Genetic analysis of ventricular arrhythmia in young German Shepherd Dogs.

BACKGROUND: Ventricular arrhythmias (VA) and sudden death are inherited in German Shepherd Dogs (GSDs). OBJECTIVES: To estimate the genetic parameters (heritabilities and correlations) of 3 traits of VA (single premature ventricular complexes (PVCs), 2 consecutive PVCs (couplets), and 3 or more consecutive PVCs-ventricular tachycardia [VT]). ANIMALS: Three hundred and ninety-eight GSDs. METHODS: Prospective, observational, case control study. Dogs were phenotyped by 24-hour ambulatory ECG from 6 to 45 weeks of age. Edited ECG records included the number of incidents of (1) single PVCs, (2) couplets, and (3) VT. RESULTS: A data set of 1,239 Holter records from 398 GSDs was used to estimate genetic variables. Phenotypic correlations for affectedness (binarily coded 0/1) of the 3 traits ranged from 0.55 to 0.74, whereas correlations for severity (continuous values of 24-hour VA counts) ranged from 0.26 to 0.39. Estimates of genetic correlation among the severity traits were 0.06 to 0.27. Estimated heritabilities were 0.54, 0.54, and 0.46 for affectedness and 0.33, 0.69, and 0.69 for severity of PVCs, couplets, and VT, respectively. Month and year of birth and age at ECG recording had significant effects on all 3 traits. Season of ECG recording had a significant effect on the number of single PVCs, but not couplets or incidents of VT. Age of onset differed, with single PVCs appearing an average of 4 days earlier than couplets and VT. CONCLUSION: These results imply a strong genetic component for this disease but suggest that differences in the 3 traits should be taken into consideration in studies to identify the underlying genes.

Multifactorial genetics of exencephaly in SELH/Bc mice.

BACKGROUND: The SELH/Bc mouse strain has 10-30% exencephaly and is an animal model for human neural tube closure defects. This study examined the number of causative genes, their dominance relationships, and linkage map positions. METHODS: The SELH/Bc strain (S) was crossed to the normal LM/Bc strain (L) and frequencies of exencephaly were observed in the F(1), BC(1), and F(2) generations. 102 F(2) males were individually testcrossed by SELH/Bc. The extremes, the 10 highest and 10 zero exencephaly-producing F(2) sires, were typed for 109 SSLP marker loci in a genome screen. Next, the resultant five provisional chromosomal regions were tested for linkage in 31 F(2) exencephalic embryos. Finally, 12 males, SS or LL for the Chr 13 region on an LM/Bc background, were testcrossed by SELH/Bc. RESULTS: The exencephaly frequencies in the F(1) (0.3%), BC(1) (4.4%), and F(2) (3.7%), and the distribution of F(2) males' testcross values (0-15.5%), indicated that the high risk of exencephaly in SELH/Bc is due to the cumulative effect of two or three loci. Linkage studies indicated the location of semidominant exencephaly-risk genes on Chr 13 near D13Mit13 (P < 0.001), Chr 5 near D5Mit168 (P < 0.025), and possibly Chr 11 near D11Mit10 (P < 0.07). The gene on Chr 13, Exen1, and the strong role of other loci were confirmed by the congenic males. CONCLUSIONS: The high risk of exencephaly in SELH/Bc mice is caused by the cumulative effect of two to three semidominant genes. Candidate genes include Msx2, Madh5, Ptch, and Irx1 (Chr 13) and Actb and Rac1 (Chr 5).

Molecular and phenotypic analysis of Attractin mutant mice.

Mutations of the mouse Attractin (Atrn; formerly mahogany) gene were originally recognized because they suppress Agouti pigment type switching. More recently, effects independent of Agouti have been recognized: mice homozygous for the Atrn(mg-3J) allele are resistant to diet-induced obesity and also develop abnormal myelination and vacuolation in the central nervous system. To better understand the pathophysiology and relationship of these pleiotropic effects, we further characterized the molecular abnormalities responsible for two additional Atrn alleles, Atrn(mg) and Atrn(mg-L), and examined in parallel the phenotypes of homozygous and compound heterozygous animals. We find that the three alleles have similar effects on pigmentation and neurodegeneration, with a relative severity of Atrn(mg-3J) > Atrn(mg) > Atrn(mg-L), which also corresponds to the effects of the three alleles on levels of normal Atrn mRNA. Animals homozygous for Atrn(mg-3J) or Atrn(mg), but not Atrn(mg-L), show reduced body weight, reduced adiposity, and increased locomotor activity, all in the presence of normal food intake. These results confirm that the mechanism responsible for the neuropathological alteration is a loss--rather than gain--of function, indicate that abnormal body weight in Atrn mutant mice is caused by a central process leading to increased energy expenditure, and demonstrate that pigmentation is more sensitive to levels of Atrn mRNA than are nonpigmentary phenotypes.

A biochemical function for attractin in agouti-induced pigmentation and obesity.

Agouti protein, a paracrine signaling molecule normally limited to skin, is ectopically expressed in lethal yellow (A(y)) mice, and causes obesity by mimicking agouti-related protein (Agrp), found primarily in the hypothalamus. Mouse attractin (Atrn) is a widely expressed transmembrane protein whose loss of function in mahogany (Atrn(mg-3J)/ Atrn(mg-3J)) mutant mice blocks the pleiotropic effects of A(y). Here we demonstrate in transgenic, biochemical and genetic-interaction experiments that attractin is a low-affinity receptor for agouti protein, but not Agrp, in vitro and in vivo. Additional histopathologic abnormalities in Atrn(mg-3J)/Atrn(mg-3J) mice and cross-species genomic comparisons indicate that Atrn has multiple functions distinct from both a physiologic and an evolutionary perspective.

Secreted and membrane attractin result from alternative splicing of the human ATRN gene.

Attractin, initially identified as a soluble human plasma protein with dipeptidyl peptidase IV activity that is expressed and released by activated T lymphocytes, also has been identified as the product of the murine mahogany gene with connections to control of pigmentation and energy metabolism. The mahogany product, however, is a transmembrane protein, raising the possibility of a human membrane attractin in addition to the secreted form. The genomic structure of human attractin reveals that soluble attractin arises from transcription of 25 sequential exons on human chromosome 20p13, where the 3' terminal exon contains sequence from a long interspersed nuclear element-1 (LINE-1) retrotransposon element that includes a stop codon and a polyadenylation signal. The mRNA isoform for membrane attractin splices over the LINE-1 exon and includes five exons encoding transmembrane and cytoplasmic domains with organization and coding potential almost identical to that of the mouse gene. The relative abundance of soluble and transmembrane isoforms measured by reverse transcription-PCR is differentially regulated in lymphoid tissues. Because activation of peripheral blood leukocytes with phytohemagglutinin induces strong expression of cell surface attractin followed by release of soluble attractin, these results suggest that a genomic event unique to mammals, LINE-1 insertion, has provided an evolutionary mechanism for regulating cell interactions during an inflammatory reaction.

Mahogany/attractin: en route from phenotype to function.

The mouse mahogany mutation affects melanocortin signaling pathways that regulate energy homeostasis and hair color. The gene mutated in mahogany mice encodes attractin, a large transmembrane protein that is broadly expressed and conserved among multicellular animals. Mouse attractin is likely to have additional roles outside melanocortin signaling, and cloning of the gene provides information that can be used to form testable hypotheses about its biochemical function.

Distribution of Mahogany/Attractin mRNA in the rat central nervous system.

The Mahogany/Attractin gene (Atrn) has been proposed as a downstream mediator of Agouti signaling because yellow hair color and obesity in lethal yellow (A(y)) mice are suppressed by the mahogany (Atrn(mg)) mutation. The present study examined the distribution of Atrn mRNA in the brain and spinal cord by in situ hybridization. Atrn mRNA was found widely distributed throughout the central nervous system, with high levels in regions of the olfactory system, some limbic structures, regions of the brainstem, cerebellum and spinal cord. In the hypothalamus, Atrn mRNA was found in specific nuclei including the suprachiasmatic nucleus, the supraoptic nucleus, the medial preoptic nucleus, the paraventricular hypothalamic nucleus, the ventromedial hypothalamic nucleus, and the arcuate nucleus. These results suggest a broad spectrum of physiological functions for the Atrn gene product.

The mouse mahogany locus encodes a transmembrane form of human attractin.

Agouti protein and agouti-related protein are homologous paracrine signalling molecules that normally regulate hair colour and body weight, respectively, by antagonizing signalling through melanocortin receptors. Expression of Agouti is normally limited to the skin, but rare alleles from which Agouti is expressed ubiquitously, such as lethal yellow, have pleiotropic effects that include a yellow coat, obesity, increased linear growth, and immune defects. The mahogany (mg) mutation suppresses the effects of lethal yellow on pigmentation and body weight, and results of our previous genetic studies place mg downstream of transcription of Agouti but upstream of melanocortin receptors. Here we use positional cloning to identify a candidate gene for mahogany, Mgca. The predicted protein encoded by Mgca is a 1,428-amino-acid, single-transmembrane-domain protein that is expressed in many tissues, including pigment cells and the hypothalamus. The extracellular domain of the Mgca protein is the orthologue of human attractin, a circulating molecule produced by activated T cells that has been implicated in immune-cell interactions. These observations provide new insight into the regulation of energy metabolism and indicate a molecular basis for crosstalk between melanocortin-receptor signalling and immune function.

Molecular pharmacology of Agouti protein in vitro and in vivo.

Agouti protein and Agouti-related protein (Agrp) are paracrine signaling molecules that act by antagonizing the effects of melanocortins, and several alternatives have been proposed to explain their mechanisms of action. Genetic crosses in a sensitized background uncover a phenotypic difference between overexpression of Agouti and loss of Mc1r function, demonstrate that a functional Mc1r is required for the pigmentary effects of Agouti, and suggest that Agouti protein can act as an agonist of the Mc1r in a way that differs from alpha-MSH stimulation. In vitro, Agouti protein inhibits melanocortin action by two mechanisms: competitive antagonism that depends on the carboxyterminus of the protein, and downregulation of melanocortin receptor signaling that depends on the aminoterminus. Our findings provide evidence of a novel signaling mechanism whereby alpha-MSH and Agouti protein function as independent ligands that inhibit each other's binding and transduce opposite signals through a single receptor.

Genetic studies of the mouse mutations mahogany and mahoganoid.

The mouse mutations mahogany (mg) and mahoganoid (md) are negative modifiers of the Agouti coat color gene, which encodes a paracrine signaling molecule that induces a swithc in melanin synthesis from eumelanin to pheomelanin. Animals mutant for md or mg synthesize very little or no pheomelanin depending on Agouti gene background. The Agouti protein is normally expressed in the skin and acts as an antagonist of the melanocyte receptor for alpha-MSH (Mc1r); however, ectopic expression of Agouti causes obesity, possibly by antagonizing melanocortin receptors expressed in the brain. To investigate where md and mg lie in a genetic pathway with regard to Agouti and Mc1r signaling, we determined the effects of these mutations in animals that carried either a loss-of-function Mc1r mutation (recessive yellow, Mc1re) or a gain-of-function Agouti mutation (lethal yellow, Ay). We found that the Mc1re mutation suppressed the effects of md and mg, but that md and mg suppressed the effects of Ay on both coat color and obesity. Plasma levels of alpha-MSH and of ACTH were unaffected by md or mg. These results suggest that md and mg interfere directly with Agouti signaling, possibly at the level of protein production or receptor regulation.

Exencephaly and cleft cerebellum in SELH/Bc mouse embryos are alternative developmental consequences of the same underlying genetic defect.

SELH/Bc inbred mice have ataxia in 5-10% of young adults and exencephaly in 10-20% of newborns. SELH/Bc mice also have a high rate of spontaneous mutation and therefore it could not be assumed that these two abnormalities share the same genetic cause. Previously, we have shown that the liability to exencephaly in SELH/Bc mice is multifactorial, involving two to three loci, and that all the ataxics have a midline cleft cerebellum. The purpose of the present study was to resolve the genetic relationship between liability to exencephaly and liability to cleft cerebellum. We tested whether these traits were transmitted together by segregating F2 males; cotransmission would indicate that both traits are probably caused by the same genes. Approximately 100 embryos from each of 25 F2 sires from a cross between SELH/Bc and the normal LM/Bc strain were scored for exencephaly and the non-exencephalic embryos were scored for cleft cerebellum. The range of exencephaly production by these 25 F2 sires was 0% to 16%; the sires had been selected to represent the extremes of the range of exencephaly production. We found that the 10 sires that produced no exencephaly also produced no cleft cerebellum and 12 of the 15 sires that produced some exencephaly also produced some cleft cerebellum. This indicated strongly that the two traits are transmitted together (Fisher's exact test, P < 0.0002). Furthermore, within exencephaly-producing sires, the specific frequencies of the two traits were significantly positively correlated (Spearman rs = 0.58; P < 0.05), indicating that the same multifactorial risk factors influence both traits. All SELH/Bc embryos omit one normal initiation site of cranial neural tube closure, Closure 2. In a previous study, absence of the Closure 2 initiation site of cranial neural tube closure has been shown to be genetically correlated with liability to exencephaly. In the second part of the present study, the same Closure 2 data from eight of the F2 sires were observed to be significantly positively correlated with liability to cleft cerebellum (Spearman rs = 0.83; P < 0.05). The results of this genetic approach have supported the hypothesis, based on observation of embryos, that one basic multifactorial genetic defect in SELH mice leads to an abnormal cranial neural tube closure mechanism, to exencephaly to cleft cerebellum, and to ataxia.

Genetically determined absence of an initiation site of cranial neural tube closure is causally related to exencephaly in SELH/Bc mouse embryos.

The SELH/Bc mouse strain (SELH) has a high frequency of the lethal neural tube closure defect, exencephaly, in newborns and embryos. Previous work has shown that all SELH mouse embryos have an abnormal mechanism of rostral neural tube closure. They lack initiation of contact and fusion of the cranial neural tube at the prosencephalon/mesencephalon boundary [Closure 2), and undergo closure by extension of a more rostral site of fusion. This process fails in 10-20% of embryos, where the mesencephalic folds remain unelevated, resulting in exencephaly. Previous work has also shown that the cause of liability to exencephaly in SELH mice is multigenic, involving a small number of loci. The purpose of the present study was to test the hypothesis that the genes causing the lack of Closure 2 also cause the liability to exencephaly in SELH, by observation of their joint transmission from genetically segregating animals. A concurrent mapping study provided the necessary genetic material, a segregating F2 generation from a cross of SELH with the normal LM/Bc strain. The genetic liability to exencephaly transmitted by individual F2 sires had been measured by the frequencies of exencephalic day 14 embryos they produced in test-crosses with SELH females. A selected subset of 13 of these test-crossed F2 sires was bred with a second set of SELH females, and the embryos were examined earlier, during the period of neural tube closure, on days 8 and 9 of gestation, to determine the presence of Closure 2. Six F2 sires were among the highest exencephaly producers (6-11%), six were among the lowest (0%), and one was intermediate (5%). Among embryos at the appropriate stage for scoring, the presence of Closure 2 was observed to be inversely correlated with the later risk of exencephaly, being present in 93% (71/76) from the low-risk sires and 35% (36/103) from the high-risk sires. In each case, the remaining embryos had a closure mechanism like that of SELH embryos. Among the individual intermediate- and high-risk sires, there was also a clear correlation between the frequency of exencephaly in older embryos and the frequency of lack of Closure 2 in early embryos (rs = 0.88; P < 0.05). This study demonstrates that high liability to exencephaly and absence of Closure 2 are genetically transmitted together. That is, the cause of the lack of Closure 2 in SELH mice is shown to be also the probable cause of the high liability to exencephaly.

Development of the cerebellar defect in ataxic SELH/Bc mice.

In SELH/Bc mice, 5-10% of young adults are ataxic, due to a midline cleft in the cerebellum. An additional 10-20% of SELH/Bc embryos have exencephaly and die at birth. All SELH/Bc embryos omit a normal step in cranial neural tube closure, initiation of fusion at Closure 2. In the 80-90% that complete cranial neural tube closure, the last region of closure, on late D9, is the region of the prospective cerebellum, and its closure is late. We postulated that the cleft cerebellum in ataxic SELH/Bc mice derives from this delay in neural tube closure and predicted that we would see evidence of a cerebellar midline cleft in all earlier stages after cranial neural tube closure is normally complete. In the present study we show that the cerebellum is cleft in a 7-9% proportion of SELH/Bc D16 fetuses (2/28) and D11 embryos (15/167), and that the defect is detectable on D10. In these abnormal D16 fetuses, D11 and D10 embryos, there is a gap in midline continuity of cerebellar neuroepithelium, a finding consistent with our hypothesis that the neuroepithelium in this region fails to complete fusion in those embryos. We also show that cerebella of adult SELH/Bc ataxic mice have no obvious deficiency of lobules, or disorganization of tissue as in the Wnt-1 mutants.

Histological study of the cranial neural folds of mice genetically liable to exencephaly.

The SELH/Bc (SELH) inbred stock of mice has a high liability to the neural tube closure defect, exencephaly. All SELH embryos close their cranial neural tubes by an abnormal mechanism, lacking elevation and initiation of fusion in the posterior prosencephalon/anterior mesencephalon region. Most embryos complete closure of the cranial neural tube by extension of a more rostral site of fusion, but in 10-20% this process fails, and the embryos are subsequently exencephalic. In this study, transverse histological sections of the cranial neural folds of SELH embryos at the 3-5, 6-8, and 9-11 somite stages were compared to those of two strains with normal neural tube closure, ICR/Bc and LM/Bc. At all stages, consistent morphological differences were observed between SELH and the two normal strains. In 3-5 somite SELH embryos, the divergence of the folds from the neural groove is more angular, the folds are flatter, and their lateral tips appear "hooked" downward. In 6-8 somite SELH embryos, the lateral tips of the folds appear more elongated and in the prosencephalon they are less elevated than in the normal strains. The boundary between neuroepithelium and mesenchyme or surface ectoderm tends to be less clear than normal in SELH lateral tips. In 9-11 somite SELH embryos, divergence of the folds from the neural groove continues to be angular and the lateral folds are splayed horizontally. In addition, the lateral surface ectoderm is abnormally indented and the neuroepithelium/surface ectoderm boundary is more ventral and lateral in SELH than in ICR/Bc and LM/Bc. The hypothesis that the defect in SELH cranial neural folds might involve the cytoskeleton was tested using a fluorescent probe for filamentous actin in 7 somite SELH and ICR/Bc embryos. The actin staining pattern in SELH embryos was like that of normal ICR/Bc embryos, with a strongly staining apical concentration in the neuroepithelium. This suggests that there is no gross cytological abnormality within the neuroepithelium, but does not rule out more subtle defects, such as those involving cytoskeletal function.

Ataxia and a cerebellar defect in the exencephaly-prone SELH/Bc mouse stock.

SELH/Bc inbred mice have an abnormal mechanism of anterior neural tube closure and 10-20% of embryos have a lethal neural tube closure defect, exencephaly. Our previous studies have focused on this multifactorial threshold trait. However, SELH mice are also characterized by another trait that also shows non-Mendelian transmission ratios, an ataxia recognized in juvenile and adult mice. Here we report our first genetic and morphological studies of the ataxia trait. Recent pedigree records for the SELH colony showed that 7% of the 467 weaned progeny from normal breeding pairs were ataxic; 17 of the 20 pairs produced ataxic progeny. This result was statistically consistent with the hypothesis that all SELH mice have the ataxic genotype, which is expressed in only 7% of them. Genetic studies of an outcross to a normal strain and the subsequent F2 and testcross of the F2 were also done. The results were consistent with a one or two gene locus cause of liability to ataxia in SELH mice. The genetic correlation between exencephaly production and ataxia production for a sample of nine F2 males was 0.35, as expected if both traits are caused by the same genes, but was not statistically significant. In another approach, we examined the morphology of brains from normal and ataxic adult SELH mice. All 20 brains from non-ataxic SELH mice were morphologically normal. In all 18 brains from ataxic SELH mice the cerebellum was abnormal, lacking the vermis, and characterized by a midline fissure. This phenotype in mice has previously been known in Mendelian mutants at the Wnt-1 locus.(ABSTRACT TRUNCATED AT 250 WORDS)

Further genetic studies of the cause of exencephaly in SELH mice.

We have developed an inbred stock of mice called SELH that has a high frequency of the neural tube defect exencephaly at birth. A previous genetic study indicated that the exencephaly is due to two to three additive loci differing between SELH and a closely related normal strain, ICR/Bc, but this analysis was not designed to detect genetic maternal effects. Recently, we demonstrated that there is genetic polymorphism among normal mouse strains leading to differences in site of initiation of closure of the cranial neural tube. In the present study, an inbred substrain of SELH mice, with 24% exencephaly among embryos, was crossed with an unrelated normal strain, SWV/Bc, and the frequency of exencephaly in subsequent generations used to extend our understanding of the genetic cause of exencephaly in SELH mice. The purposes of the genetic studies reported here were twofold. First, based on the influence of genetic maternal effects on other genetically complex birth defects in mice, we hypothesized that the exencephaly of SELH mice would exhibit strong genetic maternal effects. This hypothesis was tested by comparisons among the four possible reciprocal backcrosses to SELH. The result was an overall frequency of 2.3% exencephaly in first backcross embryos with no difference among the four crosses and no evidence of genetic maternal effects. Second, the frequency of exencephaly recovered in the backcross and F1 embryos was compared with the previous genetic study and with various genetic models. The frequencies were similar to those obtained from the cross to ICR/Bc mice and were compatible with a hypothesis of additive gene action at a few loci.(ABSTRACT TRUNCATED AT 250 WORDS)