Loss of heterozygosity in polycystic kidney disease with a missense mutation in the repeated region of PKD1

Abstract

Loss of heterozygosity (LOH) is a molecular phenomenon that denotes the loss of one of the two alleles at a specific locus. It is frequently associated with tumour suppressor genes in various cancers and also with hyperproliferative disorders, although not exclusively. Interestingly, in conditions where there is an inherited germline mutation, the lost allele is always the functional one, thereby rendering a phenotypically dominant disease of recessive character at the cellular level. A disease more recently shown to be associated with LOH is polycystic kidney disease type 1, a systemic disorder characterized by significant pleiotropy. The main pathology is from renal cyst formation that eventually leads to end-stage renal failure during adult life. We describe the identification of a missense mutation in the repeated part of the PKD1 gene, exon 31, that substitutes valine for methionine. The mutation, M3375V, cosegregates with the disease phenotype in a large Cypriot family. During transplantation of one patient, one of the polycystic kidneys was removed and DNA was isolated from cystic epithelial cells. In 3 of 17 cysts examined with intragenic and flanking polymorphic markers on chromosome 16 we detected LOH, since the wild-type allele was lost, thereby rendering the affected kidneys of mosaic character. The degree of LOH was extensive and varied among the three cysts, supporting the multiplicity of expression of the phenomenon on different occasions. No LOH was detected for other selected loci examined. Our work further supports the hypothesis that the rate-limiting step in cyst formation may be the occurrence of a second somatic hit, although other factors may be also involved. The high frequency of mutations at this locus may, to a great extent, explain the variability in phenotype observed among patients in the same families, and the relatively high frequency of the disease worldwide.

Loss of heterozygosity in renal and hepatic epithelial cystic cells from ADPKD1 patients

“…Evidence of loss of heterozygosity (LOH) at the PKD1 and PKD2 loci in cystic epithelia has been reported. [6][7][8][9][10][11][12] suggesting that cytogenesis in ADPKD results from the inactivation of the normal copy of the gene by a second somatic mutation. On the other hand, there is also evidence that the majority of cysts show strong immunoreactivity with antibodies directed against both polycystins.…”

Novel PKD1 deletions and missense variants in a cohort of Hellenic polycystic kidney disease families

“…30 The LR-PCR encompassing exons 23 ± 34 was performed according to the conditions described. 15 Briefly, PCR buffer consisted of 20 mM Tris.HCl pH 8.75, 16 mM (NH4) 2 SO 4 , bovine serum albumin 150 mg/ml, 8% glycerol, 2.5 mM MgCl 2 and 100 mM each dNTP. DMSO to a final concentration of 7% and 1.5 ml of 2 M Trizma-base were also included in the 100 ml PCR reaction mix.…”

“…SSCP analysis, DNA cloning and sequencing Exon-by-exon screening for mutations was performed on the LR-PCR products by SSCP analysis. 15,31 Amplification was performed with TaqExpress (GenPak, Brighton, UK). LR-PCR products were diluted 1 : 1000 and 1 ml of this was used as a template for all subsequent PCR reactions.…”

“…This dilution was sufficient to exclude detectable genomic carry-over contamination, since amplification of exons 43 ± 44 with primer pair 3A3C1/E44R failed. 15 Sequences of primers and conditions for PCR amplification of the region covering exons 16 ± 21, are shown in Table 1. Primers used for LR-PCR and for PCR-SSCP analysis of sequences in exons 23 ± 46 were published previously.…”

Mutations of the PKD1 gene among Japanese autosomal dominant polycystic kidney disease patients, including one heterozygous mutation identified in members of the same family

“…PCR was performed according to the method of Peral et al (1996) with slight modifications. Long-range PCR (Expand Long Template PCR system, Roche, Mannheim, Germany) was performed by using primer set E23F4 (nt 38145-38165) (Koptides et al 1998) in exon 23 and primer set E34R1 (nt 44403-44422) (Watnick et al 1997) in exon 34. PCR was performed according to the manufacturer's buffer system 3 method with slight modifications.…”