Approach to the diagnosis of thin basement membrane nephropathy in females with the use of antibodies to type IV collagen

CONTEXT: Thin basement membrane nephropathy is recognized by a diffusely thin glomerular basement membrane (GBM) ultrastructurally. In contrast to Alport syndrome (AS), there is no GBM thickening, lamellation, or granular inclusions. Morphologically, there is overlap between thin basement membrane nephropathy and AS in female patients in whom there might be only thin GBM and no pathognomonic findings of AS. OBJECTIVE: To determine if the use of antibodies to collagen IV is helpful in making the distinction between thin basement membrane nephropathy and AS in female patients with primarily thin GBMs. DESIGN: We examined renal biopsies from 9 adult female patients with thin GBMs for the presence of alpha1, alpha3, alpha4, and alpha5 chains of type IV collagen by immunofluorescence. RESULTS: In 2 patients with segmental GBM staining, no suggestion for AS was found on physical examination or in their family history. In the remaining 7 patients with normal GBM staining, 4 had family members with end-stage renal disease of unknown etiology, raising the suspicion of X-linked or autosomal-recessive AS. Three patients were presumed to have thin basement membrane nephropathy. CONCLUSION: Segmental GBM staining for alpha3, alpha4, and alpha5 chains of type IV collagen raises the suspicion of AS in the presence of adequate controls and other supporting evidence. Normal GBM staining for alpha3, alpha4, and alpha5 chains of type IV collagen, however, does not exclude AS.     

The clinical spectrum of type IV collagen mutations

Clinical manifestations of type IV collagen mutations can vary from the severe, clinically and genetically heterogeneous renal disorder, Alport syndrome, to autosomal dominant familial benign hematuria. The predominant form of Alport syndrome is X-linked; more than 160 different mutations have yet been identified in the type IV collagen α5 chain (COL4A5) gene, located at Xq22-24 head to head to the COL4A6 gene. The autosomal recessive form of Alport syndrome is caused by mutations in the COL4A3 and COL4A4 genes, located at 2q35–37. Recently, the first mutation in the COL4A4 gene was identified in familial benign hematuria. This paper presents an overview of type IV collagen mutations, including eight novel COL4A5 mutations from our own group in patients with Alport syndrome. The spectrum of mutations is broad and provides insight into the clinical heterogeneity of Alport syndrome with respect to age at renal failure and accompanying features such as deafness, leiomyomatosis, and anti-GBM nephritis. Hum Mutat 9:477–499, 1997. © 1997 Wiley-Liss, Inc.     

Thin Glomerular Basement Membranes in Patients with Hematuria and Minimal Change Disease

A detailed morphometric analysis of glomerular basement membrane (GBM) thickness was carried out on biopsies from 16 patients exhibiting normal histology and unremarkable immunofluorescence. Eleven of these patients presented with proteinuria, 8 in the nephrotic syndrome range, while 5 had hematuria as well. The remaining 5 patients presented with hematuria only. Eight patients had an initial diagnosis of minimal change disease, 4 were diagnosed as thin-membrane nephropathy, 2 had Alport syndrome, and the remaining 2 had hypertensive nephropathy. Quantitative morphometric analysis of GBM identified 3 subsets of patients. The first subset consisted of 6 patients: 5 adults, with an average GBM width of 361+/- 34 nm, and 1 child. The second subset included 8 patients with thin GBMs and a mean thickness of 253+/- 15 nm. The last subset comprised 2 patients with Alport syndrome showing marked variability in GBM thickness. This study has confirmed the presence of thin GBMs in hematurics, but has also revealed GBM thinning in 50% of patients with a diagnosis of minimal change disease.     

A human-mouse chimera of the alpha3alpha4alpha5(IV) collagen protomer rescues the renal phenotype in Col4a3-/- Alport mice

Collagen IV is a major structural component of basement membranes. In the glomerular basement membrane (GBM) of the kidney, the alpha3, alpha4, and alpha5(IV) collagen chains form a distinct network that is essential for the long-term stability of the glomerular filtration barrier, and is absent in most patients affected with Alport syndrome, a progressive inherited nephropathy associated with mutation in COL4A3, COL4A4, or COL4A5 genes. To investigate, in vivo, the regulation of the expression, assembly, and function of the alpha3alpha4alpha5(IV) protomer, we have generated a yeast artificial chromosome transgenic line of mice carrying the human COL4A3-COL4A4 locus. Transgenic mice expressed the human alpha3 and alpha4(IV) chains in a tissue-specific manner. In the kidney, when expressed onto a Col4a3(-/-) background, the human alpha3(IV) chain restored the expression of and co-assembled with the mouse alpha4 and alpha5(IV) chains specifically at sites where the human alpha3(IV) was expressed, demonstrating that the expression of all three chains is required for network assembly. The co-assembly of the human and mouse chains into a hybrid network in the GBM restores a functional GBM and rescues the Alport phenotype, providing further evidence that defective assembly of the alpha3-alpha4-alpha5(IV) protomer, caused by mutations in any of the three chains, is the pathogenic mechanism responsible for the disease. This line of mice, humanized for the alpha3(IV) collagen chain, will also provide a valuable model for studying the pathogenesis of Goodpasture syndrome, an autoimmune disease caused by antibodies against this chain.     

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.     

Hereditary nephritis mimicking immune complex-mediated glomerulonephritis

The defining ultrastructural features of hereditary nephritis are "basket weave" lamellation or thinning of glomerular basement membranes. Electron-dense deposits are not seen and immunofluorescence (IF) is generally negative. In this study, we report 5 cases of hereditary nephritis in which substantial amounts of glomerular electron-dense deposits were identified on electron microscopy, with corresponding positive IF staining in 4 cases, suggesting immune complex-mediated glomerulonephritis. However, no case had histological evidence of glomerular endocapillary or extracapillary proliferation or leukocyte infiltration typical of active glomerulonephritis. Four cases were diagnosed at outside institutions simply as forms of glomerulonephritis without considering the possibility of hereditary nephritis and were sent for consultation in contemplation of possible immunosuppressive therapy. All patients had negative serologies and no known underlying infectious or autoimmune disease; 4 patients had family history of hematuria or renal disease. The glomerular electron-dense deposits were predominantly mesangial (4 cases) and intramembranous (4 cases), as well as subepithelial (2 cases) or subendothelial (1 case). Corresponding IF positivity for immune reactants was identified in 4 cases, and IgG was the predominant immunoglobulin deposited. A characteristic feature was the tendency for deposits to form between the complex layers of glomerular basement membrane material, favoring a process of nonspecific entrapment of immune reactants within the thickened, lamellated basement membrane. In all cases, a diagnosis of hereditary nephritis was confirmed by demonstration of the characteristic loss of immunoreactivity for the alpha5 subunit of collagen IV (4 cases) or Goodpasture's antigen (1 case) in renal or epidermal basement membranes. These cases expand the spectrum of unusual pathological findings in hereditary nephritis and emphasize the potential for hereditary nephritis to mimic immune complex glomerulonephritis.     

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.     

Clinical and pathohistological characteristics of Alport spectrum disorder caused by COL4A4 mutation c.193-2A>C: a case series

AimTo present the pathohistological and clinical characteristics of five Croatian families with Alport spectrum disorders caused by splice acceptor pathogenic variant c.193-2A>C in COL4A4 at the genomic position chr2:227985866.MethodsThe study enrolled five probands with kidney biopsy analysis and five family members. Mutation screening was performed with Illumina MiSeq platform. The pathogenic variant was confirmed with standard dye-terminator sequencing.ResultsThe only homozygous patient, aged two, had proteinuria and hematuria with preserved kidney function and no extrarenal manifestations. This patient had changes characteristic for Alport syndrome observed on electron microscopy of the kidney biopsy. In the heterozygous group, six patients had hematuria, four biopsied probands had proteinuria, and only one had moderately reduced kidney function. Heterozygous probands had variable kidney biopsy findings. Three patients had thin glomerular basement membrane nephropathy visible on electron microscopy and focal segmental glomerulosclerosis on light microscopy, two of them with focal lamellation on electron microscopy. One heterozygous patient had changes characteristic for Alport syndrome on electron microscopy without focal segmental glomerulosclerosis.ConclusionThe homozygous patient had hematuria and proteinuria with preserved kidney function. The heterozygous patients presented with reasonably mild clinical phenotype and variable pathohistological findings.

Alport syndrome (AS) is a structural disorder of the glomerular basement membrane (GBM). Its genetic basis lies in the diverse mutations of COL4A3, COL4A4, and COL4A5 genes and it phenotypically manifests as a progressive nephropathy with hematuria, ultrastructural changes of the GBM, sensorineural hearing impairment, and eye abnormalities (1-5). The most frequent mutations (85%) are COL4A5 mutations, resulting in X-linked AS (6). Individuals with autosomal recessive AS (ARAS), caused by two mutations in COL4A3 and/or COL4A4, have similar clinical features to men with X-linked AS (7,8). The type of mutation affects disease phenotype and manifestation. The phenotype is usually severe both in men and women, with early onset of end-stage renal disease (ESRD) and frequent extrarenal disorders (9,10).The spectrum of AS disorders has recently been expanded (11). Naming and describing individuals with heterozygous COL4A3 and COL4A4 mutations is still a matter of debate (10,12,13). A number of studies showed a correlation between thin glomerular basement membrane nephropathy (TBMN) with the heterozygosity for COL4A3 or COL4A4 mutation and benign familial hematuria (2,14-25). However, a variable proportion of COL4A3 or COL4A4 carriers progress to proteinuria, hypertension, and ESRD, which raises the question of the nomenclature of autosomal dominant AS (ADAS) (14,25-31). Some scientists advocate the use of the term ADAS, others continue to use the term TBMN, while a Cyprus research group uses the term late-onset Alport nephropathy (10,12,13,32). The rationale behind ADAS nomenclature for heterozygous COL4A3 or COL4A4 patients lies in the presence of thin GBM in the kidney biopsy specimens of patients with X-linked AS and ARAS and the heterozygous carriers of COL4A3 or COL4A4 mutation (10). The authors suggest that this approach would improve clinical and diagnostic evaluation, with the possibility of ESRD rate reduction and treatment optimization (10). There are also rationales behind the use of the term TBMN. Savige et al (13) stated that most of heterozygous COL4A3 and COL4A4 carriers show either no decline in kidney function or show only mild decline with inconstant progression to ESRD and hearing impairment. The authors also argue that there is no unmistakable evidence that one mutation in COL4A3 or COL4A4 gene without disease modifying factors can be responsible for the characteristic ultrastructural signs of AS, hearing impairment, or eye abnormalities (13). Furthermore, in other genetic diseases autosomal dominant (AD) term is not used for the carriers of autosomal recessive (AR) disease because it can lead to the diagnosis of AD and AR disease in different members of the same family (13). However, there are emerging reports of autosomal dominant Alport spectrum disorders, especially in the cases that are hard to diagnose clinicopathologically (33). Here, we present the pathohistological and clinical characteristics of disorders caused by splice site mutation c.193-2A>C in COL4A4 at the genomic position chr2:227985866.     

Benign familial hematuria

Fifty children had benign familial hematuria. They were from 43 families showing neither deafness, heavy proteinuria, nor chronic renal failure and had a nonprogressive course. Light microscopy of renal biopsy specimens showed little or no glomerular changes. Immunofluorescence showed no significant glomerular deposits of immunoglobulins or complement components, but deposition of C3 in the arteriolar walls was observed in 21 of the 39 patients examined. Electron microscopy demonstrated widespread attenuation of the glomerular basement membrane (GBM) in 19 patients, focal attenuation in 22, and normal GBM in nine. These observations suggest that patients with benign familial hematuria are heterogeneous and that the thin GBM may be related to hematuria.     

Thin glomerular basement membrane disease: light microscopic and electron microscopic studies.

Benign recurrent hematuria usually indicates a good prognosis. This condition is associated with abnormally thin glomerular basement membranes. Of 680 renal biopsy cases in which lower urinary tract disease had been excluded by careful study, 25 cases from seven children and eighteen adults met the criteria for thin glomerular basement membrane disease, placing the incidence of the disease at 3.7%. The mean patient age was 32.4 years and the male to female ratio was 1 to 5.3. The primary finding was microscopic hematuria in eighteen patients and gross hematuria in five patients. Among eighteen patients who had microscopic hematuria, one patient also exhibited proteinuria and one patient suffered from acute renal failure due to acute drug-induced interstitial nephritis. Proteinuria was only found in one patient. All of the patients had normal renal function, with the exception of one who suffered from acute renal failure. The duration of hematuria from the time of detection to the date of biopsy ranged from 3 months to 30 years with a mean interval of 56.6 months. No apparent evidence of familial hematuria in any patient was noted. Under light microscopy most glomeruli were normal. However, five cases showed focal global sclerosis. Under immunofluorescence microscopy seventeen cases were negative for all immunoglobulins, for complement, and for fibrinogen. Eight cases showed nonspecific mesangial deposition of fibrinogen and/or IgM. Ultrastructurally, extensive diffuse thinning of the GBM was a constant finding. The mean thickness of the GBM was 203.2 +/- 28.3 nm (n = 25); the thickness in adult (201.4 +/- 27.5 nm; n = 18) did not differ from that in children (208.1 +/- 32.0 nm; n = 7).     

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).     

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.     

Consensus statement on standards and guidelines for the molecular diagnostics of Alport syndrome: refining the ACMG criteria

The recent Chandos House meeting of the Alport Variant Collaborative extended the indications for screening for pathogenic variants in the COL4A5, COL4A3 and COL4A4 genes beyond the classical Alport phenotype (haematuria, renal failure; family history of haematuria or renal failure) to include persistent proteinuria, steroid-resistant nephrotic syndrome, focal and segmental glomerulosclerosis (FSGS), familial IgA glomerulonephritis and end-stage kidney failure without an obvious cause. The meeting refined the ACMG criteria for variant assessment for the Alport genes (COL4A3–5). It identified ‘mutational hotspots’ (PM1) in the collagen IV α5, α3 and α4 chains including position 1 Glycine residues in the Gly-X-Y repeats in the intermediate collagenous domains; and Cysteine residues in the carboxy non-collagenous domain (PP3). It considered that ‘well-established’ functional assays (PS3, BS3) were still mainly research tools but sequencing and minigene assays were commonly used to confirm splicing variants. It was not possible to define the Minor Allele Frequency (MAF) threshold above which variants were considered Benign (BA1, BS1), because of the different modes of inheritances of Alport syndrome, and the occurrence of hypomorphic variants (often Glycine adjacent to a non-collagenous interruption) and local founder effects. Heterozygous COL4A3 and COL4A4 variants were common ‘incidental’ findings also present in normal reference databases. The recognition and interpretation of hypomorphic variants in the COL4A3–COL4A5 genes remains a challenge.Subject terms: Diseases, Alport syndrome     

Somatic mosaicism and variant frequency detected by next-generation sequencing in X-linked Alport syndrome

X-linked Alport syndrome (XLAS) is a progressive, hereditary nephropathy. Although men with XLAS usually develop end-stage renal disease before 30 years of age, some men show a milder phenotype and develop end-stage renal disease later in life. However, the molecular mechanisms associated with this milder phenotype have not been fully identified. We genetically diagnosed 186 patients with suspected XLAS between January 2006 and August 2014. Genetic examination involved: (1) extraction and analysis of genomic DNA using PCR and direct sequencing using Sanger''s method and (2) next-generation sequencing to detect variant allele frequencies. We identified somatic mosaic variants in the type VI collagen, α5 gene (COL4A5) in four patients. Interestingly, two of these four patients with variant frequencies in kidney biopsies or urinary sediment cells of ≥50% showed hematuria and moderate proteinuria, whereas the other two with variant frequencies of <50% were asymptomatic or only had hematuria. De novo variants can occur even in asymptomatic male cases of XLAS resulting in mosaicism, with important implications for genetic counseling. This is the first study to show a tendency between the variant allele frequency and disease severity in male XLAS patients with somatic mosaic variants in COL4A5. Although this is a very rare status of somatic mosaicism, further analysis is needed to show this correlation in a larger population.     

Molecular aspects of Alport's syndrome

Summary We review the recent progress achieved on the understanding of the molecular basis of Alport's syndrome. This inherited disease is defined as progressive nephritis with sensorineural hearing loss. In 80%–85% of the families, inheritance is compatible with X-linked dominant transmission, whereas in the remaining cases autosomal dominant transmission is assumed. Histology studies demonstrated that the main defect is within the glomerular basement membrane (GBM). In addition, evidence for an altered GBM antigenicity came from immunofluorescence studies which showed a reduced or absent binding of anti-GBM autoantibodies or monoclonal antibodies to the Goodpasture antigen in some families. Subsequent studies added substantial evidence that Alport's syndrome is a type IV collagen disease. Genetic linkage analyses coherently identified an Alport locus at the X-chromosomal region Xg21.3–22. Recently, a previously unknown 5 chain of type IV collagen was identified, and the corresponding gene was also mapped to Xg22. Subsequent studies on Alport families by various groups identified more than 25 COL4A5 lesions. Segregation in linkage with the Alport phenotype could be shown in large kindreds. Mainly deletions and only a few point mutations were described. Most lesions reported so far are heterogeneous. We were able to identify two deletions and one point mutation involving a 3 splice site in 20 Alport families from Germany. One of the patients with a COL4A5 deletion and the patient with the splice site mutation developed anti-GBM antibodies after renal transplantation. In contrast, no COL4A5 lesions have been found in 2 further patients with posttransplant anti-GBM nephritis. The posttransplant anti-GBM antibodies mainly react with the NC domain of the 3(IV) chain, which has been shown to carry the target epitopes of Goodpasture autoantibodies. The results are discussed comprehensively.Abbreviations GBM glomerular basement membrane - AS Alport's syndrome - PCR polymerise chain reaction - DGGE denaturing gradient gel electrophoresis     

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.     

Genotype-phenotype correlations in 17 Chinese patients with autosomal recessive Alport syndrome

Autosomal recessive Alport syndrome (ARAS) results from mutations in the COL4A3 or COL4A4 gene. We analyzed the genotype and phenotype of 17 unrelated Chinese patients with ARAS. Clinical data were reviewed. All coding exons of COL4A3 and COL4A4 genes were PCR-amplified and sequenced from genomic DNA. We identified pathologic mutations in all patients, giving a mutation detection rate of 100%, with 82% in COL4A3 gene and 18% in COL4A4 gene. Sixteen novel mutations in COL4A3 gene and four novel mutations in COL4A4 gene were identified. Furthermore, a previously reported in-frame deletion mutation (40_63del24) in exon 1 of the COL4A3 gene was found in four patients in our study. A single 40_63del24 mutation in COL4A3 seems to result in mild or no renal manifestations, whereas the homozygous state of 40_63del24 in COL4A3 gene or compound heterozygous mutation of 40_63del24 plus another nonsense or frameshift mutation in COL4A3 gene seems to result severe ARAS with hearing loss. Half of the probands' parents had hematuria with or without mild proteinuria. Therefore, we recommend that ARAS be considered when a patient has a positive family history of hematuria, and screening for COL4A3 mutations firstly may be an efficient strategy for molecular diagnosis of ARAS. ? 2012 Wiley Periodicals, Inc.