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Keywords:

  • blindness;
  • cone;
  • Dachshund;
  • dystrophy;
  • nephronophthisis;
  • rod

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Cone-rod dystrophy is a progressive inherited retinal degenerative disorder that occurs in humans and dogs. The deletion in the nephronophthisis 4 (NPHP4) gene was established as a causative mutation in standard wire-haired Dachshunds. We analyzed all varieties of Dachshunds from the Czech Republic and five other dog breeds and found that the deletion in the NPHP4 (in heterozygous state) is present not only in standard-, but also in miniature wire-haired Dachshunds, but not in other varieties of Dachshunds or in other breeds.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

The cone-rod dystrophies (CRDs) represent a heterogeneous group of inherited retinal diseases of humans and dogs, which are characterized by loss of cone photoreceptors accompanied or followed by loss of rod photoreceptors.[1] Some CRDs belong to retinal ciliopathies. The ciliopathies may result in a broad spectrum of disorders including retinitis pigmentosa, macular degeneration, cone dystrophy, Leber congenital amaurosis, Joubert syndrome, and others.[2] Numerous cone-rod dystrophies in human are caused by mutations in the NPHP4 (nephronophthisis 4; nephrocystin 4) gene (http://www.genecards.org/cgi-bin/carddisp.pl?gene=NPHP4&malacards=32#malacards_diseases). It is of interest that children and young adult patients with specific mutations in the gene also developed end-stage renal disease.[3]

The human NPHP4 gene codes for a 1426-amino acid protein. The protein is evolutionarily conserved. Sequence similarities were observed in many species, including mouse, cow, pig, zebrafish, and even in Ascaris suum and Caenorhabditis elegans.[3] It was reported that NPHP4 protein interacts with several other proteins – RPGR (retinitis pigmentosa GTPase regulator), RPGRIP1(retinitis pigmentosa GTPase regulator interacting protein 1), and RPGRIP1L (RPGRIP1-like).[4]

Wiik et al.[5] studied causes of canine CRD with autosomal recessive inheritance in standard wire-haired Dachshunds. They performed a whole-genome association analysis of discordant sib-pairs using the canine single nucleotide polymorphism (SNP) array. Then, fine mapping and mutation detection followed. In the NPHP4 gene, a 180-bp deletion in exon/intron 5 was identified that caused exon skipping and early truncation of the protein. The renal disease did not occur in the afflicted dogs.

We aimed our study at finding whether the deletion in NPHP4 that causes CRD in standard wire-haired Dachshunds (SWHDs) is also present in other varieties of Dachshunds and in some other dog breeds. The Dachshunds, by the Fédération Cynologique Internationale (FCI) – The World Canine Organization, has been bred in three sizes (standard, miniature, and rabbit) and in three different kinds of coat (smooth-, wire- and long-haired). For the purpose of this study when Miniature and Rabbit Dachshunds of the same coat type were bred together and the offspring divided into miniature or rabbit based on their chest circumference (Rabbit up to 30 cm, Miniature 30–35 cm when at least 15 months of age), we grouped them both as ‘miniature’ and just divided them by coat type. The standard of the breed was accepted by FCI in 2001, but all varieties are bred separately for several decades.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

We analyzed 515 blood samples of all varieties of Dachshunds (standard wire-haired, standard long-haired, standard smooth-haired, miniature wire-haired (MWHDs), miniature long-haired and miniature smooth-haired). In addition, blood samples from five other breeds (Chinese Crested Dog, Labrador Retriever, English Cocker, Border Collie, and Sheltie; 20 dogs per breed) were also studied. For the amplification of the NPHP4 gene, fragment harboring the disease causing deletion, polymerase chain reaction (PCR) analysis was carried out using primer pair NPHP4ex5 as described by Wiik et al. (2008): forward 5′-TTCTAGGTGCCTGGGTCTTG-3′ and reverse 5′-TCCCCTCAGGCTAGTGCTTA-3′.

A direct PCR using the whole unclotted blood samples was performed using PIKO™ Thermal Cycler (Finnzymes Instruments, Oy, Espoo, Finland). The reaction set-up recommended by the producer was modified in that 8 μL of 20-times diluted blood, 10 μL Phusion™ Flash High Fidelity PCR Master Mix, and 1 μL of each primer (10 pmol/μL) were used. After initial denaturation at 95 °C for 5 min, the samples were amplified for 30 cycles at 95 °C for 20 s, 56 °C for 20 s, and 72 °C for 20 s; final extension was at 72 °C for 5 min. The amplicons were analyzed by electrophoresis in 1% agarose gel.

For sequencing, fragments prepared by standard PCR amplification were used. DNA was isolated from blood using QIAGEN Blood Mini Kit (QIAGEN, Hilden, Germany). PCR was carried out using 25 μL reaction mix of 15.3 μL H2O, 2.5 μL LA buffer, 2 μL 25 mm MgCl2, 0.5 μL dNTP (200 μmol each), 0.5 μL Primer F, 0.5 μL Primer R (5 pmol each), 0.2 μL (1 unit) LA polymerase (Top Bio, Prague, Czech Republic), and 100 ng DNA. Samples were denatured for 2 min at 95 °C followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s and 68 °C for 40 s, and finally 68 °C for 7 min. The amplicons were analyzed by electrophoresis in 1% agarose gel. The presence of the deletion (del) was confirmed in three samples (1 SWHD and 2 MWHDs) by sequencing of the excised fragments of PCR products from agarose gel. For purification of the excised fragments, Min Elute Gel Extraction Kit (QIAGEN, Hilden, Germany) was used.

Additionally, two samples of wild-type (wt/wt) homozygotes (1 SWHD and 1 MWHD) were sequenced. Residual amplification primers and dNTPs were removed from the PCR product using QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany). The purified fragments were sequenced on the ABI PRISM 3130 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). For sequencing reactions, Big Dye Terminator 3.1 Cycle Sequencing Kit (Applied Biosystems, Life Technologies, NY, USA) was used.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

The deletion (del) was found only in heterozygous condition in SWHD and MWHD (Table 1), and therefore, no clinical signs were observed. However, as it was confirmed that the mutation is the same in SWHD and MWHD, we can suppose that CRD can occur in both varieties of Dachshunds. Frequency of the recessive allele, del (with deletion), was estimated to be 0.05 in both varieties. In other varieties of Dachshunds and the five other breeds (Chinese Crested Dog, Labrador Retriever, English Cocker, Border Collie and Sheltie), the deletion was not found (Table 1).

Table 1. Distribution of nephronophthisis 4 genotypes in different varieties of Dachshunds and several other dog breeds
Breed/varietyGenotypeaTotal
wt/wtwt/deldel/del
  1. a

    wt = wild -type allele; del = allele with deletion.

Standard wire-haired Dachshund199 24 0223
Standard long-haired Dachshund630063
Standard smooth-haired Dachshund790079
Miniature wire-haired Dachshund60 7 067
Miniature long-haired Dachshund300030
Miniature smooth-haired Dachshund530053
Chinese crested Dog200020
Labrador retriever200020
English cocker Spaniel200020
Border collie200020
Sheltie200020

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Wiik et al.[5] found eight carriers (heterozygotes) in 84 unrelated Norwegian SWHD; that is, frequency of del allele was approximately 0.05. They also genotyped 32 dogs from eight other breeds (Boxer, English Cocker Spaniel, English Springer Spaniel, Standard Poodle, St Bernhard, Bernese Mountain dog, Cavalier King Charles Spaniel, and Shetland Sheepdog; four dogs from each breed) and all were homozygotes wt/wt.

Because the population of the European Dachshunds is quite separated from populations in other parts of the world, there might be a difference between the presence of the deletion in the European and remaining populations of miniature wire-haired Dachshunds. Further population genetic screening with evaluation of clinical signs in recessive homozygotes (del/del) will be needed to confirm or disprove the presence of CRD in MWHDs.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

This study was supported by Institute of Animal Physiology and Genetics AS CR, v.v.i. (RVO: 67985904).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References