Scanning 20 candidate genes for association with primary cataracts in German Pinschers

4 Scanning 20 candidate genes for association with primary cataracts in German Pinschers

4.1 Abstract

Primary non-congenital cataracts (CAT) are breed related eye diseases and common in many dog breeds. In this study, we genotyped 36 cataract candidate genes flanking microsatellite markers for 20 cataract candidate genes in five German Pinscher families with a total of 45 individuals and tested them for linkage and association. For delimitation of a linked chromosome region on canine chromosome 28 (CFA28), further eight microsatellites were genotyped on this chromosome was performed. We sequenced all exons with their flanking intronic regions of PITX3.

Genome-wide significant linkage was found for PITX3-associated Markers.

4.2 Introduction

Primary hereditary cataracts are common in purebred dogs, affecting over 120 dog breeds worldwide. Cataracts are a frequent cause of visual impairment and blindness in dogs (Davidson and Nelms 1999, Gelatt and MacKay 2005). Inheritance of non-congenital cataracts has been demonstrated in several dog breeds, e.g. the Golden and Labrador Retrievers (Rubin and Flowers 1974, Curtis and Barnett 1989), German Shepherd (Barnett 1986), West Highland White Terrier (Narfström 1981), American Cocker Spaniel (Yakely 1978), Tibetan Terrier (Ketteritzsch et al. 2004), Afghan Hound (Roberts and Helper 1972), Standard Poodle (Rubin and Flowers 1972, Barnett and Startup, 1985), and Entlebucher Mountain Dog (Heitmann et al.

2005). The German Pinscher was shown as a breed predisposed to primary non-congenital cataracts (CAT) (Lepännen et al. 2001). Pedigree analysis indicated a monogenic autosomal recessive mode of inheritance for CAT (Menzel and Distl, 2010). The prevalence of CAT in the German Pinscher population in Germany has been estimated at 15.33% (Menzel and Distl, 2010). The majority of the affected German Pinschers develop bilateral cataracts, which are mostly located in the

Scanning 20 candidate genes for association with primary cataracts in German Pinschers

anterior cortical part of the lens. First signs of CAT in the German Pinscher population in Germany were registered at a mean age of 3.8 ± 1.6 years.

Because of the relatively late onset of CAT, it is difficult to exclude either CAT susceptible affected animals early in life from breeding or to ascertain unaffected carriers. A DNA test, which shows whether the dog is homozygous for a CAT-causing mutation or a heterozygous carrier or free from CAT-causing mutations, would be very helpful. Combined with an adequate breeding program, the prevalence of CAT could be effectively decreased in this breed.

To date, more than 20 genes that have to be considered as possible candidate genes for primary cataracts in dogs because these genes were found to be associated with hereditary cataracts in humans or mice (Reddy et al. 2004, Graw 2004, Hunter et al. 2006, Mellersh et al. 2006, Müller and Distl 2009). These genes encode structural or membrane transport proteins of the lens, transcription factors which are involved in eye and lens development, or enzymes which are necessary for lens metabolism (Graw 2004). Here, we investigated the 20 genes reported by Mellersh et al. (2006) and Müller and Distl (2009).

The candidate gene on CFA28, the paired-like homeodomain 3 (PITX3) gene, encodes a member of the RIEG/PITX homeobox family, which is in the bicoid class of homeodomain proteins. Members of this family act as transcription factors. This protein is involved in lens formation during eye development. Mutations of this gene have been associated with anterior segment mesenchymal dysgenesis and congenital cataracts in humans and mice (Semina et al. 1997, Semina et al. 1998, Rieger et al. 2001, Medina-Martinez et al. 2009). The PITX3 gene is conserved in chimpanzee, dog, cow, mouse, and rat. These observations made PITX3 a candidate for CAT in dogs.

4.3 Material and Methods

Animals, phenotypic data and DNA specimens

Ophthalmological data for the German Pinschers were provided by the Dortmunder Kreis (DOK), the German panel of the European Eye Scheme for diagnosis of

inherited eye diseases in animals. The ophthalmological examinations of the investigated dogs were carried out by veterinary specialists of the DOK and in accordance with the rules of the European College of Veterinary Ophthalmologists (ECVO). Only primary cataracts, and not secondary cataracts caused by diabetes or trauma, were recorded.

The Pinscher-Schnauzer-Klub e.V. (PSK) supplied pedigree data and we identified pedigrees with multiple CAT-affected dogs. For the present analysis, we chose 45 dogs from five different German Pinscher families (Figure 1). Altogether this study included 15 CAT-affected German Pinschers. In most of the affected dogs included in this analysis, the opafication of the lens was located in the anterior cortex. Thirteen (86.67%) of these 15 dogs had an anterior cortical cataract. Both eyes were affected in 11 animals (73.33%), while alterations were found only in the lens of the left or the right eye in the other four. Most of the dogs (about 70%) were examined two or three times. At least one unaffected dog was investigated from each family. Table 1 shows the distribution of localization and status of CAT for the 5 German Pinscher families included in our analyses. The 14 unaffected dogs were over 4.6 years old at the last ophthalmological examination. Unaffected dogs with the last ophthalmological examination at an age <4.5 years were classified as dogs with unknown phenotype (Table 1).

We also tested three unaffected dogs from other breeds as control animals. Two milliliters EDTA blood (BIOTA) was obtained from each dog, and DNA was extracted using QIAamp 96 DNA Blood kit (Qiagen, Hilden, Germany).

Genotyping of microsatellites

The microsatellites were in a distance to the particular candidate genes of less than one megabase (Mb). For the investigation of the 20 candidate genes we used microsatellites and PCR conditions according to Mellersh et al. (2006) and Müller and Distl (2009). We employed the microsatellites LIM2_1_107.53, LIM2_1_108.39, FTL_1_109.84, FTL_1_109.88, CRYAB_5_24.38, MAF_5_74.57, MAF_5_75.09, HSF4_5_85.24, HSF4_5_85.60, CRYBA1_9_35.59, CRYBA1_9_36.14, MIP_10_3.58, MIP_10_3.77, FOXE3_15_16.23, FOXE3_15_16.33, GJA8_17_61.25,

Scanning 20 candidate genes for association with primary cataracts in German GCNT2_35_13.04, GCNT2_35_13.64, CRYGA_37_19.28 and CRYGA_37_19.59.

For fine mapping of the linked region on canine chromosome 28 (CFA28), we chose three additional microsatellites from the Minimal Screening Set 2 (Guyon et al. 2003;

Clark et al. 2004) (C28176, FH2585, REN51i12) and five additional markers from the dog genome assembly 2.1 (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi):

CF28_11.36, CF28_16.26, CF28_17.26, CF28_17.89 and CF28_19.99.

The PCR primers and conditions are shown in Table 2. The PCR for genotyping of the microsatellites started at 94°C for 4 min, followed by 38 cycles at 94°C for 30 sec, optimum annealing temperature for 1 min, 72°C for 30 sec, and at 4°C for 10 min. All PCR reactions were performed in 11.5-µl reactions using 6 pmol of each primer, 0.2 µl dNTPs (100 µM) and 0.1 µl Taq-DNA-Polymerase (5 U/µl) (Q-Biogen, Heidelberg, Germany) in the reaction buffer supplied by the manufacturer for 2 µl template DNA.

The forward primers were labelled fluorescently with IRD700 or IRD800. For the analysis of the marker genotypes, PCR products were size-fractionated by gel electrophoresis on an automated sequencer (LI-COR, Lincoln, NE, USA) using 4%

polyacrylamide denaturing gels (Rotiphorese Gel40, Carl Roth, Karlsruhe). Allele sizes were detected using an IRD700- and IRD800-labeled DNA ladder; the genotypes were assigned by visual examination.

Non-parametric linkage analysis

A non-parametric multipoint linkage analysis was employed for the German Pinscher families using the MERLIN 1.1.2 (Abecasis et al. 2002). This analysis is based on allele sharing among affected individuals by identical-by-descent methods (Kong and Cox 1997). Haplotypes were estimated using MERLIN 1.1.2 with the option “best”. A case-control analysis based on χ²-tests for genotypes, alleles and trend of the most prevalent allele was also performed for the German Pinschers. The CASECONTROL and ALLELE procedures of SAS/Genetics were used for association tests, tests for

Hardy-Weinberg equilibrium of genotype frequencies and the estimation of allele frequencies (SAS Institute, 2010).

Structural and mutation analysis of PITX3

We searched the dog-expressed sequence tag (EST) archive (http://www.ncbi.nlm.nih.gov/genome/seq/CfaBlast.html) for ESTs by cross-species BLAST searches with the corresponding human reference mRNA sequences for PITX3 (NM_005029.3). We found a canine EST (DN379690.1) isolated from dog synovial tissue with 90% identity to the human PITX3 mRNA sequence. A significant match to this canine EST was identified on canine chromosome 28 by means of BLASTN searches of the canine EST against the dog genome assembly (Dog genome assembly 2.1). The genomic structure of the canine PITX3 gene was determined with the Spidey mRNA-to-genomic alignment program (http://www.ncbi.nlm.nih.gov/IEB/Research/Ostell/Spidey/index.html).

We sequenced all exons and the flanking intronic regions of the canine PITX3 gene for 6 affected and 4 unaffected German Pinschers out of the five families. PCR primers were designed using the Primer3 program (http://frodo.wi.mit.edu/cgi-bin/primer3_www.cgi) based on the canine EST (DN379690.1) and the genomic sequence for canine PITX3 (LOC 486856). The PCR primers are listed in Table 3. All PCRs were performed in 50-µl reactions using 20 pmol of each primer, 40 µM dNTPs, 0.5 U KappG-Robust-DNA-Polymerase (PeqLab, Erlangen, Germany) in the reaction buffer supplied by the manufacturer, 5x PCR Enhancer 1 (PeqLab, Erlangen, Germany), and 5% DMSO for 3 µl template DNA. The PCR conditions were: 95°C for 5 min followed by 38 cycles of 95 °C for 30 s, optimum annealing temperature for 30 s, 72°C for optimum elongation time, and 4°C for 10 min. All PCR products were cleaned using the Nucleo-Fast PCR purification kit (Macherey-Nagel) and directly sequenced with the DYEnamic ET Terminator kit (GE healthcare, München, Germany) and a MegaBACE 1000 capillary sequencer (GE Healthcare).

Sequence data were analysed with Sequencher version 4.7 (GeneCodes, Ann Arboer, MI, USA).

Scanning 20 candidate genes for association with primary cataracts in German Pinschers

4.4 Results and Discussion

Non-parametric linkage analysis

Table 4 shows the results of the non-parametric linkage analysis for all candidate gene flanking microsatellites. The highest and the only significant LOD score of 0.64 was obtained for the marker PITX3_28_16.97 which is located next to the candidate gene PITX3 on CFA28. We genotyped eight additional microsatellite markers on CFA28 to verify the linkage of these markers and to delimit the linked region on CFA28. After that, the highest LOD scores were obtained for the markers CF28_16.26, PITX3_28_16.97, CF28_17.26 and CF28_17.89, which are located in a region of 0.5 Mb proximal and 1.1 Mb distal of the PITX3 gene (Table 5). The maximum achievable Zmean was 7.05 and the corresponding value for the LOD score was 2.50 indicating that the power of the analysis was high enough to detect genome-wide significant linkage. The error probabilities for linked markers ranged from 0.015 to 0.02. The polymorphism information content of the individual markers was between 60.92 and 77.54%.

Structural and mutation analysis of PITX3

The canine EST for PITX3 (DN379690.1), which was found by cross-species BLAST searches with the corresponding human reference mRNA sequences, mapped to the same position as the annotated canine gene for PITX3 (LOC486856) and revealed an open reading frame of 1029 bp predicting a protein of 302 amino acids (Fig. 2).

We performed a mutation analysis for the PITX3 gene due to the significant linkage of the flanking markers. The canine PITX3 gene consists of four exons interrupted by one long (intron 1) and two short introns. The sequencing of exon 2 and 3 failed partly despite repeated attempts with different PCR conditions, the cause could be the abundance GC. The search for sequence variations within exon 1 and 4 and parts of exon 2 and 3 of this gene revealed a total seven single nucleotide polymorphisms (SNPs) as shown in table 6. Of these seven SNPs, five were located in the exon 3 sequence of PITX3 (g322T>C, g.324G>C, g.334T>C, g.336G>C, g.352G>C), while the others were located in intron 2 next to 5’ of exon 3 (g.309T>C,

g.311G>C). Two of the exonic SNPs (g322T>C, g.334T>C) were T/C transitions and cause an amino acid change from serin (S) to tryptophan (W) compared to the dog reference sequence (boxer). In the protein sequences of human and mice, the same protein sequence as in the German Pinscher were found (Fig. 6). The other exonic SNPs (g.324G>C, g.336G>C, g.352G>C) were transitions without cause of amino acid change. We found no polymorphisms within the German Pinscher breed.

Sequencing of the complete exons 2 and 3 for all animals should be performed in order to detect polymorphisms in these regions which could be possibly associated with the CAT phenotype in the German Pinscher.

Another approach is the analysis of cDNA of the PITX3 gene. Lens tissue of German Pinscher affected by CAT should be collected in connection with cataract surgery, for example with the phacoemulsification method with ultrasound. After removal from the eye, the lens tissue could be conserved using RNA-later solution.

To date, there are no other cataract candidate genes known in the linked region, and we could not find any possible functional cataract candidate genes by searching this region in the current dog genome assembly 2.1 (http://www.ncbi.

nlm.nih.gov/mapview/mapsearch.cgi?taxid=9615).

4.6 Acknowledgements

The authors express their gratitude to the Pinscher-Schnauzer-Klub 1895 e.V. for providing the pedigree data and the blood samples. The authors would like to thank the veterinary ophthalmologists of the Dortmunder Kreis (DOK) for providing the ophthalmological data.

Scanning 20 candidate genes for association with primary cataracts in German Pinschers

4.7 References

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Barnett KC. Hereditary cataract in the German Shepherd Dog. Journal of Small Animal Practice 1986; 27: 387-395.

Barnett KC, Startup FG. Hereditary cataract in the Standard Poodle. The Veterinary Record 1985 a; 117: 15-16.

Clark LA, Tsai KL, Steiner JM, Williams DA, Guerra T, Ostrander EA, Galibert F, Murphy KE. Chromosome-specific microsatellite multiplex sets for linkage studies in the domestic dog. Genomics 2004; 84: 550–554.

Curtis R, Barnett KC. A survey of cataracts in Golden and Labrador Retrievers.

Journal of Small Animal Practice 1989; 36: 277-286.

Davidson MG, Nelms SG. Diseases of the lens and cataract formation. In: Veterinary Ophthalmology 3^rd edition (ed. Gelatt KN), Williams & Wilkins: Philadelphia, 1999;

797-826.

Gelatt KN, MacKay EO. Prevalence of primary breed-related cataracts in the dog in North America. Veterinary Ophthalmology 2005; 8: 101-111.

Graw J. Congenital hereditary cataracts. International Journal of Developmental Biolology 2004; 48: 1031-1044.

Guyon R, Lorentzen TD, Hitte C, Kim L, Cadieu E, Parker HG, Quignon P, Lowe JK, Renier C, Gelfenbeyn B, et al. A 1-Mb resolution radiation hybrid map of the canine genome. Proceedings of the National Academy of Sciences of the United States of America 2003; 100: 5296–5301.

Heitmann M, Hamann H, Brahm R, Grußendorf H, Rosenhagen CU, Distl O. Analysis of prevalences of presumed inherited eye diseases in Entlebucher Mountain Dogs. Veterinary Ophthalmology 2005; 8: 145-151.

Hunter LS, Sidjani DJ, Johnson JL, Zangerl B, Galibert F, Andre C, Kirkness E, Talamas E, Acland GM, Aguirre GD. Radiation hybrid mapping of cataract genes in the dog. Molcular Vision 2006; 12: 588-596.

Ketteritzsch K, Hamann H, Brahm R, Grußendorf H, Rosenhagen CU, Distl O.

Genetic analysis of presumed inherited eye diseases in Tibetan Terriers. The Veterinary Journal 2004; 168: 151-159.

Kong A, Cox NJ. Allele-sharing models: LOD scores and accurate linkage tests. The American Journal of Human Genetics 1997; 61: 1179-1188.

Leppänen M, Martenson J, Mäki K. Results of ophthalmologic screening examinations of German Pinscher in Finland – a retrospective study. Veterinary Ophthalmology 2001; 4: 165-169.

Medina-Martinez O, Shah R, Jamrich M. Pitx3 controls multiple aspects of lens development. Developmental Dynamics 2009; 238: 2193-2201.

Mellersh CS, Pettitt L, Forman OP, Vaudin M, Barnett KC. Identification of mutations in HSF4 in dogs of three different breeds with hereditary cataracts. Veterinary Ophthalmology 2006; 9: 369-378.

Narfström K. Cataract in the West Highland White Terrier. Journal of Small Animal Practice 1981; 22: 467-471.

Reddy MA, Francis PJ, Berry V, Bhattacharya SS, Moore AT. Molecular genetic basis of inherited cataract and associated phenotypes. Survey of Ophthalmology 2004; 49: 300-315.

Rieger DK, Reichenberger E, McLean W, Sidow A, Olsen BR. A double-deletion mutation in the Pitx3 gene causes arrested lens development in aphakia mice.

Genomics 2001; 72: 61-72.

Roberts SR, Helper L. Cataracts in the Afghan Hounds. Journal of the American Veterinary Medical Association 1972; 160: 427-432.

Rubin LF, Flowers RD. Cataract in Golden Retrievers. Journal of the American Veterinary Medical Association 1974; 165: 457-458.

Rubin LF, Flowers RD. Inherited cataract in a family of Standard Poodles. Journal of the American Veterinary Medical Association 1972; 161: 207-208.

SAS Institute. SAS/Genetics, Version 9.1.3. Cary, NC, USA, 2005

Semina EV, Reiter RS, Murray JC. Isolation of a new homeobox gene belonging to the Pitx/Rieg family: expression during lens development and mapping to the

Scanning 20 candidate genes for association with primary cataracts in German Pinschers

aphakia region on mouse chromosome 19. Human Molcular Genetics 1997; 6:

2109-2116.

Semina EV, Ferrell RE, Mintz-Hittner HA, Bitoun P, Alward WL, Reiter RS, Funkhauser C, Daack-Hirsch S, Murray JC. A novel homeobox gene PITX3 is mutated in families with autosomal-dominant cataracts and ASMD. Nature Genetics 1998; 19: 167-170.

Yakely WL. A study of heritability of cataracts in the American Cocker Spaniel.

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4.8 Appendix

Table 1: Distribution of localisation and status of CAT for the five German Pinscher families (* unaffected dogs with the last ophthalmological examination at an age <4.5 years were classified as dogs with unknown phenotype)

Localisation of the

Scanning 20 candidate genes for association with primary cataracts in German Pinschers

Table 2: PCR primers with their product size range and annealing temperature (Ta) for the amplification of the additional microsatellites on canine chromosome 28 (CFA28).

Table 3: PCR primers, their position on canine chromosome 28 (CFA28), their product size and annealing temperature (Ta) for the amplification of the genomic sequence of the canine PITX3 gene

Primer Position on

Table 4: Non-parametric test statistics Zmean and LOD Score, their error probabilities (PZ, PL), polymorphism information content (PIC), χ²-tests for allele and genotype distribution of the case-control analysis, degrees of freedom (DF) and their corresponding error probabilities (P) for the candidate gene flanking microsatellite markers in the German Pinscher

Test for linkage Test for association Gene Marker Position

Scanning 20 candidate genes for association with primary cataracts in German Pinschers

FOXE3_15_16.23 16.23 42.11 -0.29 0.6 -0.04 0.7 1.50 3 0.68 1.23 3 0.53 CRYBB2_26_23.50 23.50 48.55 -0.60 0.7 -0.10 0.7 5.15 7 0.64 4.39 4 0.35 CRYBB2

(CFA26) CRYBB2_26_23.59 23.59 48.21 -0.60 0.7 -0.10 0.7 6.68 6 0.35 3.74 3 0.32 PITX3

Scanning 20 candidate genes for association with primary cataracts in German Pinschers

GCNT2_35_13.04 13.04 52.60 0.06 0.5 0.00 0.5 7.71 7 0.35 1.26 4 0.86 GCNT2

(CFA35) GCNT2_35_13.64 13.64 53.22 0.11 0.4 0.00 0.4 0.45 4 0.97 0.32 3 0.95 CRYGA_37_19.28 19.28 58.66 0.87 0.2 0.38 0.09 2.68 5 0.74 0.43 2 0.80 CRYGA

(CFA37) CRYGA_37_19.59 19.59 39.66 0.86 0.2 0.38 0.09 1.53 5 0.90 0.73 3 0.86

Scanning 20 candidate genes for association with primary cataracts in German Pinschers

corresponding error probabilities (P) for all microsatellite markers on canine chromosome 28 (CFA28) in the German Pinscher

Tests for linkage Tests for association Gene Marker Position

e genes for association with primary cataracts in German Pinschers

Scanning 20 candidate genes for association with primary cataracts in German Pinschers

Table 6: Single nucleotide polymorphisms (SNPs) within the German Pinscher for the canine PITX3 gene

SNP LOC486856 Location Effects on protein sequence g.309T>C Intron 2 no effects

g.311G>C Intron 2 no effects

g322T>C Exon 3 S>W

g.324G>C Exon 3 no effects

g.334T>C Exon 3 S>W

g.336G>C Exon 3 no effects g.352G>C Exon 3 no effects

Figure 1: Pedigrees of the five German Pinscher families which were used for the candidate genes scan for association with primary cataracts.

Haplotypes for the three linked microsatellite markers and their two flanking markers were given over the symbols for each proband.

First line: CF28_16.26 Second line: PITX3_28_16.97 Third line: CF28_17.26 Fourth line: CF28_17.89 Fifth line: CF28_19.99 Family 1:

Scanning 20 candidate genes for association with primary cataracts in German Pinschers

Family 2:

Family 3:

Scanning 20 candidate genes for association with primary cataracts in German Pinschers

Family 4:

Family 5:

Scanning 20 candidate genes for association with primary cataracts in German Pinschers

Figure 2: Alignment of the canine PITX3 protein (302 amino acids) of two different dog breeds (boxer and German Pinscher (GP)) with the known orthologous protein sequences. The sequences were derived from GenBank entries with the association nos. NP_032878 (mouse) and NP_005020 (human). Identical residues to the German Pinscher are indicated by asterisks. Amino acids 40 (SNP g.322T>C) and 44 (SNP g.334T>C) are indicated by a grey underlay).

GP MEFGLLSEAEARSPALSLSDAGTPHPPLPEHGCKGQEHSDSEKASASLPG 50 boxer ****************************************W***W***** 50 mouse ******G******************************************* 50 human **************************Q*********************** 50

GP GSPEDGSLKKKQRGQRTHFTSQQLQELEAPFQRNRYPDMSTREEIAVWTN 100 boxer *************G************************************ 100 mouse *****************************T******************** 100 human *****************************T******************** 100

GP LTEARVGVWFKNRRAKWRKRERSQQAELCKGGFAAPLGGLVPPYEEVYPG 150 boxer ************************************************** 150 mouse ******R******************************************* 150 human ******R************************S****************** 150

GP YSYGNWPPKALGPPLAAKTFPFAFNSVNVGPLASQPVFSPPSSIAASMVP 200 boxer ************************************************** 200 mouse ***********A************************************** 200 human ***********A************************************** 200

GP SAAAAPGTVPGPGALQGLGGGPPGLAPAAVSSGAVSCPYASAAAAAAAAA 250 boxer ************************************************** 250 mouse ********************A***************************** 250 human ************************************************** 250

GP SSPYVYRDPCNSSLASLRLKAKQHASFSYPAVPGPPPAANLSPCQYAVER 300 boxer ************************************************** 300 mouse ************************************************** 300 human ********************************H***************** 300

GP PV 302 boxer ** 302 mouse ** 302 human ** 302

CHAPTER 6

General discussion

6 General discussion

In our population analysis for persistent right aortic arch (PRAA) in the German Pinscher, which was performed at the beginning of the present thesis, we discovered a rare combination of vascular anomalies that has only been described in two isolated cases of other dog breeds before (House et al., 2005). In the German pinscher, the occurrence of any form of PRAA was not previously known. We can conclude from our analyses that PRAA is an inherited disease in the German Pinscher. This study did not analyze the mode inheritance of PRAA in the German Pinscher but excluded specific forms of inheritance. However, PRAA in other dog breeds is believed as a complex polygenetic trait (Patterson, 1989). The proof of the mode of inheritance of PRAA is difficult because of the lack of profound knowledge of

Im Dokument Population and molecular genetic analyses of persistent right aortic arch and primary cataracts in the German Pinscher (Seite 78-116)