Sixteen novel mutations identified in COL4A3, COL4A4, and COL4A5 genes in Slovenian families with Alport syndrome and benign familial hematuria

Alport syndrome (ATS) and benign familial hematuria (BFH) are type IV collagen inherited disorders. Mutations in COL4A5 are generally believed to cause X-linked ATS, whereas mutations in COL4A3 and COL4A4 genes can be associated with the autosomal-recessive and -dominant type of ATS or BFH. In view of the wide spectrum of phenotypes, an exact diagnosis is sometimes difficult to achieve. This study involved screening each exon with boundary intronic sequences of COL4A3, COL4A4, and COL4A5 genes by optimized polymerase chain reaction-single-stranded conformational polymorphism analysis in 17 families with ATS and in 40 families diagnosed as having BFH. Twelve different mutations were found in the COL4A5 gene in ATS patients, comprising nine missense mutations, a splice site mutation, a mutation causing frameshift, and a nonsense mutation. One of the missense mutations (p.G624D) was present not only in one family with ATS but also in five families with suspected BFH. Three heterozygous mutations in the COL4A3 gene (two missense and one frameshift) and four heterozygous mutations in COL4A4 (two splice site, one in-frame deletion, and one missense) were identified in patients with BFH. Sixteen mutations are to the best of our knowledge new and private.     

Evidence for genetic heterogeneity in benign familial hematuria.

Benign familial hematuria (BFH: MIM141200) is an autosomal-dominant disease accounting for one-fifth of all hematuria of unknown cause in children. Previous observations suggest that BFH may be allelic to recessive Alport syndrome (AS: MIM 203780) with a mutation in the COL4A3/COL4A4 locus. However, it is not clear whether all cases of BFH are due to heterozygous mutation of COL4A3/COL4A4 genes. We report here the exclusion of linkage between BFH and COL4A3/COL4A4 loci at 2q35-37 in a restricted population from Sicily (Italy). Total lod score is -9.6 at theta 0. Furthermore, in some cases exclusion of linkage is evident even considering single families. We conclude that BFH is genetically heterogeneous.     

Alport syndrome and benign familial hematuria (thin basement membrane disease) in two brothers of a family with hematuria

Alport syndrome (AS) and benign familial hematuria (BFH) are inherited disorders of the glomerular basement membrane, which are sometimes difficult to differentiate at the early stage without type IV collagen staining of the renal basement membrane. Previous studies have indicated that mutation of type IV collagen alpha4 gene may be responsible for both BFH and AS. We report here a Japanese family with consanguinity, in which autosomal-recessive AS and BFH were separately identified in two brothers on the basis of findings of electron microscopy and type IV collagen chain staining of the renal biopsy specimens. Their parents, being first cousins, paternal uncle and grandmothers were found to have hematuria. Our observations suggest that BFH patients were heterozygous carriers of autosomal-recessive AS.     

Successful renal transplantation in a family with a novel mutation in COL4A3 gene and autosomal recessive Alport syndrome

Alport syndrome (AS) is an inherited disorder of basement membranes caused by mutations affecting specific proteins of the type IV collagen family, presenting with nephropathy and extrarenal manifestations such as sensorineural deafness and ocular anomalies. Ten percentage to 15% of the patients with AS have autosomal recessive (ARAS) due to mutation in either COL4A3 or COL4A4 gene. We report a novel mutation in the COL4A3 gene in an Indian family with ARAS. The above‐mentioned genetic anomaly was a missense variation in exon 26 of the COL4A3 gene (chr2:228137797G>A; c.1891G>A) that resulted in the amino acid substitution of Arginine for Glycine at codon 631 (p.Gly631Arg) that was present in the heterozygous state in the asymptomatic parents and homozygous state in the male offspring who presented with early‐onset end‐stage renal disease, lenticonus and hearing loss. The patient (male offspring) underwent successful renal transplantation with his mother as a donor.     

COL4A3/COL4A4 mutations: from familial hematuria to autosomal-dominant or recessive Alport syndrome

COL4A3/COL4A4 mutations: From familial hematuria to autosomal-dominant or recessive Alport syndrome. BACKGROUND: Mutations of the type IV collagen COL4A5 gene cause X-linked Alport syndrome (ATS). Mutations of COL4A3 and COL4A4 have been reported both in autosomal-recessive and autosomal-dominant ATS, as well as in benign familial hematuria (BFH). In the latter conditions, however, clinical features are less defined, few mutations have been reported, and other genes and non-genetic factors may be involved. METHODS: We analyzed 36 ATS patients for COL4A3 and COL4A4 mutations by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and direct sequencing. Sporadic patients who had tested negative for COL4A5 mutations were included with typical cases of autosomal recessive ATS to secure a better definition of the phenotype spectrum. RESULTS: We identified seven previously undescribed COL4A3 mutations: in two genetic compounds and three heterozygotes, and one in COL4A4. In agreement with the literature, some of the mutations of compound heterozygotes were associated with microhematuria in healthy heterozygous relatives. The mutations of heterozygous patients are likely dominant, since no change was identified in the second allele even by sequencing, and they are predicted to result in shortened or abnormal chains with a possible dominant-negative effect. In addition, both genes showed rare variants of unclear pathogenicity, and common polymorphisms that are shared in part with other populations. CONCLUSIONS: This study extends the mutation spectrum of COL4A3 and COL4A4 genes, and suggests a possible relationship between production of abnormal COL IV chains and dominant expression of a continuous spectrum of phenotypes, from ATS to BFH.     

常染色体隐性遗传Alport 综合征一家系COL4A3及COL4A4基因突变分析

目的 通过对有近亲婚配史的Alport综合征一家系Ⅳ型胶原α3和α4链的 COL4A3/COL4A4 基因分析，明确常染色体隐性遗传Alport综合征的基因突变,为该病的基因诊断和家系遗传咨询提供更为全面的理论基础｡ 方法 PCR扩增先证者DNA COL4A3/COL4A4 基因的共98个外显子，经直接测序，寻找突变位点，对有意义的突变经限制性内切酶AvaⅡ酶切在家系中分析验证｡ 结果 在该患者中共发现1个错义突变和10个序列变异｡其中在COL4A3 基因上发现一个位于42号外显子上的错义突变 G3725A,导致蛋白质Gly1242Asp的突变｡错义突变在患者中是纯合子，携带者中是杂合子，其他正常家系成员及筛查100条正常人染色体，未发现该突变｡10个序列变异为单核苷酸多态性改变｡ 结论 报道了一个国内较少见的常染色体隐性遗传Alport 综合征家系，同时经基因突变筛查发现Ⅳ型胶原α3链的一个新的致病性的基因突变｡     

COL4A4 mutation in thin basement membrane disease previously described in Alport syndrome

BACKGROUND: Carriers of autosomal-recessive and X-linked Alport syndrome often have a thinned glomerular basement membrane (GBM) and have mutations in the COL4A3/COL4A4 and COL4A5 genes respectively. Recently, we have shown that many individuals with thin basement membrane disease (TBMD) are also from families where hematuria segregates with the COL4A3/COL4A4 locus. This study describes the first COL4A4 mutation in an individual with biopsy-proven TBMD who did not have a family member with autosomal-recessive or X-linked Alport syndrome, inherited renal failure, or deafness. METHODS: The index case and all available family members were examined for dysmorphic hematuria> 50,000/mL using phase contrast microscopy and for segregation of hematuria with the COL4A3/COL4A4 and COL4A5 loci using DNA satellite markers. COL4A4 exons from the index case were then studied using the enzyme mismatch cleavage method, and exons that demonstrated abnormal cleavage products were sequenced. RESULTS: Hematuria in this family segregated with a haplotype at the COL4A3/COL4A4 locus (P = 0.031) but not with haplotypes at the COL4A5 locus. A mutation in COL4A4 that changed C to T resulting in an arginine residue being replaced by a stop codon (R1377X) was demonstrated in exon 44, which encodes part of the alpha 4(IV) collagen sequence close to the junction with the noncollagenous domain. This mutation was present in all five family members with hematuria, but not in the four unaffected family members, 33 unrelated individuals with TBMD, or 22 nonhematuric normals. CONCLUSIONS: R1377X has been described previously in a compound heterozygous form of autosomal-recessive Alport syndrome. Our observation is evidence that TBMD can represent a carrier state for autosomal-recessive Alport syndrome in at least some individuals.     

Mutation detection of COL4An gene based on mRNA of peripheral blood lymphocytes and prenatal diagnosis of Alport syndrome in China

Aim: Alport syndrome (AS) is a progressive renal disease characterized by haematuria and progressive renal failure. An accurate genetic diagnosis of AS is very important for genetic counselling and even prenatal diagnosis. Methods: We detected mutation of COL4An by amplifying the entire coding sequence mRNA of peripheral blood lymphocytes using polymerase chain reaction (PCR) in five Chinese AS families who asked for genetic counselling and prenatal diagnosis, then performed prenatal genetic diagnosis for four families. Mutation analysis of the foetus was made using DNA extracted from amniocytes. Foetus sex was determined by PCR amplification of SRY as well as karyotype analysis. Maternal cell contamination was excluded by linkage analysis. Results: Four different COL4A5 gene variants and two COL4A3 gene variants were detected in the five families. Because there was a de novo mutation in family 2, prenatal diagnosis was performed for the other four families. Results showed a normal male foetus for family 1 and family 4, respectively. Results showed an affected male foetus for families 3 and 5, and the pregnancies were terminated. Conclusion: An easier, faster and efficacious method for COL4An gene mutation screening based on mRNA analysis from peripheral blood lymphocytes was established. Prenatal genetic diagnosis was performed in four AS families in China.     

A Japanese family with Alport syndrome associated with esophageal leiomyomatosis: genetic analysis of COL4A5 to COL4A6 and immunostaining for type IV collagen subtypes

BACKGROUND: In some families, X-linked Alport syndrome (AS) is associated with diffuse leiomyomatosis. We describe clinical, pathologic and molecular-genetic findings in a Japanese family with this inheritance mode of AS in association with leiomyomatosis. PATIENT: AS was diagnosed in a one-year-old boy with recurrent aspiration pneumonia caused by esophageal stenosis from leiomyomatosis. Diagnosis was confirmed by electron microscopy coupled with type IV collagen chain subtype staining in a renal biopsy specimen. His mother, who exhibited esophageal leiomyomatosis and is heterozygous for AS, showed a discontinuous staining pattern for collagen alpha5(IV) chain along the epidermal basement membrane in a skin biopsy specimen. Genetic analysis in the boy revealed the deletion of the first two exons of COL4A6 together with deletion of the 5' end of COL4A5. Despite administration of cyclosporin A, massive proteinuria has persisted in the boy, although renal function otherwise remains normal. CONCLUSION: Identification of an AS patient during infancy is extremely rare. Clinical manifestations, including macroscopic hematuria, cataracts and leiomyomatosis caused by the large deletion involving COL4A5 to COL4A6, led to early presentation with AS.     

Autosomal dominant Alport syndrome linked to the type IV collage &agr;3 and &agr;4 genes (COL4A3 and COL4A4)

Background: Alport syndrome is a hereditary nephritis that may lead to end-stage renal disease (ERSD) in young adult life and is often associated with sensorineural deafness and/or ocular abnormalities. The majority of families are X-linked due to mutations in the COL4A5 gene at X122. Autosomal forms of the disease are also recognized with recessive disease, having been shown to be due to mutations in the COL4A3 and COL4A4 genes on chromosome 2. Familial benign haematuria has also been mapped to this region in some families.Subjects and methods: We describe a large family with autosomal dominant Alport syndrome in which males and females are equally severely affected and one member with a mild sensorineural deafness reached ESRD aged 35 years. Renal biopsy in four affected patients demonstrated characteristic thickened and split glomerular basement membranes on electron-microscopy. Results: Genetic linkage analysis using markers on chromosome 2q demonstrated co-segregation of the disease with the markers D2S351 and D2S401 with a maximum lod score of 3.4 at zero recombination. Linkage to the COL4A4 gene was confirmed using an intragenic COL4A4 polymorphism. Mutation analysis has revealed a missense Leu36Pro mutation in exon 5 of the adjacent COL4A3 gene in the unaffected mother, which may lead to a more severe phenotype in affected family members carrying this mutation. Conclusion: Mutations in the COL4A3 and COL4A4 cause a spectrum of glomerular basement membrane disease ranging from autosomal recessive Alport syndrome to autosomal dominant Alport syndrome and familial benign haematuria.     

薄基底膜肾病并发局灶节段性肾小球硬化症一家系的COL4A3和COL4A4基因突变分析

目的 探讨薄基底膜肾病(TBMN)合并局灶节段性肾小球硬化症(FSGS)的遗传学机制.方法 对一病理学诊断为TBMN合并FSGS患者及其家系的COL4A3和COL4A4基因突变,应用与COL4A3和COL4A4基因连锁的微卫星标记连锁分析方法进行分析.PCR扩增COIAA3和COL4A4全部98个外显子后,直接测序筛查突变.同时测序排除已为公认的FSGS相关基因NPHS1、NPHS2、WT1、TRPC6、ACTN4、CD2AP突变导致FSGS的可能.结果 微卫星标记连锁分析显示此家系与COL4A3和COL4A4基因连锁.直接测序在此家系中发现疾病患者COL4A4基因1214位的鸟嘌呤突变为腺嘌呤,导致Ⅳ型胶原α4链第405位甘氨酸突变为谷氨酸,并且发现COL4A3基因一多态性IVS1-4C＞T.此多态性随疾病分布,可能与致病相关.未发现FSGS相关基因的突变.结论 此家系是在TBMN的基础上发生FSGS.Ⅳ型胶原α4链突变及随疾病分布的基因多态性是否导致TBMN合并FSGS或使其易感性增加尚待更多家系进一步研究.     

Novel X-linked glomerulopathy is associated with a COL4A5 missense mutation in a non-collagenous interruption

A novel COL4A5 mutation causes rapid progression to end-stage renal disease in males, despite the absence of clinical and biopsy findings associated with Alport syndrome. Affected males have proteinuria, variable hematuria, and an early progression to end-stage renal disease. Renal biopsy findings include global and segmental glomerulosclerosis, mesangial hypercellularity and basement membrane immune complex deposition. Exon sequencing of the COL4A5 locus identified a thymine to guanine transversion at nucleotide 665, resulting in a phenylalanine to cysteine missense mutation at codon 222. The phenylalanine at position 222 is absolutely conserved among vertebrates. This mutation was confirmed in 4 affected males and 4 female obligate carriers, but was absent in 6 asymptomatic male family members and 198 unrelated individuals. Immunostaining for α5(IV) collagen in renal biopsies from affected males was normal. This mutation, in a non-collagenous interruption associated with severe renal disease, provides evidence for the importance of this structural motif and suggests the range of phenotypes associated with COL4A5 mutations is more diverse than previously realized. Hence, COL4A5 mutation analysis should be considered when glomerulonephritis presents in an X-linked inheritance pattern, even with a presentation distinct from Alport syndrome.     

Alport syndrome associated with diffuse leiomyomatosis: COL4A5-COL4A6 deletion associated with a mild form of Alport nephropathy.

BACKGROUND: The X-linked Alport syndrome (AS) is an inherited nephropathy due to mutations in the COL4A5 gene, encoding the alpha5 chain of type IV collagen, a major component of the glomerular basement membrane (GBM). Here, we report a new kindred with the rare association of X-linked AS and diffuse leiomyomatosis (DL), which is a tumourous process involving smooth muscle cells of the oesophagus, the tracheobronchial tree and, in females, the genital tract. For this syndrome, an almost constant association of large COL4A5 rearrangements with a severe juvenile form of nephropathy has been described for male patients. METHODS: DNA rearrangement at the COL4A5-COL4A6 locus was studied in several members of this family using polymerase chain reaction and pulsed field gel electrophoresis. Furthermore, immunohistochemical staining of tumour and skin samples was performed. RESULTS: The affected patients in this family carry a 120 kb deletion by which the COL4A5 exon 1 and COL4A6 exons 1, 1', and 2 are removed. Immunohistochemical investigation of a skin biopsy of an affected male patient confirmed the absence of both the alpha5 and the alpha6 chains of type IV collagen in the basement membrane of the skin. Surprisingly, both affected male patients had a rather mild renal phenotype. CONCLUSIONS: This report shows that, contrary to what has been reported to date, patients suffering from AS associated with DL can be associated with a late onset renal failure (adult) form of nephropathy.     

Autosomal-dominant Alport syndrome: natural history of a disease due to COL4A3 or COL4A4 gene

BACKGROUND: Alport syndrome is a clinically and genetically heterogeneous nephropathy. The majority of cases are transmitted as an X-linked semidominant condition due to COL4A5 mutations. In this form males are more severely affected than females. Less than 10% of cases are autosomal recessive due to mutation in either COL4A3 or COL4A4. In this rarer form, both males and females are severely affected. Only two cases of autosomal-dominant Alport syndrome have been reported, one due to a COL4A3 mutation and the other due to a COL4A4 mutation. Because of the paucity of the reported families, the natural history of autosomal-dominant Alport syndrome is mostly unknown. METHODS: Four families with likely autosomal-dominant Alport syndrome were investigated. COL4A3 and COL4A4 genes were analyzed by denaturing high-performance liquid chromatography (HPLC). Automated sequencing was performed to identify the underlying mutation. RESULTS: Two families had a mutation in the COL4A4 gene and two in the COL4A3. Accurate clinical evaluation of family members showed interesting results. Affected individuals (22 persons) had a wide range of phenotypes from end-stage renal disease (ESRD) in the fifth decade to a nonprogressive isolated microhematuria. Finally, three heterozygous individuals (90, 22 and 11 years old, respectively) were completely asymptomatic. CONCLUSION: This paper demonstrated that patients affected by autosomal-dominant Alport syndrome have a high clinical variability. Moreover, a reduced penetrance of about 90% (3 of 25) may be considered for the assessment of recurrence risk during genetic counseling of these families.     

Phenotypic and genotypic features of Alport syndrome in Chinese children

Chinese Alport syndrome (AS) was analyzed in 44 unrelated patients who were screened for mutations in the COL4A5 gene by polymerase chain reaction (PCR)-single-strand conformation polymorphism analysis or PCR direct sequencing in 30 of the 44 patients. The clinical data showed that all patients had hematuria; 25 of 29 male patients (86%) and 9 of 15 female patients (60%) had proteinuria; 11 of 29 male patients (38%) and 1 of 15 female patients (7%) had nephrotic-level proteinuria; 10 of 21 male patients examined (48%) and 1 of 12 female patients examined (8%) had hearing abnormalities. Renal function remained normal despite hearing abnormalities, and ocular lesions occurred in 10%. Among 30 of 44 patients who had a family history of end-stage renal disease (ESRD), 80% (24/30) belonged to X-linked juvenile kindreds, and 20% (6/30) patients to adult kindreds. Of the 44 patients, 14 did not have a family history of ESRD, while 11 of 14 patients diagnosed with X-linked AS did. DNA analysis revealed four missense mutations, two silent mutations, one substitution, and one in-frame deletion. PCR along with Southern hybridization analysis revealed a large deletion of the paired COL4A5 and COL4A6 genes. Chinese AS patients were characterized clinically with hematuria, heavy proteinuria, and more juvenile forms. Mutations in these patients were usually small mutations, while a large deletion involving the 5' part of both COL4A5 and COL4A6 genes was identified.     

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.     

Meta-analysis of genotype-phenotype correlation in X-linked Alport syndrome: impact on clinical counselling.

BACKGROUND: Alport syndrome (AS) is a hereditary nephropathy characterized by progressive renal failure, hearing loss and ocular lesions. Numerous mutations of the COL4A5 gene encoding the alpha 5-chain of type IV collagen have been described, establishing the molecular cause of AS. The goal of the present study was to identify the genotype-phenotype correlations that are helpful in clinical counseling. COL4A5-mutations (n=267) in males were analysed including 23 German Alport families. METHODS: Exons of the COL4A5 gene were PCR-amplified and screened by Southern blot, direct sequencing or denaturing gradient gel electrophoresis. Phenotypes were obtained by questionnaires or extracted from 44 publications in the literature. Data were analysed by Kaplan-Meier statistics, chi(2) and Kruskal-Wallis tests. RESULTS: Genotype-phenotype data for 23 German Alport families are reported. Analysis of these data and of mutations published in the literature showed the type of mutation being a significant predictor of end-stage renal failure (ESRF) age. The patients' renal phenotypes could be grouped into three cohorts: (1) large rearrangements, frame shift, nonsense, and splice donor mutations had a mean ESRF age of 19.8+/-5.7 years; (2) non-glycine- or 3' glycine-missense mutations, in-frame deletions/insertions and splice acceptor mutations had a mean ESRF age of 25.7+/-7.2 years and fewer extrarenal symptoms; (3) 5' glycine substitutions had an even later onset of ESRF at 30.1+/-7.2 years. Glycine-substitutions occurred less commonly de novo than all other mutations (5.5% vs 13.9%). However, due to the evolutionary advantage of their moderate phenotype, they were the most common mutations. The intrafamilial phenotype of an individual mutation was found to be very consistent with regards to the manifestation of deafness, lenticonus and the time point of onset of ESRF. CONCLUSIONS: Knowledge of the mutation adds significant information about the progress of renal and extrarenal disease in males with X-linked AS. We suggest that the considerable prognostic relevance of a patient's genotype should be included in the classification of the Alport phenotype.