This preliminary, single centre, small scale prospective study was designed to evaluate the use of routine PKD2 mutation testing in a nephrology outpatient clinic. The clinic does not see those who have already developed ESRF. Since it is known that PKD2 status identifies those with a milder clinical phenotype and delayed progression to ESRF by up to 20 years , this information would be valuable to provide patients and their families with improved prognostic information in the earlier stages of their disease and to determine whether there were other simple, patient-reported variables that could be used to predict genotype and prognosis. Therefore additional information about PKD1 mutation status was not required, as assignment to the PKD2 group did not require this. Similarly, the role of reduced-penetrance PKD1 alleles on the expression of PKD2-linked disease is not known and has not been evaluated in other studies [19, 20]. Patients carrying both PKD1 and PKD2 pathogenic mutations are also very rare .
We demonstrated that a fifth of an unselected ADPKD patient cohort from a single clinic that sees patients only with eGFR > 15 mL/min/1.73 m2 had a likely pathogenic PKD2 mutation. Our patient characteristics are very similar to those reported by Garcia-Gonzalez et al. (2007), although the number reaching end-stage was lower in our study (10% vs. 21%) . In its design, our study was likely to favour a higher PKD2 detection rate, as testing was not offered to individuals with CKD5 or receiving RRT. In addition, defining the proportion of all patients with ADPKD harbouring a PKD2 mutation was not a primary aim of this study. Consistent with this, other studies have shown a detection rate of ~15% when all prevalent cases have been included. Therefore the conclusions of this study can only be applied to similar clinical cohorts. As patients with ADPKD and CKD1-4 are typically seen in the nephrology out-patient clinic setting that does not include those with CKD5 and ESRF, this should not present significant clinical difficulty.
Interestingly, there appeared to be no overall difference between the two groups in the risk of developing, or age of development of, CKD3 or CKD4, or in the rate of functional deterioration (eGFR) once CKD had supervened. It is unclear whether the former was due to the cohort size or selection of patients with a milder phenotype due to the exclusion of those that presented with CKD5. Post-hoc power calculations demonstrated that the sample had 60% power to detect a renal survival difference for CKD3. For 80% power a sample size of 169 patients would be required (34 with a PKD2 mutation and 135 without). Therefore the relatively small sample size will limit some of the inferences that can be drawn from our data. Although the proportion of patients developing CKD3/4 in the two groups were not statistically different, we recognise that our study was sub-optimally powered to detect such a difference, but the direction of the effect of non-PKD2 status would be consistent with previous reports of worse renal outcomes in this patient group. There was greater heterogeneity in the renal phenotype of non-PKD2 patients, with a minority experiencing early-onset CKD (Figure 3). Further well- powered longitudinal studies in the same clinical setting, using multi-centre registries of genotyped patients, are therefore required to address this including the contribution of other factors such as age, gender, hypertension and albuminuria.
Currently, genic variation in ADPKD is the main factor predicting renal outcome, ESRF occurring up to 15–20 years later in patients with a PKD2 mutation when compared to those with a PKD1 mutation . MRI imaging data from the CRISP study suggests that this is due to a lower number of cysts being present in PKD2-related disease, rather than differences in the rate of cyst growth or renal enlargement [23, 24]. Although not generally in clinical use, measurement of single or serial renal volumes may better predict disease severity, with larger kidneys predicting a more rapid rate of decline in GFR . However, routine PKD2 mutation testing that does not require the patient to be present, will become more economical and identifies a group of patients likely to have preserved renal function for longer .
The use of direct mutation testing rather than linkage analysis also has the benefit of being available to individuals rather than extended families. About a third of our cohort was not aware of a FH of ADPKD at the time of clinic attendance and would therefore not have been suitable for linkage analysis. This most likely represents a combination of factors including new mutation, undiagnosed disease in relatives and unrevealed diagnoses rather than a higher than expected new mutation rate alone. Whilst the lack of a FH means that the Ravine criteria for the diagnosis of ADPKD cannot be strictly employed it is likely that other causes of PKD, such as renal cysts and diabetes (RCAD, OMIM 137920) which can phenocopy ADPKD, were not sufficiently common to account for the discrepancy with the published data. Similarly the role of hypomorphic alleles in modifying disease presentation remains unknown although is unlikely to account for significant non-penetrance. In the study by Barua et al. , the new mutation rate determined following parental ultrasound examination was ~10%, suggesting that in more than a fifth of our patients, a diagnosis of ADPKD is not being communicated or is undiagnosed. Wider use of parental screening after appropriate counselling is therefore justified to clarify disease risks for family members. Further work will be required to explore how this may be more generally implemented in non-specialist clinics and in primary health care.
The detection rate of ~ 20% PKD2 mutations is in broad agreement with other studies (range 15-36%; Table 1) [5, 6, 12, 13, 22, 25, 26]. However, each of these studies employed different inclusion and exclusion criteria and sampled different populations, making ascertainment bias likely. The estimate of 36% in one study may represent the true frequency when population screening for ADPKD is carried out, and would likely represent the ascertainment of asymptomatic, undiagnosed and therefore mild cases.
Unlike direct PKD1 mutation testing, where it remains difficult to assign pathogenicity to some variants, the majority of PKD2 sequence variants can be assigned as likely or highly likely to be pathogenic [12, 13]. Sensitivity of mutation detection is therefore likely to be very high when using direct sequencing, although this needs to be formally tested in families where linkage data are also available . In the study by Veldhuisen et al. (1997), mutations were found in 57% of 35 families linked to the PKD2 locus when 80% of the gene was screened by single strand conformation polymorphism analysis and direct sequencing . With newer methods and complete gene sequencing and dosage analysis, a higher detection rate is likely. Our figure of ~ 20% may therefore still slightly underestimate the true number of PKD2 mutations in this cohort of patients with CKD1-4. Dosage analysis is now available and should be included in future studies. However, the PKD Mutation Database suggests that large deletions and duplications in PKD2 are rare. Only a single deletion among 115 pathogenic PKD2 mutations reported here and in the database would have been missed without dosage analysis. Thus a > 98% detection rate by screening coding exons and their splice sites can be achieved. This represents the best current method for identifying individuals and families that harbor a PKD2 mutation and may therefore have a better prognosis.
Although molecular testing for ADPKD is now available in the UK (http://www.ukgtn.org.uk), it has not been routinely used in nephrology out-patient clinics for diagnostic, predictive or prognostic testing. At present, diagnosis and family screening are almost exclusively carried out using imaging combined with a FH. The main utility of genetic testing to date has been in research to define the natural history of the disease by genotype, and to identify genotype/phenotype correlations . Indications for clinical molecular testing include establishment of the correct diagnosis if there is no family history and if there are atypical features on imaging; exclusion testing for potential living-related donors and prenatal/pre-implantation genetic diagnosis . Of these, the ~ 20% detection rate of PKD2 mutations is likely to be most clinically useful in screening potential living-related donors, particularly for those with normal ultrasounds below age 40 . Non-PKD2 status is assumed here to represent PKD1-linked disease, i.e. in this context the test is 100% specific and sensitive.
Of the clinical variables recorded in this study, few had significant predictive power to identify patients likely to have a PKD2 mutation. In the multivariate model adjusting for gender and hypertension, point estimates suggested that non-PKD2 disease, male gender and the presence of hypertension may together be associated with earlier onset of CKD3 (Additional file 2: Table S2), although formal significance levels were not reached in this cohort due to its size. However, the directional nature of the effect supports previous reports indicating a milder renal phenotype with PKD2 mutations .
The lack of difference in progression from CKD3 between the two groups was unexpected, but may be due to a number of factors. Firstly, although 16 non-PKD2 cases had developed CKD4 earlier than the youngest PKD2 case with CKD4 (59.6 years), 25% (5/20) of non-PKD2 cases had not yet reached CKD3 by the age of 62, compared to 17% (1/6) of PKD2 cases, further highlighting that likely PKD1 mutations may be associated with mild disease . Secondly, our analysis may underestimate progression of CKD in non-PKD2 cases due to ascertainment bias through exclusion of patients with existing CKD5/ESRD, since our results suggest a predominance of non-PKD2 in patients in this group. Thirdly and most likely, our available sample may be underpowered to detect differences in progression to CKD3. This further emphasises the continued need to conduct long-term studies on large, genotyped cohorts.
However, the comparable rate of decline in eGFR after the onset of CKD3 between the two groups is similar to that observed in previous studies where no significant differences between genotypes were found, suggesting that the underlying disease process is independent of the causal mutation . This is also supported by previously observed similar rates of change in renal volume between PKD1 and PKD2 groups, although the PKD2 group have fewer cysts and smaller kidneys at a given age .
The predictive value of the FH was strong . PKD2 patients were more likely to have a FH of ADPKD without progression of CKD, and the average age of a family member with ESRF was significantly higher than the non-PKD2 group (Table 3). This reflects the ascertainment of all degrees of disease among family members and is in keeping with the previously reported milder clinical phenotype of PKD2-linked disease . A FH of ESRF before age 50 was highly predictive of non-PKD2 status (PPV 100%, sensitivity 0.71). Therefore, in PKD patients with relatively preserved renal function, the disease course of affected family members can usefully be used to direct genetic testing, with PKD2 testing targeted to those patients with a FH of ESRF occurring only over 50 yrs. Similarly, a FH of ESRF occurring at or after age 70 was strongly predictive of a PKD2 mutation (PPV 88%, sensitivity 0.29). However, sensitivity was low, as some individuals with apparent PKD1-linked disease (non-PKD2) can have very mild disease , as observed in our cohort (Figure 3). Our results confirm those of Barua et al. who found that the presence of at least one family member who developed ESRF before 55 years was highly predictive of PKD1 (positive predictive value 100%, sensitivity 72%) . Having at least one family member with ESRF after 70 years of age was also highly predictive of PKD2 in that study. FH alone has limitations if additional family information is not available, or if an individual represents a new mutation (i.e. if both parents have normal renal imaging). Such patients are suitable for genetic analysis.
Importantly, a positive PKD2 test allows other family members to consider predictive genetic testing. Therefore, whilst a detailed FH has some predictive value and patients with a FH of ESRF before age 50 can be excluded from PKD2 testing, genotyping offers the best means to provide prognostic information in the absence of other discriminant clinical parameters. In summary, we suggest that PKD2 screening can be routinely offered to ADPKD patients except where they or their family members have developed ESRF under age 50. Additional studies in larger cohorts or using pooled data analysis would also be of value in refining the current indications and criteria for PKD gene testing.