Excess transforming growth factor (TGF)-beta present in asthmatic airways may cause immune suppression, as well as transdifferentiate fibroblasts to myofibroblasts, thereby augmenting proinflammatory responses after rhinovirus infection. After rhinovirus infection we examined virus replication and host cell immune responses in airway fibroblasts in the presence of TGF-beta1 and in myofibroblasts. Primary culture fibroblasts were pretreated with TGF-beta1 or transdifferentiated into myofibroblasts, and then infected with rhinovirus. Viral replication, virus release, chemokine production, and interferon (IFN) responses were measured over 72 hours. Rhinovirus replication and virus release into supernatants were enhanced in fibroblasts incubated with TGF-beta1 and in fibroblasts obtained from patients with asthma. Myofibroblasts also showed more rhinovirus replication, and infected myofibroblasts produced excess neutrophil chemokines. Examination of innate responses revealed blunting of type I IFN reactions with dissociated viral RNA and IFN mRNA responses. Addition of type I IFN restituted antiviral responses, and the effect of TGF-beta1 appeared to be mediated via actions on IFN regulatory factor-3 pathways. These data demonstrate that TGF-beta1 mediates enhanced virus replication and proinflammatory responses in airway cells. TGF-beta may act as an endogenous immunosuppressant promoting virus replication and inflammation during the evolution of acute severe asthma associated with rhinovirus infection.

T-cell cytokine profiles are altered in childhood asthma exacerbation.

BACKGROUND AND OBJECTIVE: Stable asthma is characterized by the production of Th2 cytokines, although Th1 cytokines may play a key role in aspects such as airway hyper-responsiveness. This study explored cytokine profiles associated with asthma exacerbation. METHODS: Intracellular T-cell cytokine production was measured in 16 children with acute severe asthma (emergency department), after convalescence (6 weeks, n = 13), with stable disease (after 6 months, n = 7) and in 14 age-matched hospital controls. Flow cytometry was used to identify CD4+ and CD8+ cells and to quantify intracellular T-cell production of the cytokines interferon (IFN)-gamma, IL-4 and IL-13. Cytokine production was compared using analysis of variance and random-effects generalized linear models and associations were examined using Pearson's correlation. RESULTS: Cytokine production was evident in CD4+ and CD8+ cells, and compared with asthmatic children, non-asthmatics had a higher percentage of IFN-gamma+CD4+ cells (P = 0.01). The percentage of CD8+IFN-gamma+ cells was increased in the convalescent phase compared with acute (P = 0.009) and stable asthma (P = 0.004). IL-4+ cells were not significantly altered. IL-13 levels were higher in acute disease than in stable asthma (P = 0.009 in CD4+ cells) and IFN-gamma/IL-13 ratios indicated a Th2 profile during exacerbation (P = 0.005 in CD4+ cells). CONCLUSIONS: IL-13, rather than IL-4, may play a pro-inflammatory role during acute severe asthma, whereas IFN-gamma responses were associated with recovery from acute severe asthma. These results suggest that altered T-cell cytokine profiles may contribute to the pathogenesis of and recovery from asthma exacerbations.

The Chemical Chaperone, PBA, Reduces ER Stress and Autophagy and Increases Collagen IV α5 Expression in Cultured Fibroblasts From Men With X-Linked Alport Syndrome and Missense Mutations.

INTRODUCTION: X-linked Alport syndrome (OMIM 301050) is caused by COL4A5 missense variants in 40% of families. This study examined the effects of chemical chaperone treatment (sodium 4-phenylbutyrate) on fibroblast cell lines derived from men with missense mutations. METHODS: Dermal fibroblast cultures were established from 2 affected men and 3 normals. Proliferation rates were examined, the collagen IV α5 chain localized with immunostaining, and levels of the intra- and extracellular chains quantitated with an in-house enzyme-linked immunosorbent assay. COL4A5 mRNA was measured using quantitative reverse transcriptase polymerase chain reaction. Endoplasmic reticulum (ER) size was measured on electron micrographs and after HSP47 immunostaining. Markers of ER stress (ATF6, HSPA5, DDIT3), autophagy (ATG5, BECN1, ATG7), and apoptosis (CASP3, BAD, BCL2) were also quantitated by quantitative reverse transcriptase polymerase chain reaction. Measurements were repeated after 48 hours of incubation with 10 mM sodium 4-phenylbutyrate acid. RESULTS: Both COL4A5 missense variants were associated with reduced proliferation rates on day 6 (P = 0.01 and P = 0.03), ER enlargement, and increased mRNA for ER stress and autophagy (all P values < 0.05) when compared with normal. Sodium 4-phenylbutyrate treatment increased COL4A5 transcript levels (P < 0.01), and reduced ER size (P < 0.01 by EM and P < 0.001 by immunostaining), ER stress (p HSPA5 and DDIT3, all P values < 0.01) and autophagy (ATG7, P < 0.01). Extracellular collagen IV α5 chain was increased in the M1 line only (P = 0.06). DISCUSSION: Sodium 4-phenylbutyrate increases collagen IV α5 mRNA levels, reduces ER stress and autophagy, and possibly facilitates collagen IV α5 extracellular transport. Whether these actions delay end-stage renal failure in men with X-linked Alport syndrome and missense mutations will only be determined with clinical trials.

The collαgen III fibril has a "flexi-rod" structure of flexible sequences interspersed with rigid bioactive domains including two with hemostatic roles.

Collagen III is critical to the integrity of blood vessels and distensible organs, and in hemostasis. Examination of the human collagen III interactome reveals a nearly identical structural arrangement and charge distribution pattern as for collagen I, with cell interaction domains, fibrillogenesis and enzyme cleavage domains, several major ligand-binding regions, and intermolecular crosslink sites at the same sites. These similarities allow heterotypic fibril formation with, and substitution by, collagen I in embryonic development and wound healing. The collagen III fibril assumes a "flexi-rod" structure with flexible zones interspersed with rod-like domains, which is consistent with the molecule's prominence in young, pliable tissues and distensible organs. Collagen III has two major hemostasis domains, with binding motifs for von Willebrand factor, α2ß1 integrin, platelet binding octapeptide and glycoprotein VI, consistent with the bleeding tendency observed with COL3A1 disease-causing sequence variants.

Three novel COL4A4 mutations resulting in stop codons and their clinical effects in autosomal recessive Alport syndrome.

Autosomal recessive Alport syndrome is caused by mutations in the COL4A3 and COL4A4 genes which code for the alpha3 and alpha4 chains of type IV collagen. These mutations result in haematuria, progressive renal impairment and often hearing loss, lenticonus and retinopathy. We describe here the mutations demonstrated by screening the 47 coding exons of the COL4A4 gene in six families with autosomal recessive Alport syndrome using PCR-single stranded conformational polymorphism (SSCP) analysis. Six sequence variants were identified. These included three novel mutations (2846delG, 2952delG and S969X) in exons 30 - 32 that all resulted in premature stop codons. These mutations were demonstrated in the heterozygous form in 3 families, and the S969X mutation was also present in the homozygous form in one of the two consanguinous families. These three mutations accounted for 40% (4/10) of the total mutant alleles in the six families studied. Six of the seven (86%) individuals with autosomal recessive Alport syndrome who had these mutations in the compound heterozygous or homozygous forms developed renal failure in adulthood, as well as hearing loss and ocular abnormalities. Haematuria was present in 15 of the 17 (88%) heterozygous mutation carriers. The other non-pathogenic sequence variants noted in COL4A4 included a nonglycine missense variant (L1004P), an intronic variant (4731-8 T>C) and a neutral polymorphism (V1516V).

Clinical, histopathologic, and genetic studies in nine families with focal segmental glomerulosclerosis.

BACKGROUND: Familial forms of focal segmental glomerulosclerosis (FSGS) are caused by mutations in genes at 1q25-31 (gene for steroid-resistant nephrotic syndrome 2 [NPHS2]), 11q21-22, 19q13 (gene for alpha-actinin 4 and NPHS1), and at additional unidentified chromosomal loci. METHODS: We describe clinical and histopathologic features and results of linkage analysis in nine consecutive index cases with familial FSGS who, together with their families, were referred for genetic studies. RESULTS: Two of the index cases presented in childhood (22%) and seven cases presented in adolescence or adulthood (78%). Six of their families (67%), including the two cases with childhood-onset disease, showed probable autosomal recessive inheritance. FSGS segregated at the 1q25-31 locus in two of these families and at the 11q21-22 locus in four families. None had disease caused by mutations in genes at the 19q13 locus, and no locus was identified in the three remaining families. Clinical features of proteinuria, minimal hematuria, hypertension, preeclampsia, and progressive renal impairment were usually present with autosomal recessive or dominant inheritance and with disease that segregated at the different loci. Eighteen renal biopsies from affected members of eight families showed a strong correlation between tubulointerstitial damage and percentage of obsolescent glomeruli (rho = +0.76; P < 0.01). None of the 13 patients from eight families who underwent transplantation developed recurrent FSGS in their grafts. In general, carriers of autosomal recessive disease had no distinctive clinical features apart from the development of preeclampsia in successive pregnancies. CONCLUSION: Familial forms of FSGS are not uncommon, and presentation frequently is in adolescence or adulthood, even when inheritance is autosomal recessive. Furthermore, carriers of autosomal recessive FSGS often have no distinctive phenotype.

Rhinovirus detection: comparison of real-time and conventional PCR.

Rhinoviruses are important human respiratory viruses and the major causative agents of the common cold. Historically, detection of rhinovirus has been by virus culture and this was significantly improved by the use of PCR assays. Recently real-time PCR was developed but to date there have been no reported comparisons of conventional and real-time PCR assays for detection of rhinovirus. In this study, we first compared real-time PCR (SYBR Green I) to conventional PCR for the detection of rhinovirus in serially diluted standard DNA and rhinovirus stock to determine the limits of detection. Next, assays were compared for sensitivity to detect rhinovirus in cell culture with a known number of infected cells. Finally, the assays were compared using clinical samples known to contain rhinovirus. Real-time PCR was 10-fold more sensitive than conventional PCR to detect rhinovirus in standard DNA and in virus stock and >10-fold more sensitive to detect rhinovirus in cultured cells. Real-time PCR was significantly superior for detection of rhinovirus in patients' nasal aspirates (sensitivity 72% versus 39%, P < 0.05). In summary, we found that real-time PCR was more sensitive than conventional PCR and reduced post-PCR processing. Hence, real-time PCR is suitable for both research and clinical purposes.

Characterization of the peripheral retinopathy in X-linked and autosomal recessive Alport syndrome.

BACKGROUND: Alport syndrome is an inherited disease resulting in kidney failure, hearing loss and ocular abnormalities. Alport syndrome is however often unrecognized, and the aim of this study was to characterize the associated but rarely described peripheral retinopathy and determine whether its demonstration was diagnostically helpful. METHODS: Index cases were diagnosed with Alport syndrome on renal biopsy in themselves or a family member. Inheritance and affected status were determined using microsatellite markers at the COL4A5 and COL4A3/COL4A4 loci, respectively. Participants' eyes were dilated, and examined with direct and indirect ophthalmoscopy, and slit lamp biomicroscopy by an expert ophthalmologist who was unaware of the patients' disease status. RESULTS: Ten males and nine females with X-linked Alport syndrome and seven with autosomal recessive disease were studied. Of the 26 patients, 16 had central retinopathy (62%), and 19 patients had peripheral retinopathy (74%). The peripheral changes occurred in both males and females with X-linked and autosomal recessive Alport syndrome, and were more common when renal failure, hearing loss, lenticonus and the central changes were present, but were also noted in 3 X-linked carriers with normal renal function. CONCLUSIONS: The peripheral retinopathy occurs in X-linked and autosomal recessive Alport syndrome even when the central retinopathy is absent. Careful retinal examination and photography that includes the periphery is a safe and inexpensive method that may help in the diagnosis of Alport syndrome especially in carriers of X-linked disease.

Vascular endothelial growth factor induction by rhinovirus infection.

Vascular participation manifested by a runny nose (rhinorrhea) is a prominent feature of the acute consequences of rhinovirus infection. Vascular endothelial growth factor (VEGF) is an angiogenic factor that also induces potent increases in vascular permeability; it is a candidate mediator of rhinorrhea in response to rhinovirus infection as well as contributing to enhanced vascular leakage in rhinovirus-linked asthma exacerbations. It has been shown that rhinovirus induces significant increases in both VEGF protein and mRNA in primary airway fibroblasts [Ghildyal et al. (2005): J Med Virol 75:608-615]. The current studies assessed VEGF responses to rhinovirus in primary culture airway epithelium, in epithelial and fibroblast cell lines and in rhinovirus-infected nasal secretions. Epithelial and fibroblast cells were infected with rhinovirus serotype 16 and VEGF protein and isoforms assessed by ELISA and RT-PCR, respectively. VEGF protein was released by both epithelial and fibroblast cell lines and primary airway epithelial cells in culture but was not increased following rhinovirus infection. PCR products coding for four or five of the six known VEGF isoforms were produced (121, 145, 165 and 183, and/or 189 amino acids) in cell lines and primary culture cells, but no specific isoform was linked to rhinovirus infection. Nasal VEGF was also measured in a cohort of asthmatics with verified rhinovirus and respiratory syncytial virus (RSV) infection. VEGF was not raised following rhinovirus infection alone, but was increased significantly if concomitant RSV infection was present. The data suggest that fibroblasts rather than the epithelium may play a key role in VEGF mediated vascular responses after rhinovirus infection. This may aid recruitment of inflammatory cells and contribute to airway inflammation and bronchial obstruction.

Rhinovirus infects primary human airway fibroblasts and induces a neutrophil chemokine and a permeability factor.

The events linking rhinovirus (RV) infection to airway symptoms are poorly understood. The virus initially infects airway epithelium followed by a vigorous inflammatory response that may entail spread of RV from epithelium to other cells in the airway wall. However, RV has fastidious growth characteristics and to date reproductive infection of primary cells other than human airway epithelium has not been confirmed. Airway fibroblasts are adjacent to and in contact with epithelial cells, play a key role in innate immune responses, and may participate in the evolution of inflammation. To investigate fibroblast actions, we first determined whether RV could infect and replicate in primary culture human lung fibroblasts. RV serotype 16 (RV16) was used to infect fibroblasts grown from lung tissue, and virus infection with replication was demonstrated by a combination of techniques. RT-PCR was used to show an increase in RV transcription; confocal microscopy demonstrated colocalization of the replicative form of RV genome (double-stranded RNA) and RV16 proteins; infectious virus was also recovered from the culture supernatant of infected fibroblasts. Functional consequences of RV infection were next examined. RV infection of fibroblasts was followed by an increase in epithelial neutrophil-activating peptide-78 (ENA-78) mRNA and protein. The permeability factor vascular endothelial growth factor (VEGF) was also induced over a similar time course. These data suggest that interactions between RV and human fibroblasts are feasible, may coordinate neutrophil chemoattraction with enhanced vascular permeability and that fibroblasts may contribute to inflammatory responses following RV infections.

Thin basement membrane nephropathy.

Thin basement membrane nephropathy. Thin basement membrane nephropathy (TBMN) is the most common cause of persistent glomerular bleeding in children and adults, and occurs in at least 1% of the population. Most affected individuals have, in addition to the hematuria, minimal proteinuria, normal renal function, a uniformly thinned glomerular basement membrane (GBM) and a family history of hematuria. Their clinical course is usually benign. However, some adults with TBMN have proteinuria >500 mg/day or renal impairment. This is more likely in hospital-based series of biopsied patients than in the uninvestigated, but affected, family members. The cause of renal impairment in TBMN is usually not known, but may be due to secondary focal segmental glomerulosclerosis (FSGS) or immunoglobulin A (IgA) glomerulonephritis, to misdiagnosed IgA disease or X-linked Alport syndrome, or because of coincidental disease. About 40% families with TBMN have hematuria that segregates with the COL4A3/COL4A4 locus, and many COL4A3 and COL4A4 mutations have now been described. These genes are also affected in autosomal-recessive Alport syndrome, and at least some cases of TBMN represent the carrier state for this condition. Families with TBMN in whom hematuria does not segregate with the COL4A3/COL4A4 locus can be explained by de novo mutations, incomplete penetrance of hematuria, coincidental hematuria in family members without COL4A3 or COL4A4 mutations, and by a novel gene locus for TBMN. A renal biopsy is warranted in TBMN only if there are atypical features, or if IgA disease or X-linked Alport syndrome cannot be excluded clinically. In IgA disease, there is usually no family history of hematuria. X-linked Alport syndrome is much less common than TBMN and can often be identified in family members by its typical clinical features (including retinopathy), a lamellated GBM without the collagen alpha3(IV), alpha4(IV), and alpha5(IV) chains, and by gene linkage studies or the demonstration of a COL4A5 mutation. Technical difficulties in the demonstration and interpretation of COL4A3 and COL4A4 mutations mean that mutation detection is not used routinely in the diagnosis of TBMN.

Mutations in the COL4A4 gene in thin basement membrane disease.

BACKGROUND: Patients with thin basement membrane disease (TBMD) are often from families where hematuria segregates with the COL4A3 and COL4A4 genes. These genes also are affected in autosomal recessive Alport syndrome. The aim of this study was to demonstrate COL4A4 mutations in TBMD. METHODS: Forty-eight unrelated individuals with TBMD who had no family members with autosomal recessive Alport syndrome were examined for COL4A4 mutations. The diagnosis of TBMD had been confirmed by renal biopsy (43/48, 90%) or by a family history of hematuria but without a renal biopsy (5/48, 10%). The 47 coding exons of COL4A4 were screened for mutations with the methods of enzyme mismatch cleavage or single stranded conformational polymorphism (SSCP) analysis, and exons that demonstrated electrophoretic abnormalities were sequenced. RESULTS: Nine variants that altered the coding sequences were identified. These were nonsense and frameshift mutations that resulted in stop codons (N = 3), and glycine (N = 3) and non-glycine missense variants (N = 3). Four intronic variants and three neutral polymorphisms were also detected. In total, four variants were considered 'pathogenic' principally because they resulted in stop codons or were not present in non-hematuric normal subjects. Three variants were considered 'possibly pathogenic' but two of these were each present in one of 46 non-hematuric normal subjects. CONCLUSIONS: Pathogenic COL4A4 mutations were demonstrated in three of the nine (33%) families in whom hematuria segregated with the COL4A3/COL4A4 locus. Two stop codons (R1377X and 2788/91delG) and a glycine substitution (G960R) resulted in hematuria in all 16 members who were tested from these three families. The S969X mutation described here in TBMD for the first time, as well as the R1377X mutation, also occur in autosomal recessive Alport syndrome.