Deciphering Variability of PKD1 and PKD2 in an Italian Cohort of 643 Patients with Autosomal Dominant Polycystic Kidney Disease (ADPKD)

Abstract

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common hereditary kidney disease. We analysed PKD1 and PKD2, in a large cohort of 440 unrelated Italian patients with ADPKD and 203 relatives by direct sequencing and MLPA. Molecular and detailed phenotypic data have been collected and submitted to the PKD1/PKD2 LOVD database. This is the first large retrospective study in Italian patients, describing 701 variants, 249 (35.5%) already associated with ADPKD and 452 (64.5%) novel. According to the criteria adopted, the overall detection rate was 80% (352/440). Novel variants with uncertain significance were found in 14% of patients. Among patients with pathogenic variants, in 301 (85.5%) the disease is associated with PKD1, 196 (55.7%) truncating, 81 (23%) non truncating, 24 (6.8%) IF indels, and in 51 (14.5%) with PKD2. Our results outline the high allelic heterogeneity of variants, complicated by the presence of variants of uncertain significance as well as of multiple variants in the same subject. Classification of novel variants may be particularly cumbersome having an important impact on the genetic counselling. Our study confirms the importance to improve the assessment of variant pathogenicity for ADPKD; to this point databasing of both clinical and molecular data is crucial.

Figure 1

(A) In this family, the proband (III-1, black arrow) with ESRD, inherited from the father (II-2) and shared only with affected relatives (III-3, III-4) the intronic PKD1 variant: c.1202-9G>A. None of the analyzed healthy subjects in the family (II-1, III-2, III-5, III-6, IV-1) had this variant; (B) Sequence chromathogram; abolishing of the nearby splice acceptor site was predicted in silico (www.fruitfly.org). Genetic testing was important to support classification of the intronic variant as highly likely pathogenic and to identify the III-2 subject (red arrow) as a potential donor for kidney transplantation in the patient. Unfortunately it was not possible to collect the proper samples for RT-PCR. Filled symbol: ADPKD; empty symbol: asymptomatic; (+) sign indicates a subject heterozygous for the c.1202-9G>A variant; (−) sign indicates a subject without the variant.

Figure 2

(A) Pedigree. In this family, the proband (arrowed) inherited a synonymous PKD1 variant (c.2097G>T, p. Ser699=) from his mother. (B) RT-PCR from whole blood RNA. In the control sample (Wt), only the expected 585 bp fragment was present. In the patient sample (P), an additional 1038 bp abnormal fragment was amplified. (C) Sequencing of the regular and abnormal fragments from the patient sample. In the upper panel, the sequence of the 585 bp normal c.2097G allele cDNA showing the adjacent exons 10 and 11, correctly spliced; in the lower panel, the sequence of the abnormal 1038 bp cDNA showing that in the mutated allele c.2097T, intron 10 has been completely retained in the transcript. (D) A scheme of the spliced regions in the normal and mutant alleles. Filled symbol: ADPKD; empty symbol: healthy subject.

Figure 3. In this family, the proband has inherited from the mother a truncating variant, classified as definitely pathogenic, and from the asymptomatic father a del/ins in-frame variant, classified as highly likely pathogenic with the adopted criteria.

The proband developed renal insufficiency much earlier than her mother; a RMI renal examination was advised to the father to evaluate a possible subclinical phenotype. Filled symbol: ADPKD; empty symbol: asymptomatic.

Figure 4

(A) MLPA result suggesting the presence of heterozygous deletion of exon 3 in PKD1; (B) PCR amplification of the deletion borders. In lane 2 the lower fragment is generated by the deleted allele; 2: patient; 3: healthy control; bl: no DNA; 1, 4: molecular weight markers; (C) schematic representation of the 5′ region of the PKD1 gene in normal (upper) and deleted (lower) alleles. Primer F-2FLR-5′-ATTTTTTGAGATGGAGCTTCACTCTTGCAGG; primer R-4R 5′-AGCCCTGCCCAGTGTCT. (D) Sequence of the deletion boundaries. Direct sequencing revealed the presence of a 1431 bp deletion, extending from nucleotide c.216-793 to c.429 (c.216-793_429del143; http://www.hgvs.org/mutnomen/). The deletion starts 793 bp upstream of exon 2 and stops at position 429 in exon 4, thus causing a frameshift with the production of a truncated putative protein (p.Leu72fs).

Figure 5. In this family, the proband and his sister are the only ADPKD affected subjects; the proband has kidney and liver multiple cysts, his sister had kidney and liver transplantation at 44 years of age, the parents are healthy and negative to US abdominal examination.

Genetic analysis in the family revealed that they did not inherited the truncating variant p.Tyr610*, suggesting the presence of germinal mosaicism in one of the parents. Paternity was confirmed. Filled symbol: ADPKD; empty symbol: healthy.

Figure 6. Allelic influence on renal survival.

A significant difference in renal survival between patients harboring a PKD1 pathogenic variant and patients with a PKD2 pathogenic variant was observed. Log rank test: Chi-square 5.47, p = 0.0194.