Histopathology,ultrastructure, and clinical phenotypes in thin glomerular basement membrane disease variants

Recent genetic studies indicate that Alport syndrome and thin glomerular basement membrane disease (TMD) may both be due to COL4A3, COL4A4, and COL4A5 mutations, but there is continuing uncertainty concerning the diagnosis and management of patients without classic family history and symptoms. We examined kidney pathology and collagen alpha 3 to alpha 5(IV) expression in a series of 16 patients who presented with overlapping signs between TMD and Alport nephritis. All patients presented with hematuria, and 11 also had proteinuria, of whom 5 had nephrotic range proteinuria. Only 9 had family history of hematuria. In 9 of 16 (60%) we found premature glomerulosclerosis in the renal biopsies. Three of 16 had predominantly wide, lamellated glomerullar basement membranes (GBM), and in these, alpha 3 to alpha 5(IV) was absent in glomeruli or skin, diagnostic of Alport nephritis. One patient (12) had a very wide GBM with intramembranous lucencies but no lamellation. Skin biopsy was collagen alpha 5(IV) positive. Nine of 16 patients had predominantly thin GBM by electron microscopy, and 3 had thin and slightly lamellated GBM. Collagen alpha 3 to alpha 5(IV) expression in the kidney or skin biopsy was present in all of the latter 12 patients. Three patients had end-stage renal disease, 7 patients had hypertension, and 1 patient had chronic renal failure. We found that of the 16 patients with presumed TMD, 3 had X-linked Alport nephritis, 2 appeared to have autosomal recessive Alport nephritis, and the remaining patients had either an Alport or a TMD variant. The latter had histologic and/or clinical evidence of progressive renal disease, including premature glomerulosclerosis, hypertension, sustained proteinuria, and either thin or slight GBM lamellation focally, and preserved alpha 3 to alpha 5(IV) expression. These patients have a TMD variant, but an Alport variant with a potentially transmissible severe defect different from benign hematuria cannot be excluded.     

Frequent COL4 mutations in familial microhematuria accompanied by later‐onset Alport nephropathy due to focal segmental glomerulosclerosis

Familial microscopic hematuria (FMH) is associated with a genetically heterogeneous group of conditions including the collagen‐IV nephropathies, the heritable C3/CFHR5 nephropathy and the glomerulopathy with fibronectin deposits. The clinical course varies widely, ranging from isolated benign familial hematuria to end‐stage renal disease (ESRD) later in life. We investigated 24 families using next generation sequencing (NGS) for 5 genes: COL4A3, COL4A4, COL4A5, CFHR5 and FN1. In 17 families (71%), we found 15 pathogenic mutations in COL4A3/A4/A5, 9 of them novel. In 5 families patients inherited classical AS with hemizygous X‐linked COL4A5 mutations. Even more patients developed later‐onset Alport‐related nephropathy having inherited heterozygous COL4A3/A4 mutations that cause thin basement membranes. Amongst 62 heterozygous or hemizygous patients, 8 (13%) reached ESRD, while 25% of patients with heterozygous COL4A3/A4 mutations, aged >50‐years, reached ESRD. In conclusion, COL4A mutations comprise a frequent cause of FMH. Heterozygous COL4A3/A4 mutations predispose to renal function impairment, supporting that thin basement membrane nephropathy is not always benign. The molecular diagnosis is essential for differentiating the X‐linked from the autosomal recessive and dominant inheritance. Finally, NGS technology is established as the gold standard for the diagnosis of FMH and associated collagen‐IV glomerulopathies, frequently averting the need for invasive renal biopsies.     

Collagen type IV‐related nephropathies in Portugal: pathogenic COL4A5 mutations and clinical characterization of 22 families

Alport syndrome (AS) is caused by pathogenic mutations in the genes encoding α3, α4 or α5 chains of collagen IV (COL4A3/COL4A4/COL4A5), resulting in hematuria, chronic renal failure (CRF), sensorineural hearing loss (SNHL) and ocular abnormalities. Mutations in the X‐linked COL4A5 gene have been identified in 85% of the families (XLAS). In this study, 22 of 60 probands (37%) of unrelated Portuguese families, with clinical diagnosis of AS and no evidence of autosomal inheritance, had pathogenic COL4A5 mutations detected by Sanger sequencing and/or multiplex‐ligation probe amplification, of which 12 (57%) are novel. Males had more severe and earlier renal and extrarenal complications, but microscopic hematuria was a constant finding irrespective of gender. Nonsense and splice site mutations, as well as small and large deletions, were associated with younger age of onset of SNHL in males, and with higher risk of CRF and SNHL in females. Pathogenic COL4A3 or COL4A4 mutations were subsequently identified in more than half of the families without a pathogenic mutation in COL4A5. The lower than expected prevalence of XLAS in Portuguese families warrants the use of next‐generation sequencing for simultaneous COL4A3/COL4A4/COL4A5 analysis, as first‐tier approach to the genetic diagnosis of collagen type IV‐related nephropathies.     

A founder mutation in COL4A3 causes autosomal recessive Alport syndrome in the Ashkenazi Jewish population

Alport syndrome is an inherited progressive nephropathy arising from mutations in the type IV collagen genes, COL4A3, COL4A4, and COL4A5. Symptoms also include sensorineural hearing loss and ocular lesions. We determined the molecular basis of Alport syndrome in a non‐consanguineous Ashkenazi Jewish family with multiple affected females using linkage analysis and next generation sequencing. We identified a homozygous COL4A3 mutation, c.40_63del, in affected individuals with mutant alleles inherited from each parent on partially conserved haplotypes. Large‐scale population screening of 2017 unrelated Ashkenazi Jewish samples revealed a carrier frequency of 1 in 183 indicating that COL4A3 c.40_63del is a founder mutation which may be a common cause of Alport syndrome in this population. Additionally, we determined that heterozygous mutation carriers in this family do not meet criteria for a diagnosis of Thin Basement Membrane Nephropathy and concluded that carriers of c.40_63del are not likely to develop benign familial hematuria.     

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).     

Novel form of X-linked nonsyndromic hearing loss with cochlear malformation caused by a mutation in the type IV collagen gene COL4A6

Hereditary hearing loss is the most common human sensorineural disorder. Genetic causes are highly heterogeneous, with mutations detected in >40 genes associated with nonsyndromic hearing loss, to date. Whereas autosomal recessive and autosomal dominant inheritance is prevalent, X-linked forms of nonsyndromic hearing impairment are extremely rare. Here, we present a Hungarian three-generation family with X-linked nonsyndromic congenital hearing loss and the underlying genetic defect. Next-generation sequencing and subsequent segregation analysis detected a missense mutation (c.1771G>A, p.Gly591Ser) in the type IV collagen gene COL4A6 in all affected family members. Bioinformatic analysis and expression studies support this substitution as being causative. COL4A6 encodes the alpha-6 chain of type IV collagen of basal membranes, which forms a heterotrimer with two alpha-5 chains encoded by COL4A5. Whereas mutations in COL4A5 and contiguous X-chromosomal deletions involving COL4A5 and COL4A6 are associated with X-linked Alport syndrome, a nephropathy associated with deafness and cataract, mutations in COL4A6 alone have not been related to any hereditary disease so far. Moreover, our index patient and other affected family members show normal renal and ocular function, which is not consistent with Alport syndrome, but with a nonsyndromic type of hearing loss. In situ hybridization and immunostaining demonstrated expression of the COL4A6 homologs in the otic vesicle of the zebrafish and in the murine inner ear, supporting its role in normal ear development and function. In conclusion, our results suggest COL4A6 as being the fourth gene associated with X-linked nonsyndromic hearing loss.     

Spectrum of COL4A5 mutations in Finnish Alport syndrome patients

Alport syndrome (AS) is a hereditary kidney disorder, mainly caused by mutations in the X-chromosomal gene (COL4A5) encoding the type IV collagen a5 chain. In this study, detection of COL4A5 mutations was performed in 17 Finnish Alport syndrome families. Regions around the 51 previously known exons, as well as the two recently characterized exons 41A and 41B in COL4A5, were PCR-amplified from the patient DNA. Direct sequencing of the amplified products was performed and mutations were found in 12 families. None of the mutations involved exons 41A or 41B. Three of the mutations were potential splicing mutations, two of which were studied at the mRNA level. Seven of the mutations were single base substitutions, and two were deletions. In five families, no mutations were found.     

Type IV collagen alpha 5 chain. Normal distribution and abnormalities in X-linked Alport syndrome revealed by monoclonal antibody.

Although the evidence indicates that mutation of the gene for the alpha 5 chain of type IV collagen, alpha 5-(IV), is the primary defect in X-linked Alport syndrome, protein data for the alpha 5(IV) chain with regard to its normal distribution and its distribution in patients with Alport syndrome is lacking. We produced a rat monoclonal antibody (H51) by immunizing rats with a synthetic peptide corresponding to the nonconsensus amino acid sequence of alpha 5(IV) NC1 domain. H51 reacted by Western blotting with 26-kd cationic monomers and associated dimers of human type IV collagen NC1 domain. Immunohistochemical studies demonstrated that in normal human kidney alpha 5(IV) was present in the glomerular basement membrane and basement membranes of the Bowman's capsule and in some tubules (collecting ducts). The alpha 5(IV) chain was also detected in the basement membranes of normal skin, eye, and lung. Male patients with X-linked Alport syndrome revealed no reactivity of renal and epidermal basement membranes with H51, whereas alpha 5(IV) staining was normal in the glomerular basement membrane of patients with other types of glomerular diseases, including benign familial hematuria. The staining was also normal in the skin of nonaffected males in X-linked Alport families. Female heterozygous for Alport syndrome exhibited a discontinuous or mosaic pattern in the immunofluorescent staining of the epidermal basement membrane. These findings confirm that in patients with X-linked Alport syndrome there are abnormalities in alpha 5(IV) in renal and epidermal basement membranes at the protein level. Immunofluorescent staining of skin biopsies with this antibody may be of value in making a diagnosis of Alport syndrome, and, furthermore, may aid in detecting carrier females in whom urinary abnormalities are often mild or silent.     

Novel COL4A5, COL4A4, and COL4A3 mutations in Alport syndrome

This study summarizes 47 novel mutations identified during routine molecular diagnostics for Alport syndrome. We detected 34 in COL4A5, the gene responsible for X-linked Alport syndrome, and 13 in COL4A3 and COL4A4, the genes responsible for autosomal recessive Alport syndrome. A high detection rate of 90% was achieved among patients with typical clinical symptoms and a characteristic family history in both X-linked and autosomal recessive forms, and it can be assumed that most relevant mutations have been identified. In numerous positively tested patients, genetic variations which are unknown were detected.     

Data mining: Efficiency of using sequence databases for polymorphism discovery

Alport syndrome is a progressive renal disease leading to chronic renal failure, which often is accompanied by sensorineural deafness and ophthalmological signs in the form of anterior lenticonus. The X‐linked form of the disease is caused by mutations in the COL4A5 gene encoding the α5‐chain of type IV‐collagen. We performed mutation analysis of the COL4A5 gene by PCR‐SSCP analysis of each of the 51 exons with flanking intronic sequences in 81 patients suspected of X‐linked Alport syndrome including 29 clear X‐linked cases, 37 cases from families with a pedigree compatible with X‐linked inheritance, and 15 isolated cases. We found a mutation detection rate of 52% (42/81) (58% in males and 21% in females), and 69% (20/29) in families who clearly demonstrated X‐linked inheritance. Thirty‐six different mutations were found in 42 patients comprising 16 missense mutations, seven frameshifts, three in‐frame deletions, four nonsense mutations, and six splice site mutations. Twenty‐two of the mutations have not previously been reported. Furthermore, we found one non‐pathogenic amino acid substitution, one rare variant in a non‐coding region, and one polymorphism with a heterozygosity of 28%. Three de novo mutations were found, two of which were paternal and one of maternal origin. Hum Mutat 18:141–148, 2001. © 2001 Wiley‐Liss, Inc.     

Deletion mapping in Alport syndrome and Alport syndrome-diffuse leiomyomatosis reveals potential mechanisms of visceral smooth muscle overgrowth

Diffuse leiomyomatosis is associated with the inherited kidney disease Alport syndrome, and characterized by visceral smooth muscle overgrowth within the respiratory, gastrointestinal and female reproductive tracts. Although partial deletions of the type IV collagen genes COL4A5 and COL4A6, paired head-to-head on chromosome Xq22, are known to cause diffuse leiomyomatosis, loss of function for type IV collagen does not explain smooth muscle overgrowth. To further clarify pathogenic mechanisms, we have characterized novel deletions in patients with Alport syndrome-diffuse leiomyomatosis or Alport syndrome alone. A 27.6-kb deletion, in a female with Alport syndrome-diffuse leiomyomatosis, is marked by the most proximal, i.e. most 5', COL4A5 breakpoint described to date. By comparing this deletion to others described here and previously, we have defined a minimal overlap region, only 4.2 kb in length and containing the COL4A5-COL4A6 proximal promoters, loss of which contributes to smooth muscle overgrowth. A novel deletion in a male with Alport syndrome alone is>1.4 Mb in length, encompassing COL4A5 and COL4A6 entirely, as well as neighboring genes. We postulate that loss of the 4.2-kb region in diffuse leiomyomatosis causes misregulation of neighboring genes, contributing to smooth muscle overgrowth. Deletion of the neighboring genes themselves may afford protection from this condition.     

Detection of mutations in COL4A5 in patients with Alport syndrome

Alport syndrome (AS) can be caused by mutations in COL4A5, one of the six type IV collagen genes. For the purposes of confirming diagnoses, carrier screening and correlating genotype to phenotype, we have screened all 51 exons of this gene by SSCP analysis in 153 families with suspected AS. Mutations were identified in 77 families (of which 20 have previously been reported) and are reported with all available clinical information. All types of mutation were found (missense, nonsense, splicing, small and large deletions and insertions), with the commonest type being those affecting glycine residues in the collagen triple helix. Our 50% detection rate is similar to that of other groups and may imply the presence of mutations outside of the COL4A5 coding region or the existence of a second X-linked AS gene.     

The Inner Ear of Dogs with X-Linked Nephritis Provides Clues to the Pathogenesis of Hearing Loss in X-Linked Alport Syndrome

Alport syndrome is an inherited disorder of type IV collagen with progressive nephropathy, ocular abnormalities, and high-tone sensorineural deafness. In X-linked Alport syndrome, mutations in the COL4A5 gene encoding the alpha5 chain of type IV collagen lead to loss of the alpha3/alpha4/alpha5 network and increased susceptibility of the glomerular basement membrane to long-term damage. The molecular defects that underlie the otopathology in this disease remain poorly understood. We used a canine model of X-linked Alport syndrome to determine the expression of type IV collagen alpha-chains in the inner ear. By 1 month in normal adult dogs, the alpha3, alpha4, and alpha5 chains were co-expressed in a thin continuous line extending along the basilar membrane and the internal and external sulci, with the strongest expression along the lateral aspect of the spiral ligament in the basal turn of the cochlea. Affected dogs showed complete absence of the alpha3/alpha4/alpha5 network. The lateral aspect of the spiral ligament is populated by tension fibroblasts that express alpha-smooth muscle actin and nonmuscle myosin and are postulated to generate radial tension on the basilar membrane via the extracellular matrix for reception of high frequency sound. We propose that in Alport syndrome, the loss of the alpha3/alpha4/alpha5 network eventually weakens the interaction of these cells with their extracellular matrix, resulting in reduced tension on the basilar membrane and the inability to respond to high frequency sounds.     

Ultrastructural defects of the glomerular basement membranes associated with primary glomerular nephropathies

It is generally accepted that a glomerular basement membrane (GBM) thinner than 200 nm should be considered below normal. When this abnormality has a global and diffuse distribution, the associated clinical condition is a benign familial hematuria related to mutations of the COL4A4/COL4A3 genes, or an Alport syndrome. More often the GBM defects display a focal and segmental pattern, too small to express a thin glomerular basement membrane disease. The aim of this study is to emphasize statistical data concerning the pathogenic link between the renal glomerular diseases and the preexisting thin and very thin GBM. A series of 487 renal biopsies from adult patients has been thoroughly investigated both for nephropathologic diagnosis and the GBM ultrastructure. It has been statistically concluded that there is a close coexistence of primary glomerulonephritis and thin glomerular basement membranes with the role of a predisposing condition for immune complex deposition.     

Ultrastructural Defects of the Glomerular Basement Membranes Associated with Primary Glomerular Nephropathies

It is generally accepted that a glomerular basement membrane (GBM) thinner than 200?nm should be considered below normal. When this abnormality has a global and diffuse distribution, the associated clinical condition is a benign familial hematuria related to mutations of the COL4A4/COL4A3 genes, or an Alport syndrome. More often the GBM defects display a focal and segmental pattern, too small to express a thin glomerular basement membrane disease. The aim of this study is to emphasize statistical data concerning the pathogenic link between the renal glomerular diseases and the preexisting thin and very thin GBM. A series of 487 renal biopsies from adult patients has been thoroughly investigated both for nephropathologic diagnosis and the GBM ultrastructure. It has been statistically concluded that there is a close coexistence of primary glomerulonephritis and thin glomerular basement membranes with the role of a predisposing condition for immune complex deposition.