Abstract
Autosomal-dominant polycystic kidney disease (AD-PKD) is common in Persians and Persians-related breeds. The aims of this study were to evaluate the sensitivity and specificity of early ultrasound examination and to compare ultrasound and genetic testing for early diagnosis. Sixty-three Persians and seven Exotic Shorthairs were considered. All underwent ultrasonographic and genetic testing (polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) assay) between 2.5 and 3.5 months of age (10–14 weeks). With ultrasound, 41.4% showed renal cysts, while 37.1% were PKD positive by genetic testing and DNA sequencing. Six cats with at least one renal cyst were negative by genetic testing, while only one cat negative at ultrasound resulted positive at genetic test. DNA sequencing of three polycystic cats, negative by genetic test, revealed they were heterozygous for the mutation. Agreement was described by Cohen's kappa that resulted 0.85, considering genetic test and DNA sequencing. Sensitivity and specificity of ultrasound were 96.2% and 91%, respectively. Sensitivity was higher and specificity lower than reported previously. The higher sensitivity could be due to improved technical capabilities of ultrasound machines and transducers. Other causes of PKD could explain the lower specificity. In conclusion, ultrasound resulted in a reliable diagnostic method for feline AD-PKD1 at early age and it should always be used with genetic testing, in order to reach a complete screening programme and eventually to identify other genetic mutations.
Autosomal-dominant polycystic kidney disease (AD-PKD) is the most prevalent inherited genetic disease in cats. AD-PKD has been identified in Persians and Persian related breeds such as Exotic Shorthairs and mix-breeds. 1,2 The clinical and morphological similarity of AD-PKD in Persian cats to human disease and the autosomal dominant pattern suggest that this disease represents a good model for AD-PKD in humans. 3 The disease is progressive and may lead to irreversible renal failure. The prevalence of the disease in Persian cats in several countries worldwide ranges between 49.2% (United Kingdom) 4 and 36% (Slovenia), 5 while in other breeds it is less known.
In 2004 Lyons and collaborators identified a C>A transversion in exon 29 of the feline PKD1 gene causing a stop codon to be introduced into the mRNA and linked to AD-PKD. The mutation was found in heterozygous state in Persians and in breeds out-crossed with Persians, the homozygous state supposed to be associated to embryo death. 2,5 Easy polymerase chain reaction/restriction fragment length polymorphism assay (PCR/RFLP) 2 or real time-PCR assay 6 based genetic tests are now available which detect the AD-PKD mutation. These can be used to genotype newborn kittens and to discriminate between inherited and non-inherited cysts.
In recent studies however, a small percentage (4.5–5%) of cats with histological or ultrasonographic diagnosis of PKD were found to be wild-type, suggesting also other PKD-causing mutations in the feline population. 7,8
Renal ultrasonography is the most practical non-invasive diagnostic method for adult cats. The sensitivity of ultrasound is said to be 75% when performed at 16 weeks of age and 91% when performed at 36 weeks of age, 9 increasing with the age of the patient. Specificity is said to be 100%. 3,4,9–11 In one study, the absence of cysts on ultrasound examination at 6 months of age was correlated with the absence of PKD at necropsy. 9 A recent study compared results of ultrasound examination performed at 3 and 12 months of age and although only a small number of cats were evaluated, the resulting sensitivity of ultrasound at 3 months of age was 100%. 12
The aims of this study were to evaluate sensitivity and specificity of renal ultrasound performed at 3 months (12 weeks) of age and to compare sonographic results to an AD-PKD genetic test. In humans, the genetic test is considered the most accurate diagnostic tool for AD-PKD type 1 and type 2. 13
Materials and Methods
Seventy clinically healthy cats evaluated for ultrasonographic PKD screening at the Veterinary Teaching Hospital of the University of Parma from April 2006 to November 2007 were considered. All of them underwent both ultrasound and genetic examination for AD-PKD between 2.5 (10 weeks) and 3.5 months (14 weeks) of age (mean age 3.1±0.36 months, 12.1 weeks) and had at least one parent with ultrasonographic or genetic diagnosis of PKD.
Ultrasound examinations were all performed with a 10 MHz linear transducer (Megas GPX, Esaote, Genova, Italy). Both kidneys were scanned and each cystic lesion was evaluated with Colour Doppler flow mapping in order to distinguish a very small cystic lesion (2–3 mm in diameter) from a vessel. Cats were classified as positive when at least one cyst was found in at least one kidney. 3–5,11
PCR/RFLP genetic tests on 70 blood samples were all performed at the Department of Animal Science of the University of Milan. Genomic DNA was isolated from EDTA whole blood using the blood genomic Prep Mini Spin Kit (GE Healthcare). The PKD1 exon 29 was partially amplified by a PCR reaction producing a 559 bp amplicon, according to the Lyons et al 2 protocol. DNA restriction was performed by digestion of 5 ml of amplification product in a total volume of 10 μl reaction containing 10 U of MLY1 (New England Biolabs, Beverly, MA), NE buffer 4 (New England Biolabs, Beverly, MA) incubated at 37°C for 3 h followed by inactivation of the enzyme at 65°C for 10 min. The RFLP pattern was obtained running the complete digestion reaction on 2% agarose gel. If results were not in agreement, DNA sequencing was performed on PCR products, both strands, using the ABI Dye Terminator Sequencing Chemistry 3.1 (Applied Biosystems, Foster City, CA) and an ABI 310 analyser, according to standard protocols.
Agreement between ultrasonography and the genetic test was calculated by Cohen's kappa values. Sensitivity, specificity, positive and negative predictive values of renal ultrasound for the diagnosis of AD-PKD type 1 at 3 months of age (12 weeks) were calculated, assuming the genetic test as the gold standard. 5,13
Results
Among the 70 cats examined, 63 were Persians and seven were Exotic Shorthairs, 40 were males and 30 females.
Concerning ultrasound examination, 29 cats showed renal cysts (17 males, 12 females), with a resulting prevalence of 41.4%. Three cats showed cysts in only one kidney and among these, two kittens had only one cyst and one kitten two cysts. The other 25 cats showed more than one cyst in both kidneys.
Concerning genetic testing, 24 cats resulted AD-PKD type 1 affected (13 males and 11 females), with a resulting prevalence of 34.3% (Fig 1). Six cats (four males and two females) that showed renal cysts resulted negative at genetic test. Among these, three cats had cysts in only one kidney and three cats showed several cysts in both kidneys and two of them were siblings (Fig 2). On the other hand, only one cat that resulted negative by ultrasound, was AD-PKD type 1 positive by genetic testing.
Fig 1.
(A) Polycystic kidney of a 3-month-old Persian cat, resulted positive at AD-PKD1 genetic test. (B) RFLP typing for feline AD-PKD1 mutation. Left: Gene RulerTM 1 kb DNA Ladder (MBI Fermentas); right: heterozygous pattern showing 559 bp fragment of the wild-type allele and 316 plus 243 bp fragments of the mutated causative allele.
Fig 2.
(A) Cystic structures in a kidney of the 3-month-old Persian cat, resulted negative at genetic test. (B) RFLP typing for feline AD-PKD1 mutation. Left: Gene RulerTM 1 kb DNA Ladder (MBI Fermentas); right: homozygous pattern showing only 559 bp fragment of the wild-type allele.
The seven inconsistencies between ultrasound and genetic test were reconsidered by sequencing the AD-PKD1 mutation site. Two of the polycystic kittens negative by genetic test, were reclassified heterozygous for the mutation. All the cats that had cysts in only one kidney resulted negative by sequencing. The cat that was negative by ultrasound was confirmed positive by sequencing. Considering the PCR/RFLP and DNA sequencing results together, the prevalence of AD-PKD1 was 37.1%.
Ultrasonographically, renal cysts appeared as round, anechoic or hypoechoic cavities. The greatest number of cysts was located in the cortex or between cortex and medulla.
The agreement between renal ultrasonography and PCR/RFLP+DNA sequencing testing was described calculating Cohen's kappa, that resulted 0.85 (95% confidence interval (CI) 0.72–0.98). The sensitivity of renal ultrasonographic examination for diagnosis of AD-PKD type 1 at 3 months of age (12 weeks) resulted to be 96.2% (CI 95%: 88.8; 100), while specificity resulted to be 91% (CI 95%: 82.4; 99.4); positive predictive value was 86.2% (CI 95%: 73.7; 98.8), negative predictive value was 97.6% (CI 95%: 92.8; 100).
Discussion
Overall, the resulting prevalence of feline PKD in this study is 41.4% by ultrasound and 37.1% by genetic and sequencing testing. These data are similar to those reported in the literature 3–5,9–11,14
In the present study the resulting sensitivity of renal ultrasonography performed at 3 months of age is 96.2% and it is similar to a recent paper 12 and higher than what was reported elsewhere. 4,9–11 The increased sensitivity of early ultrasound examination could be due to improved technical developments of the ultrasound machines and higher frequencies reached by modern transducers (10–14 MHz) that give a better image resolution of superficial and small body regions.
On the other hand, the resulting specificity is 91% and it is lower than what reported in the literature, 3,4,11 in which the prevalence of solitary cysts not PKD-related is considered very low. In this report, however, 5.7% of cats with cystic kidneys were wild-type. Among these, a cat had both polycystic kidneys but resulted negative to genetic testing and sequencing. The nature of these lesions is unknown, but the authors hypothesised that they could be due to other PKD-causing mutations, as suggested in the literature 7,8 or to technical errors. Actually, cysts caused by chronic renal disease were considered less likely, due to young age of the cats.
Possibly PCR/RFLP, as any other technique, can fail in some conditions (bad sample, technical fail, human error). PCR/RFLP is used worldwide because it does not necessitate expensive supplies, it is quick and provides a clear robust result that is easy to interpret. DNA sequencing-based techniques do not provide more detailed information on the mutation site. They are more time and money consuming (especially if only small numbers of samples are performed together) and mainly cannot avoid technical and human errors or problems due to poor sample quality. In the authors' opinion more accurate, but not significantly, results obtained by sequencing are consistent with the occurrence of a re-test and not with a higher specificity of sequencing. However, it could be used additionally, if results of ultrasonography and genetic testing are discordant.
In humans, approximately 85% of the patients with AD-PKD have a mutation in the PDK1 gene, and the remaining 15% in PKD2 gene or in another unidentified genetic locus. 5 PKD1 and PKD2 genes encode for polycystin-1 and polycistin-2, respectively, that in normal kidneys control cellular proliferation and maintain the tubular cells in a state of terminal differentiation. 15 Although the different forms of AD-PKD share the same principal features, some clinical difference may distinguish them. The main difference is the overall younger age of onset of end-stage renal disease with AD-PKD type 1 than with AD-PKD type 2. 13 In humans an autosomal-recessive form of PKD exists (AR-PKD), that is a hereditary and causes severe disease of kidneys and biliary tract. AR-PKD typically presents in infancy and is caused by several mutations in PKHD1 gene that encodes for a protein named fibrocystin/polyductin, whose function in normal tissue is not yet known. 15 In cats a similar disease has been reported in six kittens from four litters of related cats, that had greatly enlarged abdomens and died before they were 7 weeks old. Kittens from two litters were necropsied and found to have bilateral polycystic renal disease and cystic bile ducts. 16
Clinical monitoring in the future months/years of the polycystic-PKD1-mutation-negative 3-month-old kitten, along with the any offspring, may partially clarify the nature of the cysts and eventually confirm the inherited pattern.
A screening programme should be essential in order to be able to document the efficiency of the eradication process. 11 Eradication of the disease should be achieved by screening all cats used for breeding and retain only unaffected cats for future breeding programmes. 10
Today the genetic test is the elective method for early diagnosis of AD-PKD type 1 in kittens under 4 months. Ultrasound is used in adult cats 5 and the genetic test is always useful to confirm the inherited PKD1 pattern.
This report points out that renal ultrasound is a reliable diagnostic tool for feline AD-PKD type 1 at early age if performed by an experienced ultrasonographer with high frequency transducers. It should not be laid apart but always used along with the genetic test. A synergic use of both tests could be useful also to identify other genetic mutations related to feline PKD, as suggested by results of this and other studies. 7,8 Ultrasound is recommended in cats resulted positive by genetic test to detect the number and size of renal cysts and to predict the severity of the disease. 5,6 Further genetic studies must be carried on in order to evaluate other mutations that could cause feline PKD. Ultrasound is still the elective diagnostic method for polycystic kidney disease not due to PKD1 mutation, until a proper genetic test will be available.
In conclusion, a paired use of ultrasound and genetic tests should be recommended in order to reach a complete medical condition of breeding cats as soon as possible, and to plan a screening programme for feline PKD.
Acknowledgments
The authors thank Mr Giuseppe Bertaccini for technical assistance and the Italian Research Fund (FIL 2006–2007).
References
- 1.Biller D.S., Chew D.J., Di Bartola S.P. Polycystic kidney disease in a family of Persian cats, J Am Vet Med Assoc 196, 1990, 1288–1290. [PubMed] [Google Scholar]
- 2.Lyons L.A., Biller D.S., Erdman C.A., et al. Feline polycystic kidney disease mutation identified in PKD1, J Am Soc Nephrol 15, 2004, 2548–2555. [DOI] [PubMed] [Google Scholar]
- 3.Beck C., Lavelle R.B. Feline polycystic kidney disease in Persian and other cats: A prospective study using ultrasonography, Aust Vet J 79, 2001, 181–184. [DOI] [PubMed] [Google Scholar]
- 4.Cannon M.J., MacKay A.D., Barr F.J., Rudorf H., Bradley K.J., Gruffydd-Jones T.J. Prevalence of polycystic kidney disease in Persian cats in the UK, Vet Rec 149, 2001, 409–411. [DOI] [PubMed] [Google Scholar]
- 5.Domanjko-Petriĉ A., Cernec D., Cotman M. Polycystic kidney disease: A review and occurrence in Slovenia with comparison between ultrasound and genetic testing, J Feline Med Surg 10, 2007, 115–119. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Helps C., Tasker S., Barr F., Wills S., Gruffydd-Jones T. Detection of the single nucleotide polymorphism causing feline authosomal-dominant polycystic kidney disease in Persian from the UK using a novel real-time PCR assy, Mol Cell Probes 21, 2007, 31–34. [DOI] [PubMed] [Google Scholar]
- 7.Helps C., Tasker S., Harley R. Correlation of the feline PKD1 genetic mutation with cases of PKD diagnosed by pathological examination, Exp Mol Pathol 83, 2007, 264–268. [DOI] [PubMed] [Google Scholar]
- 8.Kappe E.C., Hecht W., Gerwing M., Michele U., Reinacher M. Polycystic kidney disease in the German population of Persian cats. A comparative study of ultrasonographical examination and genetic testing, Tierarztl Prax Ausg K Kleintiere Heimtiere 33, 2005, 413–418. [Google Scholar]
- 9.Biller D.S., Di Bartola S.P., Eaton K.A., Pflueger S., Wellman M.L., Radin M.J. Inheritance of polycystic kidney disease in Persian cats, J Hered 87, 1996, 1–5. [DOI] [PubMed] [Google Scholar]
- 10.Barrs V.R., Gunew M., Foster S.F., Beatty J.A., Malik R. Prevalence of autosomal dominant polycystic kidney disease in Persian cats and related-breeds in Sidney and Brisbane, Aust Vet J 79, 2001, 257–259. [DOI] [PubMed] [Google Scholar]
- 11.Barthez P.Y., Rivier P., Begon D. Prevalence of polycystic kidney disease in Persian related cats in France, J Feline Med Surg 5, 2003, 345–347. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Ottesen N. Polycystic kidney disease in Persian cats: Comparison of renal ultrasonography at the age of 3 and 12 months, Vet Radiol Ultrasound 45, 2004, 600. [Google Scholar]
- 13.Nicolau C., Torra R., Badenas C., et al. Autosomal dominant polycystic kidney disease types 1 and 2: Assessment for US sensitivity for diagnosis, Radiology 213, 1999, 273–276. [DOI] [PubMed] [Google Scholar]
- 14.Bonazzi M., Volta A., Gnudi G., Bottarelli E., Gazzola M., Bertoni G. Prevalence of the polycystic kidney disease and renal and urinary bladder ultrasonographic abnormalities in Persian and Exotic Shorthair cats in Italy, J Feline Med Surg 9, 2007, 387–391. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Al-Bhalal L., Akthar M. Molecular basis of autosomal recessive polycystic kidney disease (ARPKD), Adv Anat Pathol 15, 2008, 54–58. [DOI] [PubMed] [Google Scholar]
- 16.Crowell W.A., Hubbell J.J., Riley J.C. Polycystic renal disease in related cats, J Am Vet Med Assoc 175, 1979, 286–288. [PubMed] [Google Scholar]