Spectrum of mutations in PKD1 and PKD2 genes in 102 unrelated Italian pedigrees with Autosomal Dominant Polycystic Kidney Disease (ADPKD) – Sanger sequencing vs Next Generation Sequencing (NGS)


Autosomal dominant polycystic kidney disease (ADPKD), is characterized by the development of renal cysts leading to end-stage renal failure. ADPKD is typically diagnosed by imaging but the diagnosis may be uncertain, especially in young individuals (<30 years) and in patients with a negative family history. ADPKD is caused by mutations in PKD1 or PKD2 genes encoding for policistin-1 and -2, respectively (Figure 1A, B).

The mutational analysis of ADPKD is complicated by extensive allelic heterogeneity and by the presence of six highly homologous sequences of PKD1 exons 1–33.

Although, clinical studies and case reports describing one or few ADPKD families have been reported in Italian population, to date a comprehensive molecular study is still lacking. Here, we report our comprehensive mutation analysis of PKD1 and PKD2 genes in 161 Italian ADPKD patients from 102 unrelated pedigree using both Sanger Sequencing and Next Generation Sequencing (NGS).


PKD1 and PKD2 genes were analyzed in 102 unrelated ADPKD Italian pedigree – the largest Italian cohort analyzed to date in a single study – using different approaches: Sanger sequencing, Multiplex Ligation-dependent Probe Amplification (MLPA) and NGS by multiplexing indexed paired-end libraries from long range PCR using Illumina Nextera XT kit and Illumina MiSeq instrument (Figure 2, Figure 3B). The potential pathogenicity of the newly identified variants were evaluated by combining different methods.


We identified the largest number of definitively and probable pathogenic mutations (n=88) reported in a single study in Italian population, achieving an overall detection rate of 95%; moreover, 7 novel indeterminate and likely neutral mutations were also identified. 57 mutations (53 PKD1 and 4 PKD2) have not been previously described, expanding the spectrum of known ADPKD mutations. We identified 12 de novo PKD1 mutations in 11 sporadic patients without family history, providing a definitive diagnosis of ADPKD. We found the largest number of de novo mutations reported in a single study (12/88=13,6%) demonstrating, for the first time, that the prevalence of PKD1 de novo mutations may be underestimated. A new deletion of 16 nucleotides, (c.11316_11331delCGCAGGAGGCTTCAGC) causing frameshift and a truncated protein (p.Ala3773Profs*48), was detected by Multiplex Ligation-dependent Probe Amplification (MLPA) analysis in patient 47R (Figure 3A). 7 PKD1 mutations and 4 PKD2 mutations were found in ≥2 unrelated patients from Southern Italy: a founder effect could be hypothesized for 4 novel PKD1 definitively pathogenic mutations. 29 patients were analyzed by NGS: we confirm the presence of a nonsense PKD1 mutation and the lacking of clear pathogenic mutations in 3 patients previously analyzed by Sanger sequencing, and the presence of definitively and probable pathogenic mutations in the other 26 not previously genotyped patients. All NGS results have been confirmed by Sanger sequencing, achieving sensitivity and specificity of 100% (Figure 3B. C).


Taken together, our genetic data provide important new advances in the molecular diagnostics of ADPKD Italian patients: (i) analyze the largest Italian cohort and report the largest number of new mutations in a single Italian study; (ii) describe for the first time new potential founder mutations in ADPKD patients from Southern Italy; (iii) emphasize the important role of molecular screening in ADPKD sporadic patients by reporting the largest number of de novo mutations identified in a single study and (iv) provide for the first time a new NGS method for Italian patients with a detection rate comparable to Sanger sequencing but with significantly lower cost and reduced turnaround time.