Determination of Inheritance of Single Congenital Portosystemic Shunts in Yorkshire Terriers

Introduction

Congenital portosystemic shunts (PSS) are vascular anomalies that are thought to occur with abnormal fusion or incomplete regression of embryonic abdominal vasculature.^1–3 The normal canine fetal hepatic circulation is derived from the vitelline and umbilical veins. Within the fetus, fusion and degeneration of various sections of these vessels result in formation of the hepatic sinusoids, portal system, and the fetal ductus venosus. At birth, the ductus venosus in normal dogs closes functionally within days and is structurally obliterated within 3 weeks.¹ Development of intrahepatic PSS could result from incomplete closure of the ductus venosus or retention of segments of vitelline vessels that should have degenerated.^1–5 Congenital extrahepatic shunts probably develop from abnormal anastomoses of the vitelline veins with the cardinal vessels, which form the tributaries of the caudal vena cava and its prehepatic segment.¹

Environmental and genetic factors, alone or in combination, may influence the development of congenital anomalies. When the incidence of certain diseases increases in one breed compared to that of the general population, a genetic predisposition is suspected.⁶ At the University of Tennessee College of Veterinary Medicine, 35% of all dogs diagnosed with congenital PSS over a 12-year period were Yorkshire terriers, and this percentage had been increasing over the last 10 years. Based on this information, a genetic cause for PSS in Yorkshire terriers was suspected. The specific objectives of this study were to determine the incidence of PSS in Yorkshire terriers compared to other breeds; compare the coefficient of inbreeding for Yorkshire terriers with and without PSS; determine whether a common ancestor was present in the pedigrees of Yorkshire terriers with PSS; and determine the mode of inheritance of the disease.

Materials and Methods

The Veterinary Medical Database (VMDB)^a was searched for records of dogs that presented to 24 participating veterinary teaching hospitals with single congenital PSS from 1980 to 2001. Search terms included congenital PSS, congenital portal caval shunt, congenital defect portal vein, congenital atresia portal vein, and congenital portoazygos shunt. The numbers of all dogs and of Yorkshire terriers entered into the database per year were recorded. If dogs made multiple visits, only one visit was recorded per year, and rechecks in subsequent years were not included. Incidences (shunts per year) and odds ratios for Yorkshire terriers and for all other dogs combined were calculated.

For the pedigree study, Yorkshire terriers with single congenital extrahepatic PSS (i.e., affected dogs) and unaffected Yorkshire terriers (i.e., controls) were identified through record searches at the University of Tennessee and Washington State University Veterinary Teaching Hospitals and through Internet and mail solicitations to Yorkshire terrier owners and breeders. Affected dogs were included if their PSS had been confirmed by exploratory laparotomy, contrast portography, or necropsy. Dogs were included in the control population if their liver function was normal, as evaluated by bile acid measurements. To evaluate bile acids, dogs were fasted for 12 hours before the first serum sample was taken, and then they were individually fed at least 2 tablespoons of a recovery diet^b or other high-calorie food. A serum sample was taken 2 hours after eating. Samples were obtained with a 20-gauge needle and 3-cc syringe to reduce hemolysis. Bile acids were measured by spectrophotometric analysis. Dogs were considered normal if fasting bile acids were <20 μmoles/L and postprandial bile acids were <25 μmoles/L.

Pedigrees of the normal and affected Yorkshire terriers were obtained from the American Kennel Club and entered into a data-collecting system.^c Pedigrees were included if at least five generations were listed. Pedigrees of full siblings were excluded. Wright’s coefficient of inbreeding for up to 10 generations was calculated for each dog using the same data-collecting system. Five- and eight-generation pedigrees were analyzed for common ancestors. Progenitors from each pedigree were listed for affected and normal dogs, and numbers of citings per group were recorded.

Odds ratios were calculated with an online statistical software program.^d Wright’s coefficient of inbreeding for normal and affected dogs was compared using an unpaired t-test. Presence of common ancestors was compared with a Fisher’s exact test. A P value of 0.05 was considered significant.

Two affected dogs were bred after correction of their PSS. Pre- and postprandial bile acids were measured in offspring. If bile acids were increased, the presence of a shunt would be confirmed with scintigraphy and exploratory laparotomy.

Results

From 1980 to 2001, records of 1,227,236 dogs were included in the VMDB. Of these dogs, 16,537 were Yorkshire terriers. A total of 1,847 dogs were reported to have a congenital PSS; 593 of these were Yorkshire terriers. Total percentages of all dogs and Yorkshire terriers with PSS were 0.18% and 3.6%, respectively. Total percentage of Yorkshire terriers in the VMDB with PSS in 2001 was 5%, and of all dogs combined in 2001, the total percentage was 0.35%. Over the 21-year period, the odds ratio for occurrence of congenital PSS in Yorkshire terriers compared to all other breeds combined was 35.87 (95% confidence interval, 32.53 to 39.64). The incidence of congenital PSS in Yorkshire terriers entered into the VMDB increased more than 11 times over 20 years, from 0.006 (0.6%) in 1980 to 0.07 (7%) in 2000 [see Figure], while the incidence in all other dogs combined increased from 0.0014 (0.14%) in 1980 to 0.0027 (0.27%) in 2000.

Pre- and postprandial bile acids were provided for or measured in 140 Yorkshire terriers ≥6 weeks of age and were normal in 51 dogs. The majority of the control dogs were located in the state of Tennessee at the time of sampling; however, normal dogs were also included from California, Washington, Oregon, Florida, Canada, and other areas of the country. Origins of each dog were not recorded; however, owners and breeders noted that some of the control dogs had been purchased from other areas of the country before being shipped to their final destination.

Forty-five Yorkshire terriers in the PSS group were diagnosed with single congenital extrahepatic PSS at surgery or necropsy. The origins of the affected dogs had not been recorded; most were located in the southeastern United States at the time of the surgery; however, affected dogs from Canada, California, Oregon, Washington, and other areas of the country were also included. All affected dogs had increased bile acids. Age and sex distribution of affected dogs did not differ significantly from control dogs. Two Yorkshire terriers with PSS were father and son, and two were half sisters. Complete pedigrees were available for 31 affected dogs. The pedigree of one dog was excluded from the Wright’s coefficient calculations because of lack of complete five-generation pedigree on one side. Wright’s coefficient of inbreeding tended to be greater for the 30 dogs with single congenital extrahepatic PSS (0.054±0.094) than for the 51 dogs with normal bile acids (0.028±0.042; P=0.09).

In the PSS group, a total of 1,259 different dogs were listed at least once in the 31 five-generation pedigrees. Of studs or bitches listed more than five times, one dog (dog A) was listed a total of 20 times, two dogs were listed 11 times, one dog was listed 10 times, three dogs were listed eight times, and 10 dogs were listed six times. Dog A was related to most of the dogs listed six or more times in the five-generation PSS pedigrees.

In the control group, a total of 1,047 different dogs were listed at least once in the 51 five-generation pedigrees. Dog A was listed a total of 53 times.

Three of 31 affected dogs lacked six-generation pedigrees on one side and were not included in the eight-generation pedigree analysis. Twenty-two of 28 dogs (78.6%) with PSS had dog A in their eight-generation pedigrees. Fifty of 51 dogs (98%) with normal bile acids had dog A in their eight-generation pedigrees. No other significant common ancestors were found in either group, except for those that were direct descendents or parents of dog A.

Three puppies were produced from a single mating of two Yorkshire terriers with previously corrected single congenital extrahepatic PSS. No fetal resorptions were noted during serial prenatal ultrasonographic screenings. One puppy died at birth after being entrapped in the pelvic canal; two live puppies were delivered by cesarean section. At necropsy, the ductus venosus of the dead puppy was still patent, and, therefore, the hepatic parenchyma was underperfused. No other abnormalities were detected. Pre- and postprandial bile acids of the two remaining puppies were normal at 6 and 10 weeks after birth. Both puppies are clinically normal at 6 months of age.

Discussion

Classification of a disease as inherited can be difficult if the disease is relatively rare in the population and if the phenotypic appearance of disease is not easily recognizable. In general, a genetic contribution to disease expression is suspected when the reported incidence is higher in one breed than in others; when environmental factors common to members of the breed are insufficient to account for the observed differences in incidence; and when the same or a very similar disorder is proven inherited in another species of animal or in humans.⁶ Portosystemic shunts are considered hereditary in Irish wolfhounds, based on increasing incidence and evidence of familial relationships in affected animals.^7–9 Over-representation of Australian cattle dogs and Maltese among dogs affected with single congenital PSS has also been reported.¹⁰ In humans, a recessive mode of inheritance is suspected for familial patent ductus venosus.¹¹ Genetic predispositions have also been noted in other portal vascular diseases, including muscular portal venular hypertrophy with secondary acquired portosystemic shunting in cocker spaniels and hepatic microvascular dysplasia (HMD) in Cairn terriers.¹²¹³ Based on the results of this study, Yorkshire terriers listed in the VMDB had a risk of being diagnosed with PSS that was 35 times greater than all other breeds combined. This over-representation is strongly suggestive of a genetic predisposition to the disease.

Increasing annual incidence was also detected in the Yorkshire terrier population registered with the VMDB. Incidence of PSS in Irish wolfhounds registered to the Dutch Kennel Club increased yearly from <1% in 1985 to almost 3% in 1992;⁷ prevalence was 2.3% in 1997.⁹ Incidence may increase from increased matings of carriers, inbreeding, or improvement of detection methods for disease.⁶ Incidence and odds ratio for Yorkshire terriers in the VMDB may not accurately reflect those of the general Yorkshire terrier population; because of the emphasis on referrals and advanced diagnostic medicine at veterinary teaching hospitals, there may be a bias toward diagnosis of this disease at those centers. Additionally, increasing awareness among the veterinary and lay populations may have led to more bile acid testing of Yorkshire terriers than other breeds. However, since not all of the control population in the VMDB was tested for abnormal liver function, it is possible that nonselection bias could also falsely lower these numbers.⁶

When pedigrees from all Irish wolfhounds registered to the Dutch Kennel Club from 1985 through 1992 were analyzed and divided into 14 clusters (related groups), affected dogs fell into four of these clusters. The relatedness of dogs within the affected clusters, however, did not differ from the overall relatedness of all clusters.⁸ Common ancestors significant to the affected Yorkshire terrier population could not be determined in the study reported herein, and the Wright’s coefficient of inbreeding, while twice as high for the PSS group, was not significantly different for that of the control population. Analysis of a larger number of pedigrees may provide more significant results.

In Irish wolfhounds, mode of inheritance could not be determined with pedigree analysis.⁷⁹ In a kindred of Cairn terriers with HMD, breeding of an affected male with two affected females produced six progeny, all of which had HMD.¹³ When the same male was bred to a female with PSS, four progeny were produced, two of which had HMD. When another affected male was bred to his affected daughter, one of three progeny had HMD and one had PSS. Based on this information, the mode inheritance of HMD is thought to be an autosomal dominant trait with or without variable expression, or a polygenic trait with simple or complex expression.¹³ Based on the presenting population and initial results of breeding of two affected Yorkshire terriers, PSS in Yorkshire terriers is not sex-linked, simple autosomal dominant, or simple autosomal recessive. Congenital PSS are reported in relatively equal distribution in male and female dogs.⁷¹⁴¹⁵ Dogs with sex-linked traits are usually male and result from the matings of normal parents; additionally, with sex-linked traits, all offspring of two affected parents are affected.⁶ Pure autosomal dominant traits do not skip generations, and every affected offspring has at least one affected parent.⁶¹⁶ Pure autosomal recessive traits may skip generations; however, all offspring of two affected parents are affected.⁶¹⁶

Because transmission of PSS to offspring does not follow the theories of simple Mendelian genetics, it is suspected that expression of the PSS phenotype may be limited by incomplete penetrance, variable expressivity, or multifactorial components.⁶¹⁶ Penetrance is the proportion of animals with a particular genotype that exhibit the phenotype normally associated with that genotype. Penetrance is incomplete if the phenotypic change is seen in <100% of animals with the genotype.⁶ Display of the affected phenotype may be altered by various environmental factors. For instance, development or expression of hip dysplasia may be affected by the amount of caloric intake and extent of exercise during early growth, or it may be overlooked if physical and radiographic examinations are not performed.⁶ Additionally, there may be presence of alleles at other loci that tend to produce healthy joints. A trait determined by combinations of both environmental and genetic factors is considered to be multifactorial.⁶¹⁶

A disease controlled by genes with variable expressivity may display a range of phenotypic variations. For example, matings of poodles with patent ductus arteriosus (PDA) can produce different grades of PDA, including normal puppies, puppies with partial closure of the PDA (i.e., ductus diverticulum), and puppies with complete patency of the PDA. If PDAs result from a single gene, then the existence of more than one grade of the defect indicates variable expression of that gene.⁶ Because of the similarity between PSS and HMD,¹³ it might be possible that HMD is a phenotypic variation of the gene for PSS and that development of a macroscopic shunt may also rely on additional genetic or environmental factors.

Bile acids were used as a screening test for normal dogs in this study. Concentrations of bile acids in young puppies are similar to those of adult dogs, so Yorkshire terriers in the control and breeding populations were tested as young as 6 weeks of age in this study.^17–19 Previous reports have used similar reference ranges for evaluation of liver function, although higher values can be found in normal dogs.^18–20 Interestingly, 64% of healthy Yorkshire terriers tested had bile acids greater than the reference range in this study. Because liver biopsies were not performed on these dogs, it is unknown whether the bile acids were increased from underlying hepatic disease, from other physiological variables, or from interference with the spectrophotometric assay.¹⁹²⁰ Five of the healthy dogs with postprandial bile acids >40 μmoles/L (range, 42 to 105 μmoles/L) were retested 1 to 3 months later, and four of these dogs had bile acid concentrations within the reference range. Bile acids may increase with a variety of liver dysfunctions or be altered by hemolysis, lipemia, or early or incomplete emptying of the gallbladder; therefore, reevaluation of bile acids is recommended when results of bile acid testing are questionable.¹⁹²¹

Conclusion

Although the mode of inheritance could not be determined, it is likely that the development of single congenital extrahepatic PSS in Yorkshire terriers has a genetic underpinning. Because the mode of inheritance is unknown, the most effective way in which individual breeders can control inherited disorders is to minimize the level of inbreeding in offspring by mating unaffected individuals that are as unrelated as possible.⁶⁸ While geographic distributions of affected and control animals were similar, it is possible that results of the study might vary if pedigrees from animals in different parts of the country were compared. Identification of familial tendencies and evaluation of a larger population could help determine breeding strategies for future generations. Until the mode of inheritance is determined, it is strongly recommended that bile acid testing be performed on all Yorkshire terriers, particularly if they are to be used for breeding.