Usefulness of combined genetic data in Hungarian families affected by autosomal dominant polycystic kidney disease

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Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common hereditary diseases. Mutations of two known genetic loci (PKD1: 16p13.3 and PKD2: 4q21.2) can lead to bilateral renal cysts. The PKD1 locus is the more common (∼85%), with a more severe phenotype. Because of the genetic complexity of ADPKD and the size and complexity of the PKD1 gene, pedigree-based linkage analysis is a useful tool for the genetic diagnosis in families with more than one subject affected. We tested linkage or non-linkage to the closely linked DNA markers flanking the PKD1 (D16S663 and D16S291) and one intragenic D16S3252 and PKD2 (D4S1563 and D4S2462) in 30 ADPKD-affected families, to determine the distributions of alleles and the degree of microsatellite polymorphisms (in 91 patients and 125 healthy subjects). To characterize the markers, used heterozygosity levels, polymorphism information content and LOD scores were calculated. The D16S663 marker included 12 kinds of alleles, while D16S291 had 10 alleles and D16S3252 had 8. D4S1563 had 12 alleles and D4S2462 had 11. In a search for a common ancestral relationship, we considered the patients’ alleles with the same repeat number. Only one haplotype was detected in more than one (2) unrelated families. The calculated two-point LOD scores indicated a linkage to PKD1 in 22 families (74%). In four families (13%) with a linkage to PKD2, the patients reached the end-stage renal disease after the age of 65 years. One family was linked to neither gene (3%), and in three families (10%) a linkage to both genes was possible. In the latter three families, the numbers of analyzed subjects were small (4–5), and/or some markers were only partially or non-informative. However, the elderly affected family members exhibited the clinical signs of the PKD1 form in these cases. The new Hungarian population genetic information was compared with available data on other populations.

Introduction

Polycystic kidney disease (PKD) is a hereditary (genetic) disorder in which many fluid-filled growing cysts form in the nephrons of the kidneys. These cysts can become so large and so numerous that they crowd out the normal kidney tissue, rendering the kidneys unable to function normally. The more common, autosomal dominant (AD) form of PKD (ADPKD) is age-dependent, and the cysts usually develop slowly in both number and size. The blood pressure becomes elevated early in the disease, often before the cysts appear. Cysts can also form in other organs, such as the liver (in approximately one-third of those affected), but these cysts do not affect the liver function. Fewer than 10% of the individuals have dilated cerebral blood vessels (aneurysms) or cardiac valvular abnormalities. The dilated blood vessels usually cause headaches when they expand. Many of these brain aneurysms bleed and cause strokes [1].

ADPKD is the fourth leading cause of kidney failure. Its worldwide prevalence is between 1 in 800 and 1 in 1000 people [2], irrespective of gender, race or ethnic group. Persons with ADPKD often exhibit no symptoms and in its early stages the disease can not be diagnosed by routine blood examinations. Many of those with ADPKD live for decades without developing symptoms. In contrast, in some cases cysts may form even in the very early years of life. The disease can present as early as in utero, whereas some cases may never progress to renal failure [3]. The symptoms are at times so mild that those with the disease will live their whole life without ever knowing that they have the disorder. Approximately 50% of PKD patients will eventually require renal dialysis to compensate for their reduced kidney function (end-stage renal disease, ESRD). Of the two main forms, PKD1 is the more severe disorder, with an average age at ESRD of ∼56 years, as compared with ∼71.5 years for PKD2 [4]. The most common symptoms besides the enlargement of the kidneys due to the formation of innumerable fluid-filled cysts are urinary tract infections, hematuria, high blood pressure and kidney stones. The diagnosis of ADPKD typically involves the observation of three or more kidney cysts through the use of ultrasonographic imaging, computerized tomography scans or magnetic resonance imaging. The diagnosis is strengthened by a family history of ADPKD. The chance of acquiring a dominant disease is higher than the chance of acquiring a recessive disease. In certain rare cases, ADPKD occurs spontaneously in the child soon after conception; in these cases, the parents are not the source of the disease. Genetic linkage studies have revealed at least three forms of ADPKD [3]. Mutations of two known genetic loci (16p13.3 and 4q21.2) can lead to bilateral renal cysts. The 16p13.3-linked PKD1 locus is the more common (∼85%), with a more severe phenotype. 4q21.2 is linked with PKD2. The proteins produced by these two genes are polycystin-1 and polycystin-2 [5]. It has been theorized that a PKD3 gene exists, but it has not been mapped or identified [6]. The mutation screening for the more common PKD1 has proved difficult, due to its size (12,906 bp coding sequence) and complexity (the 5′ region of the gene from upstream of exon 1 to exon 33 is repeated more than four times on the same chromosome) [7]. The PKD2 gene consists of 15 exons in the 4q21.2 region; it encodes a 5.4 kb transcript; polycystin-2 consists of 968 amino acids. A mutation in either of the genes can lead to cyst formation, but the evidence suggests that the disease development requires other factors in addition to such a mutation. Because of the genetic complexity of ADPKD and the size and complexity of the PKD1 gene, pedigree-based linkage analysis is a useful tool for the genetic diagnosis in families with more than one subject affected. The linked marker analysis test can detect the presence of the ADPKD mutations before cysts develop, though its usefulness is limited by two factors: it cannot predict the onset or the ultimate severity of the disease. However, a young person with a known presymptomatic diagnosis of ADPKD may be able to forestall the disease through the diet and blood pressure control. Family testing for evidence of linkage or non-linkage to the closely linked DNA markers flanking the PKD1 (e.g. D16S663 and D16S291 and the intragenic D16S3252) and PKD2 (D4S1563 and D4S2462) can offer new population genetic information.

Our present aim was to determine the distributions of some PKD1 and PKD2 alleles and the degree of microsatellite polymorphisms with pedigree-based linkage analysis in ADPKD-affected Hungarian families. The markers were characterized by the level of heterozygosity (HET) and polymorphism information content (PIC); LOD scores were also calculated. We wished to determine the usefulness of combined genetic data for demonstration of the mutated gene segregation in the affected families.

Section snippets

Subjects

Linkage analysis was performed on 216 members of 30 unrelated Hungarian PKD1- or PKD2-affected families (patients: 91; healthy subjects: 125). The diagnosis of ADPKD was based on the clinical history, the symptoms, the complications and the ultrasonographic finding of the presence of several renal cysts distributed in both kidneys. Each family had at least two affected and 2–3 unaffected members, although most families were considerably larger. The study was approved by the Medical Ethics

Results

The percentage frequencies of numbers of dinucleotide repeats of D16S663, D16S291 and D16S3252 and of D4S1563 and D4S2462 markers for the 30 ADPKD-affected Hungarian families are to be seen in Table 1, Table 2, Table 3. The HET contents and PIC values of the microsatellites for normal and mutated alleles are shown in Table 4.

The D16S663 (Cw2) marker included 12 kinds of alleles, while D16S291 had 10 alleles and D16S3252 (KG8) had 8 alleles. D4S1563 had 12 alleles and D4S2462 had 11 alleles.

The

Discussion

ADPKD can easily be diagnosed by sonographic examination, but the possibility of a presymptomatic clinical diagnosis is limited; molecular diagnosis is highly desirable. The genetic analysis can be performed by two different approaches: direct investigation of the disease-causing mutation, or indirect diagnosis by linkage analysis using linked DNA markers. Detection of mutation in the PKD1 gene is complicated by the fact that this is a very large gene with all but 3.5 kb repeated proximally on

Acknowledgments

This work was supported by OTKA grants T 034391 and M/036851. The authors thank Dr. Ildikó Wellinger for skillful technical help.

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