Evidence of a third ADPKD locus is not supported by re-analysis of designated PKD3 families

Abstract

Mutations to PKD1 and PKD2 are associated with autosomal dominant polycystic kidney disease (ADPKD). The absence of apparent PKD1/PKD2 linkage in five published European or North American families with ADPKD suggested a third locus, designated PKD3. Here we re-evaluated these families by updating clinical information, re-sampling where possible, and mutation screening for PKD1/PKD2. In the French-Canadian family, we identified PKD1: p.D3782_V3783insD, with misdiagnoses in two individuals and sample contamination explaining the lack of linkage. In the Portuguese family, PKD1: p.G3818A segregated with the disease in 10 individuals in three generations with likely misdiagnosis in one individual, sample contamination, and use of distant microsatellite markers explaining the linkage discrepancy. The mutation PKD2: c.213delC was found in the Bulgarian family, with linkage failure attributed to false positive diagnoses in two individuals. An affected son, but not the mother, in the Italian family had the nonsense mutation PKD1: p.R4228X, which appeared de novo in the son, with simple cysts probably explaining the mother's phenotype. No likely mutation was found in the Spanish family, but the phenotype was atypical with kidney atrophy in one case. Thus, re-analysis does not support the existence of a PKD3 in ADPKD. False positive diagnoses by ultrasound in all resolved families shows the value of mutation screening, but not linkage, to understand families with discrepant data.

Figure 1. The French-Canadian Family

A: Map of the short arm of chromosome 16 from telomere to centromere with the genetic markers(49) analyzed in the original reports (black) and those employed here (blue) indicated. The location of PKD1 is shown in red. Green scale bar shows the genomic distance. B: Top panel shows the wildtype chromatogram and bottom panel shows the c.11347_11348insACG (p.D3782_V3783insD) variant identified in affected family members. C: Multiple sequence alignment of PC-1 orthologs showing the position of insertion of the aspartic acid residue. D: Revised pedigree and haplotype data of the French-Canadian family. II-4, who was not studied in the original report, was diagnosed as affected and also found to carry the mutation. III-4 and III-5, who were originally described as affected but shown as unaffected with a repeat ultrasound, are shown in grey. Haplotype data with the segregation of microsatellite markers (KG8 and SM6), polymorphic PC-1 amino acid changes (p.A4059V, p.I4045V and p.A3512V) and the mutation p.D3782_V3783insD are shown. E: Initial and the latest clinical and molecular diagnoses of the French-Canadian family. Negative ultrasound (-ve US), Hypertensive (HTN), mutation not detected (ND).

Figure 2. The Portuguese family

A: Partial pedigree of the four-generation Portuguese family, 7001PKD redrawn from two previously published pedigrees.(18, 22) II-15, II-20, and II-21 are drawn as described in de Almeida S. et al.(18) New members were added and some original members were taken out of the fourth generation from previous publications. III-14, who was originally reported as affected, but only had two cysts and did not inherit the p.G3818A mutation is shown in grey. The haplotype shaded in blue with the microsatellite markers MC1786, KG8, SM6, 16AC2.5 and CW2 segregates with the disease. B: Direct sequencing showing the wildtype chromatogram and the PKD1: c.11453G>C nucleotide change and the corresponding p.G3818A amino acid change found in affected family members. C: Analysis of the likely pathogenicity of p.G3818A using SIFT and Align GVGD. VS: Variation score; MG: Mutation group. p.G3818A has a SIFT score of 0.000 and Align GVGD score of C55 which correspond to the Highly Likely Pathogenic mutation designation(25). D: Multiple sequence alignment of PC-1 orthologs showing the well-conserved glycine at position 3818 (red arrow) across a wide-variety of species. E: Multiple sequence alignment of PC-1-like proteins in humans, sea urchin and C. elegans compared with human PC-1, further showing the conservation of glycine at position 3818 (red arrow). F: Restriction digest analysis with EcoO109I confirms the c.11453G>C change only in affected individuals. Absence of an EcoO109I site in unaffected individuals (yellow fonts) results in a 288bp band after the restriction digestion of the PKD1-exon 41 PCR product, whereas affected individuals (red fonts) have two bands of 174 and 114bp. G: Estimated glomerular filtration rate (eGFR) in affected individuals calculated using the latest available serum creatinine measurements showing a decline in eGFR in 7001PKD family members by the late third or early fourth decade (blue squares). A reference trend line showing typical PKD1 progression was generated by analyzing 106 ADPKD patients with definitely pathogenic PKD1 mutations.

Figure 3. The Bulgarian Family

A: Top panel shows the wildtype PKD2 chromatogram and the bottom panel the frameshifting mutation c.213delC, resulting in p.A71fs45X. B: Revised pedigree and haplotype of the previously described Bulgarian family including the PKD2 SNP p.R28P. III-1 and III-3 who were previously described as affected but do not carry the mutation are shown in grey. C: Ultrasound images of the right kidney from III-3 (misdiagnosed with ADPKD) and the right kidney of III-5 (confirmed diagnosis of ADPKD), showing a few cysts (blue arrows). D: Table showing the age at renal function analysis by serum creatinine measurement and expressed as eGFR, plus the mutation status of family members. Mutation not detected (ND).

Figure 4. The Italian family

A: Top panel shows the wildtype sequence found in I-2. Bottom panel shows the c.12682C>T nucleotide change and the corresponding p.R4228X amino acid change found in II-2. B: Revised pedigree and haplotype data of the two-generation Italian family. I-2, who was originally reported as affected is shown in grey. Segregation of the genetic markers KG8, 16AC2.5, SM7 and 26.6, as previously reported, PKD1 intragenic variants (c.12570C>T and p.T2250M), and the p.R4228X mutation, are also shown. C: Allele-specific PCR to amplify the mutant p.R4228X allele amplified a 250bp product only from II-2 and not from blood, urine or buccal cell samples of I-2. D: Multiple sequence alignment of PC-1 orthologs showing position 2250, where there is a T>M change in the mother (I-2) with a few cysts and II-1. E: Cleavage analysis at the GPS domain of PC-1 in wildtype (WT), the variant found in I-2 and II-1 (T2250M) a the previously described (35) hypomorphic allele (R2220W) and the completely cleavage mutant (E2771K) (n=12 transfections). FL= full length PC-1 and CTP= C-terminal product. Statistical analysis were done using Student’s t test (***P<0.001). Error bars represent ± SD.

Figure 5. The Spanish family

A: Ultrasound image of the right kidney in II-5 showing a few cysts consistent with a diagnosis of ADPKD (blue arrows). B: Pedigree of the Spanish family with the intragenic PKD1 and PKD2 SNPs identified through direct sequencing. C: Initial and latest diagnoses in the Spanish family.