Linkage Analysis: An Important Tool for Pre-Clinical Alport's Diagnosis

Milco Ciccarese; Giancarlo Tonolo; Rossana Faedda; Maria Cossu; Michelina Spanu; Holger Luthman; Andrea Satta

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ORIGINAL ARTICLE

Year : 2002 | Volume : 13 | Issue : 4 | Page : 462-466

Linkage Analysis: An Important Tool for Pre-Clinical Alport's Diagnosis

Milco Ciccarese¹, Giancarlo Tonolo², Rossana Faedda², Maria Cossu³, Michelina Spanu², Holger Luthman⁴, Andrea Satta²
¹ Department of Molecular Medicine & CMM, Karolinska Institute, Stockholm, Sweden; Dipartimento Struttura Clinica Medica-Patologia Medica, University of Sassari, Sassari, Italy
² Dipartimento Struttura Clinica Medica-Patologia Medica, University of Sassari, Sassari, Italy
³ Servizio di Nefrologia, Ospedale Civile di Sassari, Sassari, Italy
⁴ Department of Molecular Medicine & CMM, Karolinska Institute, Stockholm, Sweden

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Abstract

The phenotypic heterogeneity of Alport's syndrome (AS) is well explained by the genetic heterogeneity. Therefore, the application of genetic techniques, such as linkage analysis (LA), could be helpful in the correct diagnosis of this disease. We performed LA in a family who manifested X-linked inheritance. We used 10 fluorescent microsatellite markers to cover the q-arms of chromosome X. The poly chain reaction (PCR) products were separated on an ABI 377 Sequencer and genotypes were identified using the ABI Genescan/Genotyper software. We confirm in this family an X-linked dominant transmission of AS. All affected individuals shared the same haplotype for the region Xq22. The genetic diagnosis of AS was confirmed in individuals aged one month and 4 years before any clinical manifestation of AS. We conclude that LA is a powerful and approachable tool, which could be used in the diagnosis of AS. An accurate genetic family study using LA may be helpful for correct classification, genetic counseling, prognosis evaluation, the assessment of the risk for kidney transplantation, and for the follow-up and therapy of AS.

Keywords: Collagen, Hematuria, Linkage analysis, Diagnosis.

How to cite this article:
Ciccarese M, Tonolo G, Faedda R, Cossu M, Spanu M, Luthman H, Satta A. Linkage Analysis: An Important Tool for Pre-Clinical Alport's Diagnosis. Saudi J Kidney Dis Transpl 2002;13:462-6

How to cite this URL:
Ciccarese M, Tonolo G, Faedda R, Cossu M, Spanu M, Luthman H, Satta A. Linkage Analysis: An Important Tool for Pre-Clinical Alport's Diagnosis. Saudi J Kidney Dis Transpl [serial online] 2002 [cited 2022 Jul 6];13:462-6. Available from: https://www.sjkdt.org/text.asp?2002/13/4/462/33099

Introduction

Alport's syndrome (AS) is a hereditary nephropathy characterized by persistent hematuria that may evolve to end-stage renal failure (ESRF). This syndrome is associated with sensorineural hearing loss, ocular abnormalities, and ultrastructural abnormalities of the glomerular basement membrane (GBM).^[1] AS shows a wide range of phenotypic expression, due to the genetic heterogeneity of the disease. In fact, in addition to the well known X-linked form, due to mutations in the COL4A5/A6,^[2] approximately 15% of all AS segregate as an autosomal trait. Mutations in COL4A3 and COL4A4 have been described in families with autosomal recessive^{[3],[4],[5],[6]} and dominant AS.^[7],[8]

The diagnosis of AS is difficult due to variability in the expression of the disease phenotype,^[9] and the high number of mutations discovered in the COL4A3/4 and COL4A5/6 genes.^[10]

In the last few years, the advance in the molecular genetics and its technology has increased our knowledge of several diseases, including AS. This resulted in better understanding of the phenotypic and genetic complexity of this disease. Moreover, the application of these genetic technologies, as linkage analysis (LA) could be helpful in the correct diagnosis of AS.

In this study, we describe a family with Xlinked AS and the use of an approachable genetic tool, such as LA, to identify affected persons before any clinical manifestation of the disease.

Materials and Methods

Selection criteria

To select families for AS, we used clinical criteria as described elsewhere.^[1] Each participating individual had a physical examination, a hearing test, and a complete screening of kidney functions. Persons were considered affected if microscopic hematuria was found on at least three occasions after exclusion of urinary infection. All subjects gave informed consent to participate in the study.

Patients and family members

The clinical characteristics of all affected members of the family in [Figure - 1] are shown in [Table - 1]. The proband (II: 1) presented with hematuria at 8 years of age. The diagnosis of AS was confirmed by renal biopsy; hemodialysis was initiated at 28 years of age, and she received a kidney graft seven years later. She died at 49 years of age from myocardial infarction. The individual I: 1 had a documented history of renal disease prior to his death at an age of 38 years from suspected myocardial infarction.

Venous blood for LA from all individuals was collected 5 years ago. At that time, the individual IV: 1 was one month old. After 4 years of follow-up, he presented with hematuria and proteinuria, with normal renal function.

Screening of the other family members (I: 2, II: 2, II: 4, II: 6, II: 7, II: 8, II: 9, II: 10, III: 3, III: 5, III: 10-13) was negative for hematuria and/or renal disease. Audiograms were normal in all individuals included in the study, and none of the family members showed evidence of eye signs or leiomyomatosis.

Linkage analysis(LA)

Genomic DNA was purified from peripheral lymphocytes and 5 ng was poly chain reaction (PCR) amplified as described in a previous report.^[7] We used 10 fluorescent microsatellite markers: (DXS7132-DXS6800DXS6789-DXS6799-DXS6797-DXS6804DXS1001-DXS1047-GATA3108-DXS1193), elected from published maps^[11] to make a dense map of the COL4A5/A6 region. The PCR products were separated on an ABI 377 Sequencer and genotypes were identified using the ABI Genescan/Genotyper software (Perkin Elmer, CA, USA).

Results

The results of the genetic analysis are shown in [Figure - 1]. We confirmed in this family an X-linked dominant transmission of AS. Moreover, recombination events in individuals III: 4, III: 6, and III: 7 allow localization of COL4A5 and COL4A6 genes between markers DXS 6800 and DXS 6804.

All affected individuals share the same haplotype for this region. In particular, AS was genetically present in individual IV: 1 five years ago, when the boy was one month old and before the development of the characteristic Alport's clinical signs.

At the same time, linkage to the other basement-type (type 4) collagen genes was excluded: COL4A1 and COL4A2 on chromosome 13q33-q34, and COL4A4/COL4A3 on chromosome 2q35-37 (data not shown).

Discussion

LA is a powerful genetic method world wide used in genome screen or in candidate gene test. The aim of this study was to evaluate the possible use of LA as an easy tool for early diagnosis of AS, in the absence of clear phenotypic manifestation of the disease, and without the use of invasive procedures as skin or renal biopsy. By using this genetic technique, we were able to identify an affected member at an early age postnatally (one month of age and four years before any clinical expression of the disease.

Alternative techniques are suitable for AS diagnosis but they all have their limitations. Such techniques include (i) immunohistochemical analysis of epidermal basement membrane (EBM) could be used in AS diagnosis, although this technique can identify COL4A5 defects in no more than 50-60% of the patients with X-linked AS;^[12] (ii) renal biopsy can be useful in AS diagnosis, but in 20% of the patients, normal staining, reported with AS, does not exclude the disease.[10] Moreover, age, advanced renal insufficiency, or coagulation defects could be contraindications for renal biopsy; (iii) sequence of collagen genes is a powerful and conclusive method that could be used in the diagnosis of AS, but is quite expensive, and it is limited by the number of exons to be screened (more than 50 for each collagen gene). Moreover, until now more than 180 mutations^{[2],[3],[4],[5],[6],[8]} have been described in COL4 A5/6 and COL4 A3/4 genes, and few of them have been recorded more than once in different families, making the search for the putative mutation quite difficult. The same applies for using alternative genetic technique, as SSCP.

LA is a powerful and approachable tool that could be used in the diagnosis of AS, solving all problems due to the phenotypic and genetic heterogeneity of this disease. In fact, as shown also by our results, the clarification of the mode of transmission,^[13] is potentially useful in the identification of asymptomatic carriers and the pre-^[14] and post-natal diagnosis of AS. This could have relevant clinical consequences in the classification, the genetic counseling, prognosis evaluation and in the assessment of the risk for kidney transplantation.

Moreover, there are interesting new advances in AS therapy. A significant decrease or disappearance of proteinuria has been obtained using cyclosporin A in eight AS patients.^[15] Angiotensin converting enzyme inhibitor treatment significantly delayed the decline of glomerular filtration rate, and signifycantly slowed the rate of increase of proteinuria in animal models.^[16] Experimental success was obtained using gene therapy in vivo the kidney perfusion system.^[17]

For these reasons, we believe that a genetic approach such as LA may be used in families with suspected AS aiming to identify the affected individuals. This could be beneficial in the application of different strategies of therapy.

References

1.	Flinter FA, Cameron JS, Chantler C, Houston I, Bobrow M. Genetics of classic Alport's syndrome. Lancet 1988;2(8618):1005-7.
2.	Lemmink HH, Schroder CH, Monnens LA, Smeets HJ. The clinical spectrum of type IV collagen mutations. Hum Mutat 1997; 9(6):477-99.
3.	Mochizuki T, Lemmink HH, Mariyama M, et al. Identification of mutations in the alpha 3(IV) and alpha 4(IV) collagen genes in autosomal recessive Alport syndrome. Nat Genet 1994;8(1):77-81.
4.	Lemmink HH, Mochizuki T, van den Heuvel LP, et al. Mutations in the type IV collagen alpha 3(COL4A3) gene in autosomal recessive Alport syndrome. Hum Mol Genet 1994;3(8): 1269-73.
5.	Boye E, Mollet G, Forestier L, et al. Determination of the genomic structure of the COLAA4 gene and of novel mutations causing autosomal recessive Alport syndrome. Am J Hum Genet 1998;63(5):1329-40.
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7.	Ciccarese M, Casu D, Ki-Wond F, et al. Identification of a new mutation in the alpha 4 (IV) collagen gene in a family with autosomal dominant alport syndrome and hypercholesterolemia. Nephrol Dial Transplant 2001;16:2008-12.
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9.	Turco AE, Renieri A, De Marchi M. Alport syndrome is there a genenotype-phenotype relationship? Nephrol Dial Transplant 1997;12(8):1551-3.
10.	Pirson Y. Making the diagnosis of alport's syndrome. Kidney Int 1999;56(2):760-75.
11.	Murray JC, Buetow KH, Weber JL, et al. A comprehensive human linkage map with centimorgan density. Cooperative Human Linkage Center (CHLC). Science 1994;265 (5181):2049-54.
12.	van der Loop FT, Monnens LA, Schroder CH, et al. Identification of COLAA5 defects in Alport's syndrome by immunohistochemistry of skin. Kidney Int 1999;55(4): 1217-24.
13.	Turco AE, Rossetti S, Bresin E, Corra S. Erroneous genetic risk assessment of alport syndrome (letter). Lancet 1995:346(8984): 1237.
14.	Turco AE, Bresin E, Rossetti S, Peterlin B, Morandi R, Pignatti PF. Rapid DNA-based prenatal diagnosis by genetic linkage in three families with alport's syndrome. Am J Kidney Dis 1997;30(2):174-9.
15.	Callis L, Vila A, Carrera M, Nieto J. Longterm effects of cyclosporine A in alport's syndrome. Kidney Int 1999;55(3):1051-6.
16.	Grodecki KM, Gains MJ, Baumal R, et al. Treatment of X-linked hereditary nephritis in Samoyed dogs with angiotensin converting enzyme (ACE) inhibitor. J Comp Pathol 1997;117(3):209-25.
17.	Heikkila P, Parpala T, Lukkarinen O, Weber M, Tryggvason K. Adenovirusmediated gene transfer into kidney glomeruli using an ex vivo and in vivo kidney perfusion system-first steps towards gene therapy of alport syndrome. Gene Ther 1996;3(1):21-7.

Correspondence Address:
Milco Ciccarese
Dipartimento Struttura Clinica, Medica-Patologia Medica, University of Sassari, V. le S. Pietro 8, 07100, Sassari, Italy