REVIEW ARTICLE

Hippokratia 2013, 17(3):207-213

Pierides A1,2, Voskarides K2, Kkolou M3, Hadjigavriel M3, Deltas C2
1Department of Nephrology, Hippocrateon Hospital, Nicosia, 2Department of Biological Sciences and Molecular Medicine Research Center, University of Cyprus, Nicosia, 3Department of Nephrology, Larnaca General Hospital, Larnaca, Cyprus

Abstract

Alport syndrome (ATS) results from X-linked, COL4A5 mutations (85%) or from autosomal recessive homozygous or compound heterozygous COL4A3/A4 mutations (15%), associated with alternate thinning and thickening as well as splitting and lamellation of the glomerular basement membranes. In contrast, familial microhematuria with thin basement membranes is thought to result from heterozygous COL4A3/A4 mutations. This absolute separation may not always be true. Renal biopsies and molecular genetics were used to study microhematuric families in the Hellenic population we serve. The COL4A5 gene was studied by PCR and direct re-sequencing for new mutations, while PCR-RFLP was used to identify more carriers of known COL4A5 and COL4A3/A4 mutations. Molecular genetics in two undiagnosed microhematuric Cypriot families, revealed COL4A5 mutation P628L indicating X-linked ATS. Of nine males, seven developed end stage kidney disease (ESKD) between 31 and 56, while two are well at 51 and 57, exhibiting microhematuria and thin basement membrane nephropathy (TBMN). COL4A5 mutation G624D was also identified in six Greek families. Seventy five members had DNA tests and 37 proved positive. Four positive males developed ESKD at 61, 51, 50 and 39 years, while the remaining and all females showed only microhematuria. A literature search revealed eight papers with six similar hypomorphic COL4A5 mutations presenting as phenocopies of TBMN. In conclusion, X-linked COL4A5 ATS mutations produce a phenotypic spectrum with a) classical ATS with early onset ESKD, neurosensory deafness and ocular defects b) males with only ESKD and late deafness and c) males due to missense mutations, such as G624D and P628L that may only exhibit microhematuria, TBMN, mild chronic renal failure (CRF) or late onset ESKD. Consequently when investigating "benign familial hematuria" these and other similar X-linked COL4A5 mutations should also be searched for.

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Key words: Alport syndrome, benign familial hematuria, COL4A3/COLA4/COL4A5 mutations, hypomorphic COL4A5 mutations, phenotypic heterogeneity, thin basement membrane nephropathy.

Corresponding author: Dr Alkis Pierides FRCP, FACP, Hippocrateon Hospital, Nicosia Cyprus, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it. and Prof. Constantinos Deltas, Molecular Medicine Research Center and Laboratory of Molecular and Medical Genetics, Department of Biological Sciences, University of Cyprus, Kallipoleos 75, 1678, Nicosia, Cyprus, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

Introduction

Patients with X-linked Alport syndrome (ATS), due to COL4A5 mutations on chromosome Xq22.3, usually present in childhood with continuous microscopic hematuria and episodes of macroscopic hematuria, often associated with pyrexia from upper respiratory tract infections (URTI) and other causes. This microscopic hematuria is usually followed in adolescence by proteinuria and hypertension that gradually progress to renal failure and end stage kidney disease (ESKD) by the 2nd or 3rd decades of life. High-tone neurosensory deafness develops in ~80% of such patients and ocular complications in ~50%1-3. Classical perimacular dot-and-fleck retinopathy commonly develops in patients that develop early ESKD, before the age of 304 and usually results from serious, large deletions, frameshift and nonsense COL4A5 mutations.

Steady progress in molecular genetics over the last 20 years, now enables confirmation of most X-linked ATS patients through identification of their underlying COL4A5 mutations. In the past however, ATS diagnosis relied entirely on renal biopsy electron microscopy (EM) findings and a thickened, split and lamellated glomerular basement membrane (GBM) was thought essential before the diagnosis of ATS could be made. Today, some 630 COL4A5 ATS mutations have been identified worldwide5 and a correlation has been attempted between the type and site of the underlying mutation and the resultant phenotype, particularly the age at which ESKD is reached1-5. It is important to realize that COL4A5 mutations are all different and while most large deletions, frameshift and nonsense mutations are characterized by early ESKD, accompanied by ocular and neurosensory hearing deficits, a small number of X-linked ATS patients, due to milder, missence mutations, develop ESKD at a much later age or not at all. These hypomorphic mutations are not totally destructive but are found on genes encoding for proteins that maintain some residual function. They were used to be called “adult type” mutations6 and affected hemizygous males did not show ocular changes, while hearing loss developed late in life, or not at all6-12. Molecular genetics have been vital in helping us understand the variable clinical phenotype of COL4A5 mutations which in some hemizygous males are characterized by mild GBM changes or even pure TBMN with only microscopic hematuria8,9,11,12. This idea was unthinkable a few years ago, when microscopic hematuria and TBMN on EM, were believed to be caused only by autosomal heterozygous COL4A3/A4 mutations. A major change in our thinking and clinical approach is now warranted and Figure 1 attempts to describe and summarize this new concept whereby X-linked COL4A5 ATS mutations are responsible for a continuous phenotypic spectrum that starts with a) classical ATS, characterized by early ESKD, ocular defects and neurosensory deafness, b) moving to milder cases with variable to late onset kidney failure and tardive neurosensory deafness without ocular defects and c) ending through some mild, hypomorphic missense mutations, such as G624D, P628L and all other mutations mentioned in references8,9,11,12, to TBMN and microscopic hematuria. These hypomorphic COL4A5 ATS mutations that encode for collagen IV molecules which retain some residual activity can lead to a phenotype in males that is identical to pure familial microhematuria, invariably produced by autosomal heterozygous COL4A3/A4 mutations. They actually present as phenocopies of TBMN.

In one of the earliest large series of 39 X-linked COL4A5 ATS mutations, published by Tryggvason’s group in 199810, two missense mutations, G624D and L1649R, stood out for failing to lead to early ESKD, ocular changes and hearing loss. Mutation L1649R, common in the western USA, had been identified even earlier7, with the onset of renal failure in the 4th and 5th decades and an even later onset of neurosensory deafness. Chen et al in a Chinese paper in 20018, Wilson et al in 2007 in New Zealand12 and Kaneko et al in Japan in 20109, all described genetically proven X-linked ATS families that demonstrated benign familial hematuria and thin membrane nephropathy from three additional COL4A5 mutations, G156A, C1638Y and G1000V respectively. In 2007, Slajpah and colleagues re-discovered missense COL4A5 mutation G624D, in six Slovenian families. One family showed members with late onset renal failure but interestingly, the remaining five families behaved more like benign familial hematuria with TBMN and no documented as yet ESKD11. Recently, we also demonstrated the presence of X-linked, ATS mutation G624D, in one Hellenic microhematuric family that exhibited a mild phenotype13. Once this was appreciated, five more clinically unclear Hellenic microhematuric families were shown to carry this same mutation, raising the number to six and rendering this G624D hypomorphic mutation, the commonest ATS COL4A5 mutation in the whole of Greece. We also identified two new Cypriot microhematuric families due to a new “adult type” X-linked COL4A5 mutation, P628L, which in affected male Cypriots, leads to a benign and variable phenotype ranging from isolated, late onset ESKD to only microscopic hematuria and TBMN.  In a recent report, we hypothesized that this milder phenotype associated with mutations G624D and P628L might be related to their position very close to the 12th natural interruption of the collagenous domain, thereby not interfering drastically with triple helix formation13.

These new findings strengthen the new concept that some milder, hypomorphic X-linked COL4A5 mutations can lead to a phenotype, similar to that of autosomal heterozygous COL4A3/A4 mutations (Figure 1). It is important to also remember that while heterozygous COL4A3/A4 mutations in both males and females are universally associated with microscopic hematuria and TBMN, up to 30% of all such patients will develop ESKD by the age of 70 years, due to progressive focal segmental glomerulosclerosis (FSGS), occasionally with sensorineural deafness14-16, most probably because of co-inherited nephrotoxic modifier genes. These patients and their families were described in the past as examples of autosomal dominant ATS.



Figure 1: This figure aims to transfer the new knowledge that different X-linked, COL4A5 ATS mutations may lead to: A) classical ATS with early ESKD, neurosensory deafness and ocular defects accompanied by widening, thickening, splitting and lamellation of the GBM, B) Hemizygous males with only late onset pure kidney failure without ocular defects and very rarely tardive neurosensory deafness with milder changes along their glomerular basement membranes, C) Lifelong microhematuria due to TBMN, occasionally accompanied with ESKD late in life or no ESKD at all. This phenotype results from some mild, missense mutations, such as G624D and P628L that maintain some residual protein activity. These hypomorphic COL4A5 mutations may also be common in patients with “benign familial hematuria”.

Variable clinical phenotypes and mixed renal biopsy electron microscopy findings are now easier to understand, thanks to  molecular genetics which has emerged as the most sensitive and accurate method for diagnosing collagen IV nephropathies. Molecular genetics testing accompanied by EM are clearly the best tools for understanding the different clinical phenotypes of ATS patients.

Missense, mild COL4A5 mutation P628L in two Cypriot families   

Figure 2A describes Cypriot family CY4206 carrying X-linked COL4A5 mutation P628L. This mutation was identified in 2009 and it helped put an end to a long discussion over 19 years, regarding the precise aetiology and pathophysiology of this familial microhematuric nephropathy. Generation II includes eight siblings, six males, four molecularly checked and affected and two sisters, one being a proven heterozygous carrier. The 5th brother, who lives in the UK, also developed ESKD, presumably from the same cause and has been successfully transplanted though no DNA studies have been carried out so far. The 6th brother has not been investigated so far. Altogether, four male and four female family members were tested positive for mutation P628L. Brothers II-8 (11018) and II-10 (11019), both presented in 1990 with microscopic hematuria, proteinuria and renal insufficiency. Renal biopsies were carried out in March and Sept 1990, before the introduction of routine EM in our renal unit and the light microscopy findings showed non-specific changes of advanced renal failure. All five affected males reached ESKD at 30, 31, 34, 44 and 56 years of age. The somewhat delayed age at which ESKD was reached together with the absence of neurosensory deafness and ocular defects were interpreted over the years as evidence against the possibility that X-linked ATS was the underlying pathology. However the clear-cut molecular genetic results in 2009, proved beyond any doubt, the presence of X-linked ATS and strengthened the new concept that mild, hypomorphic COL4A5 mutation P628L with residual protein function may lead to a benign ATS phenotype without ocular and neurosensory deficits.

Figure 2B describes Cypriot family CY4212 that had been studied clinically for over a decade before the recently identified COL4A5 mutation P628L established the correct diagnosis of X-linked ATS. Generation II consists of eight siblings, six males, four being affected and two females, one exhibiting the mutation. Altogether nine mutation carriers, four males and five females have been identified so far and all exhibit microhematuria. The four ATS males also developed proteinuria and two patients, UCY-1111 and UCY-1137 progressed to ESKD at ages 52 and 45 and died at 60 & 57 years of age respectively. The remaining two affected male patients are currently 51 and 57 year old and only show mild renal insufficiency, (creatinine: 1.6 & 1.5 mg/dl respectively). These two patients had renal biopsies that had shown well-preserved glomeruli with widespread thinning of the GBM on electron microscopy. The renal biopsies were carried out in 2002 and 2003 when no molecular genetics results were available and the precise diagnosis was not clear. Because of the presence of thin membranes in adulthood, X-linked COL4A5 ATS was thought unlikely at the time of the biopsies and the working diagnosis was TBMN due to a yet unknown heterozygous COL4A3/A4 mutation with slow progression to CRF 14,16. One of these two patients later developed C-ANCA angiitis but responded well to treatment with pulses of IV cyclophosphamide and solumedrol.  No female carriers showed any progression to CRF and all maintain normal kidney function with only microscopic hematuria. A renal biopsy with EM in one such female patient showed TBMN. The correct molecular diagnosis of X-linked COL4A5 ATS finally settled a confusing clinical picture for over a decade. What is now new and educational is the new knowledge that TBMN with microhematuria and near normal kidney function or very mild CRF in adult life may result from a COL4A5, hypomorphic, mild missense mutation, such as P628L.

Missense, mild, COL4A5 mutation G624D in six Greek families

Our Unit acts as a referral center for inherited renal diseases in Greece and following our recent publications of familial microhematuria associated with heterozygous COL4A3/A4 mutations14,16 we have received DNA samples from several Greek families exhibiting microscopic hematuria and occasional late onset renal failure. One such family with an equivocal diagnosis was initially screened for linkage to either the autosomal COL4A3/A4 genes or the X-linked COL4A5 gene. Segregation with the autosomal genes was excluded. Subsequent examination of the COL4A5 gene revealed the G624D mutation. Following this finding13,17, we screened several more such microhematuric Hellenic families for the same G624D mutation and five more large families proved positive. The latest such family (GR4217) is illustrated in Figure 2C. Fifteen members were tested, six males and nine females with 10 of them being positive, two males and eight females, all showing microhematuria. Characteristically, one hemizygous male reached ESKD at 61 and four years later, he is currently alive on hemodialysis. The other affected male, age 32, only shows microhematuria. Altogether 75 family members, 28 males (12 positive) and 47 females (25 positive) from these six families were genetically tested. Of the 12 hemizygous males in these six Hellenic families, only four have reached ESKD at ages 61, 51, 50 and 39. These families are characterized by a fairly large size, female preponderance with only microhematuria, late onset ESKD in hemizygous males at a mean age of 50 years (39-61) and complete absence of ocular signs. Only two patients showed late onset neurosensory deafness.

Figure 2: Pedigrees of two Cypriot microhematuric families (2a & 2b) with X-linked ATS, resulting from COL4A5, P628L hypomorphic mutation and a clinically similar Hellenic microhematuric family (2c) resulting from COL4A5 hypomorphic mutation G624D. Squares represent males and circles females. Filled symbols are hemizygous carrier males and a dot in the circle denotes a carrier female.

Other causes of familial microscopic hematuria (FMH) in Cyprus

Familial microscopic hematuria (FMH) has been a major clinical and research interest in our renal unit in Cyprus since its inception in 1984 and Table 1 describes the currently four major causes. These are: a) heterozygous male and female carriers of COL4A3/A4 mutations b) mesangial C3 nephropathy due to a novel, exon 2-3 duplication mutation on the CFHR5 gene in the alternative complement pathway. c) Alport syndrome due to: i) X-linked COL4A5 mutations in hemizygous males and ii) autosomal recessive homozygous or compound heterozygous COL4A3/A4 mutations in both male and female patients and d) heterozygous female carriers of COL4A5 ATS mutations.

The identification of two lifelong microhematuric Cypriot sisters that reached ESKD in their late fifties in the mid-1980’s, was a major turning point in our studies, because gradual unraveling of their family over many years, led to a very large, five generation family with 67 studied members. Among 48 at-risk members, DNA studies revealed that 31 were  positive carriers of mutation G1334E in COL4A3 and were characterized by early and lifelong microscopic hematuria, to which many years later proteinuria, hypertension, chronic kidney failure and ESKD from FSGS was added in a number of such patients (Figure 3). Several more similar Cypriot microhematuric families with very late onset FSGS were gradually identified and by 2007, after several attempts to link these families to known candidate genes of familial FSGS had failed, these families with microhematuria leading to ESKD were successfully linked to heterozygous COL4A3/A4 mutations such as G1334E on COL4A3, G871C on COL4A3, 3533delC on COL4A3 and 3854delG on COL4A414,16. Currently, 208 microhematuric patients in 22 Cypriot families are heterozygous for one of these four mutations. All carriers start with microhematuria in childhood and in all 22 families some carriers have subsequently developed proteinuria, hypertension and renal failure in their 40’s, 50’s and 60’s. Renal biopsies in 24 patients with proteinuria and renal insufficiency have all shown various stages of FSGS on top of TBMN observed in 22 biopsies where EM was available. There were no ocular ATS defects and late onset neurosensory deafness was present in only seven patients. Thus TBMN resulting from heterozygous COL4A3/A4 mutations has proven to be the commonest cause of FMH.

Figure 3: Microhematuric family CY5301 (modified from16), with 67 studied family members and 31 DNA proven heterozygous carriers of mutation COL4A3 G1334E, all showing TBMN and microhematuria. There is, however, some phenotypic variation with some mutation carriers showing persistent pure microscopic hematuria up to the end of their lives, while some other mutation carriers develop additional proteinuria, hypertension, CRF and ESKD. COL4A3/A4 heterozygous mutations are the commonest cause of familial microscopic hematuria and TBMN in Cyprus.

The second commonest cause of FMH appears to be the newly discovered familial C3/CFHR5 nephropathy that results from a novel, exon 2-3 duplication mutation on the CFHR5 gene in the alternative complement pathway. This autosomal dominant disease is present in 21 large Cypriot families with currently 136 living affected members. The histopathology is pure mesangial C3 nephropathy with similar C3 deposits along the GBM too, in the absence of any immunoglobulin deposits18. The third cause of FMH appears to be the Alport syndrome, which appears rare on the island. Only X-linked, COL4A5 mutation, P628L has been detected so far in two families with eight affected hemizygous males. This hypomorphic COL4A5, P628L mutation is of great interest because of its benign phenotype, as two adult males show only microhematuria as a result of TBMN. Autosomal recessive ATS is also unusual with only 5 COL4A3/A4 homozygous or compound heterozygous patients recognised since 1984. The fourth cause of FMH refers to the female heterozygous carriers of X-linked COL4A5 ATS with nine such female patients identified in the two Cypriot families with the P628L mutation.

Discussion

Nephrologists and physicians in general, confronted with a family in whom some male and female members, exhibit microhematuria, without ocular complications or neurosensory deafness, may not immediately think of X-linked ATS, even if a male subsequently develops late onset renal insufficiency. According to classical teaching, the idea of X-linked ATS becomes even more remote, if the renal biopsy in such an adult with microhematuria ± mild renal insufficiency shows thinning of the basement membranes. The main aim of this review study is to help change this old rigid approach and make nephrologists worldwide realize that some COL4A5 missense and mild mutations are hypomorphic and consequently they may be responsible for TBMN with benign microscopic hematuria and occasional late onset kidney failure. The eight large families described in this paper, the six families described by Slajpah11 and the other families described by other investigators throughout the world6-10,12 (Table 2)should make the nephrology community more aware of this entity, helping many such patients and their families reach the correct diagnosis early in order to receive proper medical care.  Following a recent correlation analysis in 206 ATS patients from eminent Dr Flinter’s laboratory at Guy’s Hospital, London, UK19, the recommendation was made that COL4A5 molecular studies should be requested if the patient meets at least two out of the four classical Alport diagnostic criteria. These four criteria are: a) positive family history of CRF and/or micro/macrohematuria, b) EM changes of ATS on renal biopsy, c) characteristic ocular signs and d) neurosensory deafness.  According to the study, the mutation detection rates were 18% for one criterion, 64% for two criteria, 89% for three and 81% for all four. This recommendation may indeed be valid and financially sound because conventional COL4A5 mutation screening has relatively high costs, is time-consuming and may require several weeks to complete. On the other hand, such an approach is conservative and may be the explanation why some X-linked COL4A5, ATS patients with a mild phenotype are so slow to diagnose, most probably because they are not considered as likely X-linked ATS candidates and therefore not referred early for molecular COL4A5 genetic analysis. The end result is that such families may remain undiagnosed for long periods of time. The advent of next-generation sequencing (NGS) protocols enabling easier and fast detection of all possible variants in all three COL4A3/A4/A5 genes is indeed a promising development that may help overcome this problem20. It is of interest that unlike classical ATS with early onset ESKD, the patients in these families fulfill only one of the four classical ATS criteria and are characterized by late onset CRF or ESKD in their affected males. The mutations responsible for such milder phenotypes could be viewed as hypomorphic in the sense that the collagen IV molecules maintain some residual activity and the hemizygous males are not so severely affected as similar hemizygous males due to other more serious COL4A5, ATS mutations. As a result, the mildness of the phenotype may mislead the clinician who will diagnose TBMN rather than X-linked Alport. These scenarios delay family concerns and therefore allow for bigger families with geographical clustering.

The concept of hypomorphic mutations after the 1946 Nobel Prize winner Hermann J. Muller (1890-1967) has indeed helped us to understand the variable and milder phenotype of ATS and also that of other inherited nephrological diseases like polycystic kidney disease21,22. Another probable explanation for the variable phenotypes of X-linked ATS might be the occurrence of somatic mosaicism for de novo COL4A5 mutations in sporadic ATS cases, when the mutation occurs as a somatic event post-conception and with unequal representation of the cells carrying the mutation in the various tissues and organs23,24. An interesting case of a patient with such somatic and germinal mosaicism was described for another collagen gene, COL1A1, in a patient with osteogenesis imperfect25.

Persistent isolated microscopic hematuria in the first two decades of life is a urinary finding that unfortunately does not always attract the serious attention it should, including careful family studies for an underlying hereditary nephritis. Unlike proteinuria, microscopic hematuria does not often lead to an early renal biopsy which if and when attempted, should always include good EM studies26. The main reason for not routinely carrying out renal biopsies in these young, pure microhematuric patients, is possibly the fact that pediatric nephrologists who usually see these patients first, do not often see the long term complications in these patients that usually begin in their late 30’s, 40’s and 50’s and therefore ascribe a benign nature to these microhematuric families. Indeed, as we and others have shown, several decades may elapse before additional pathological changes such as proteinuria, hypertension and renal insufficiency may develop. However, recent long term epidemiological follow up studies in Israel have confirmed that isolated microscopic hematuria in persons 16-25, was associated with a significantly increased risk of ESKD over a period of 22 years and hereditary nephritis was a major aetiological factor in these Jewish hematuric families in agreement with our studies in Cyprus27.

Conflict of Interest

None to declare.

Acknowledgments

We wish to sincerely thank all patients and relatives and the many clinicians in Greece and Cyprus who participated in this work. In particular we wish to thank Professors D. Goumenos, Th. Diamantopoulos and P. Goudas from Patras, Prof C Sombolos from Thessaloniki and Prof P Ziroyiannis in Athens, Greece for referring to us microhematuric families. Late Prof E Alexopoulos, in Thessaloniki was always a close collaborator. This research project was funded from grants from the Cyprus Research Promotion Foundation programs ΠENEK/ENIΣX/0308/08 and NEW INFRASTRUCTURE/STRATEGIC/0308/24 (co-funding by the EU Structural Funds), to CD.

References

1. Bekheirnia MR, Reed B, Gregory MC, McFann K, Shamshirsaz AA, Masoumi A, et al. Genotype-phenotype correlation in X-linked Alport syndrome. J Am Soc Nephrol. 2010; 21: 876-883.
2. Gross O, Netzer KO, Lambrecht R, Seibold S ,Weber M. Meta-analysis of genotype-phenotype correlation in X-linked Alport syndrome: impact on clinical counselling. Nephrol Dial Transplant. 2002; 17: 1218-1227.
3. Jais JP, Knebelmann B, Giatras I, De Marchi M, Rizzoni G, Renieri A, et al. X-linked Alport syndrome: natural history and genotype-phenotype correlations in girls and women belonging to 195 families: a “European Community Alport Syndrome Concerted Action” study. J Am Soc Nephrol. 2003; 14: 2603-2610.
4. Tan R, Colville D, Wang YY, Rigby L ,Savige J. Alport retinopathy results from “severe” COL4A5 mutations and predicts early renal failure. Clin J Am Soc Nephrol. 2010; 5: 34-38.
5. ALPORT (COL4A5) Database, Mutation Databases, ARUP Scientific Resource for Research and Education, University of Utah, available at: http://www.arup.utah.edu/database/ALPORT/ALPORT_display.php, accessed: May 2012.
6. Pont-Kingdon G, Sumner K, Gedge F, Miller C, Denison J, Gregory M, et al. Molecular testing for adult type Alport syndrome. BMC Nephrol. 2009; 10: 38.
7. Barker DF, Pruchno CJ, Jiang X, Atkin CL, Stone EM, Denison JC, et al. A mutation causing Alport syndrome with tardive hearing loss is common in the western United States. Am J Hum Genet. 1996; 58: 1157-1165.
8. Chen Y, Zhu S ,Zhang Y. [Thin basement membrane nephropathy: a mutation in COL4A5 gene]. Zhonghua Nei Ke Za Zhi. 2001; 40: 239-242.
9. Kaneko K, Tanaka S, Hasui M, Nozu K, Krol RP, Iijima K, et al. A family with X-linked benign familial hematuria. Pediatr Nephrol. 2010; 25: 545-548.
10. Martin P, Heiskari N, Zhou J, Leinonen A, Tumelius T, Hertz JM, et al. High mutation detection rate in the COL4A5 collagen gene in suspected Alport syndrome using PCR and direct DNA sequencing. J Am Soc Nephrol. 1998; 9: 2291-2301.
11. Slajpah M, Gorinsek B, Berginc G, Vizjak A, Ferluga D, Hvala A, et al. Sixteen novel mutations identified in COL4A3, COL4A4, and COL4A5 genes in Slovenian families with Alport syndrome and benign familial hematuria. Kidney Int. 2007; 71: 1287-1295.
12. Wilson JC, Yoon HS, Walker RJ, Eccles MR. A novel Cys1638Tyr NC1 domain substitution in alpha5(IV) collagen causes Alport syndrome with late onset renal failure without hearing loss or eye abnormalities. Nephrol Dial Transplant. 2007; 22: 1338-1346.
13. Demosthenous P, Voskarides K, Stylianou K, Hadjigavriel M, Arsali M, Patsias C, et al. X-linked Alport syndrome in Hellenic families: phenotypic heterogeneity and mutations near interruptions of the collagen domain in COL4A5. Clin Genet. 2012; 81: 240-248.
14. Pierides A, Voskarides K, Athanasiou Y, Ioannou K, Damianou L, Arsali M, et al. Clinico-pathological correlations in 127 patients in 11 large pedigrees, segregating one of three heterozygous mutations in the COL4A3/ COL4A4 genes associated with familial haematuria and significant late progression to proteinuria and chronic kidney disease from focal segmental glomerulosclerosis. Nephrol Dial Transplant. 2009; 24: 2721-2729.
15. Temme J, Peters F, Lange K, Pirson Y, Heidet L, Torra R, et al. Incidence of renal failure and nephroprotection by RAAS inhibition in heterozygous carriers of X-chromosomal and autosomal recessive Alport mutations. Kidney Int. 2012; 81: 779-783.
16. Voskarides K, Damianou L, Neocleous V, Zouvani I, Christodoulidou S, Hadjiconstantinou V, et al. COL4A3/COL4A4 mutations producing focal segmental glomerulosclerosis and renal failure in thin basement membrane nephropathy. J Am Soc Nephrol. 2007; 18: 3004-3016.
17. Deltas C, Voskarides K, Demosthenous P, Papazachariou L, Zirogiannis P, Pierides A. The power of molecular genetics in establishing the diagnosis and offering prenatal testing: The case for Alport Syndrome. Manisha Sahay (eds), Diseases of Renal Parenchyma, (open access book), In Tech publishing, 2012, 1-13.
18. Athanasiou Y, Voskarides K, Gale DP, Damianou L, Patsias C, Zavros M, et al. Familial C3 glomerulopathy associated with CFHR5 mutations: clinical characteristics of 91 patients in 16 pedigrees. Clin J Am Soc Nephrol. 2011; 6: 1436-1446.
19. Hanson H, Storey H, Pagan J, Flinter F. The value of clinical criteria in identifying patients with X-linked Alport syndrome. Clin J Am Soc Nephrol. 2011; 6: 198-203.
20. Artuso R, Fallerini C, Dosa L, Scionti F, Clementi M, Garosi G, et al. Advances in Alport syndrome diagnosis using next-generation sequencing. Eur J Hum Genet. 2012; 20: 50-57.
21. Rossetti S, Kubly VJ, Consugar MB, Hopp K, Roy S, Horsley SW, et al. Incompletely penetrant PKD1 alleles suggest a role for gene dosage in cyst initiation in polycystic kidney disease. Kidney Int. 2009; 75: 848-855.
22. Harris PC, Rossetti S. Determinants of renal disease variability in ADPKD. Adv Chronic Kidney Dis. 2010; 17: 131-139.
23. Krol RP, Nozu K, Nakanishi K, Iijima K, Takeshima Y, Fu XJ, et al. Somatic mosaicism for a mutation of the COL4A5 gene is a cause of mild phenotype male Alport syndrome. Nephrol Dial Transplant. 2008; 23: 2525-2530.
24. Plant KE, Boye E, Green PM, Vetrie D, Flinter FA. Somatic mosaicism associated with a mild Alport syndrome phenotype. J Med Genet. 2000; 37: 238-239.
25. Constantinou-Deltas CD, Ladda RL, Prockop DJ. Somatic cell mosaicism: another source of phenotypic heterogeneity in nuclear families with osteogenesis imperfecta. Am J Med Genet. 1993; 45: 246-251.
26. Deltas C, Pierides A, Voskarides K. The role of molecular genetics in diagnosing familial hematuria(s). Pediatr Nephrol. 2012; 27: 1221-1231.
27. Vivante A, Afek A, Frenkel-Nir Y, Tzur D, Farfel A, Golan E, et al. Persistent asymptomatic isolated microscopic hematuria in Israeli adolescents and young adults and risk for end-stage renal disease. JAMA. 2011; 306: 729-736.