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Tosso Leeb

Publications and source records attributed to Tosso Leeb.

At least 19 recordsLinked to original sources

A SINE-like insertion in intron 13 of the ATP7A gene is associated with a mild form of Menkes-like disease in a Cavalier King Charles Spaniel.

A 7-month-old intact male Cavalier King Charles Spaniel was presented for persistent glucosuria despite normoglycemia, failure to thrive, chronic diarrhea, and cerebellar ataxia. Fanconi syndrome was diagnosed, but the neurologic abnormalities were not fully explained. As a consequence of the early onset Fanconi syndrome, a hereditary process was suspected. Whole genome sequencing identified a private hemizygous SINE-like insertion into the ATP7A gene, at the end of intron 13, near the start of exon 14. In humans, variants in ATP7A are associated with Menkes disease, a disorder of copper metabolism associated with a spectrum of clinical signs including progressive neurodegeneration and connective tissue abnormalities. Clinically affected dogs with variants in ATP7A have not been reported previously. Although this case appears to represent a mild phenotypic presentation of Menkes-like disease, it raises the possibility that copper disorders aside from copper-associated hepatitis might exist in dogs. Further genetic screening and phenotypic characterization of rare genetic variants associated with copper metabolism would be beneficial to expand our knowledge of copper disorders in dogs and allow potential early intervention and modeling for metabolic diseases in humans.

Animals↗

The Animal Variant Classification Guidelines v2: An Update With New Criteria and Improved Clarifications.

The Animal Variant Classification Guidelines (AVCG) were developed to standardize and objectify the classification of putative disease-causing variants. These guidelines are sufficiently reproducible and are used to classify previously published and new disease-causing variants across species. Here, the guidelines are updated (AVCG.v2), based on a three-phase decision process. Overall, four new criteria and seven clarifying comments were added. The number of criteria has increased from 23 to 27, with three new criteria supporting pathogenicity and one new criterion supporting benign classification. Pharmacogenomic variants were determined to fall within the scope of the guidelines. These updated guidelines are being used by the Variant Pathogenicity Working Group (VPWG), part of the Animal Genetic Testing Standardization standing committee, which is a committee of elected members of the International Society for Animal Genetics (ISAG). Under the auspices of ISAG, the VPWG retrospectively classifies published putative disease-causing variants. The pathogenicity label for a variant will be presented in the variant tables of Online Mendelian Inheritance in Animals (OMIA; https://omia.org/). The AVCGv.2 criteria and recommendations were developed by the expertise of the animal genetics community and the ISAG Executive Committee through the Animal Genetics Testing Standardization Committee endorses and strongly encourages their use to evaluate the evidence supporting pathogenicity of putative disease-causing variants.

Animals↗

OCA2 splice site variant in German Spitz dogs with oculocutaneous albinism.

We investigated a German Spitz family where the mating of a black male to a white female had yielded three puppies with an unexpected light brown coat color, lightly pigmented lips and noses, and blue eyes. Combined linkage and homozygosity analysis based on a fully penetrant monogenic autosomal recessive mode of inheritance identified a critical interval of 15 Mb on chromosome 3. We obtained whole genome sequence data from one affected dog, three wolves, and 188 control dogs. Filtering for private variants revealed a single variant with predicted high impact in the critical interval in LOC100855460 (XM_005618224.1:c.377+2T>G LT844587.1:c.-45+2T>G). The variant perfectly co-segregated with the phenotype in the family. We genotyped 181 control dogs with normal pigmentation from diverse breeds including 22 unrelated German Spitz dogs, which were all homozygous wildtype. Comparative sequence analyses revealed that LOC100855460 actually represents the 5'-end of the canine OCA2 gene. The CanFam 3.1 reference genome assembly is incorrect and separates the first two exons from the remaining exons of the OCA2 gene. We amplified a canine OCA2 cDNA fragment by RT-PCR and determined the correct full-length mRNA sequence (LT844587.1). Variants in the OCA2 gene cause oculocutaneous albinism type 2 (OCA2) in humans, pink-eyed dilution in mice, and similar phenotypes in corn snakes, medaka and Mexican cave tetra fish. We therefore conclude that the observed oculocutaneous albinism in German Spitz is most likely caused by the identified variant in the 5'-splice site of the first intron of the canine OCA2 gene.

Albinism, Oculocutaneous↗

Polymorphic variants of the multidrug resistance gene Mdr1a and response to antiepileptic drug treatment in the kindling model of epilepsy.

Allelic variants of the human P-glycoprotein encoding gene MDR1 (ABCB1) are discussed to be associated with different clinical conditions including pharmacoresistance of epilepsy. However, conflicting data have been reported with regard to the functional relevance of MDR1 allelic variants for the response to antiepileptic drugs. To our knowledge, it is not known whether functionally relevant genetic polymorphisms also occur in the two genes (Mdr1a/Abcb1a, Mdr1b/Abcb1b) coding for P-glycoprotein in the brain of rodents. Therefore, we have started to search for polymorphisms in the Mdr1a gene, which governs the expression of P-glycoprotein in brain capillary endothelial cells in rats. In the kindling model of temporal lobe epilepsy, subgroups of phenytoin-sensitive and phenytoin-resistant rats were selected in repeated drug trials. Sequencing of the Mdr1a gene coding sequence in the subgroups revealed no general differences between drug-resistant and drug-sensitive rats of the Wistar outbred strain. A comparison between different inbred and outbred rat strains also gave no evidence for polymorphisms in the Mdr1a coding sequence. However, in exon-flanking intron sequences, four genetic variants were identified by comparison between these rats strains. In conclusion, the finding that Wistar rats vary in their response to phenytoin, while having the same genetic background, argues against a major impact of Mdr1a genetics on pharmacosensitivity to antiepileptic drugs in the amygdala kindling model.

ATP Binding Cassette Transporter, Subfamily B↗

A human-horse comparative map based on equine BAC end sequences.

In an effort to increase the density of sequence-based markers for the horse genome we generated 9473 BAC end sequences (BESs) from the CHORI-241 BAC library with an average read length of 677 bp. BLASTN searches with the BESs revealed 4036 meaningful hits (E <or= 10(-5)) in the human genome that provide useful markers for the human-horse comparative map. The 4036 BLASTN hits allowed the anchoring of 3079 BAC clones to the human genome, on average one corresponding equine BAC clone per megabase of human DNA. We used the BLASTN anchored BESs for an in silico prediction of the gene content and chromosome assignment of comparatively mapped equine BAC clones. As a first verification of our in silico mapping strategy we placed 19 equine BESs with matches to HSA6 onto the RH map. All markers were assigned to the predicted localizations on ECA10, ECA20, and ECA31, respectively.

Animals↗

Sequence analysis of a 212 kb defensin gene cluster on ECA 27q17.

Defensins are a family of evolutionary ancient antimicrobial peptides consisting of three sub-families: alpha-, beta- and theta-defensins. This investigation was focused on the genomic characterization of equine beta-defensins and the investigation of the potential clustering of beta-defensin genes in the equine genome. Six genomic BAC clones were isolated from the CHORI-241 library and one of these was mapped by FISH to ECA 27q17. This location was confirmed by RH-mapping. The contiguous 212 kb sequence of this clone was determined. Sequence analysis revealed the identification of ten pseudogenes and nine genes, six of which were highly homologous to human beta-defensin DEFB4. Clustering of the beta-defensin genes was confirmed and the order of the genes on the analyzed BAC was related to the corresponding defensin cluster on HSA 8. The knowledge about the sequence and the genomic structure of the equine beta-defensin genes will improve the classification of different paralogous defensin genes and is a prerequisite for subsequent functional studies. Additionally, the first alpha-defensin-like sequence outside the groups of primates, lagomorphs and rodents (glires) was identified.

Animals↗

Molecular characterization of the porcine deleted in malignant brain tumors 1 gene (DMBT1).

The human gene deleted in malignant brain tumors 1 (DMBT1) is considered to play a role in tumorigenesis and pathogen defense. It encodes a protein with multiple scavenger receptor cysteine-rich (SRCR) domains, which are involved in recognition and binding of a broad spectrum of bacterial pathogens. The SRCR domains are encoded by highly homologous repetitive exons, whose number in humans may vary from 8 to 13 due to genetic polymorphism. Here, we characterized the porcine DMBT1 gene on the mRNA and genomic level. We assembled a 4.5 kb porcine DMBT1 cDNA sequence from RT-PCR amplified seminal vesicle RNA. The porcine DMBT1 cDNA contains an open reading frame of 4050 nt. The transcript gives rise to a putative polypeptide of 1349 amino acids with a calculated mass of 147.9 kDa. Compared to human DMBT1, it contains only four N-terminal SRCR domains. Northern blotting revealed transcripts of approximately 4.7 kb in size in the tissues analyzed. Analysis of ESTs suggested the existence of secreted and transmembrane variants. The porcine DMBT1 gene spans about 54 kb on chromosome 14q28-q29. In contrast to the characterized cDNA, the genomic BAC clone only contained 3 exons coding for N-terminal SRCR domains. In different mammalian DMBT1 orthologs large interspecific differences in the number of SRCR exons and utilization of the transmembrane exon exist. Our data suggest that the porcine DMBT1 gene may share with the human DMBT1 gene additional intraspecific variations in the number of SRCR-coding exons.

Amino Acid Sequence↗

The mutation causing the black-and-tan pigmentation phenotype of Mangalitza pigs maps to the porcine ASIP locus but does not affect its coding sequence.

The gene for agouti signaling protein (ASIP) is centrally involved in the expression of coat color traits in animals. The Mangalitza pig breed is characterized by a black-and-tan phenotype with black dorsal pigmentation and yellow or white ventral pigmentation. We investigated a Mangalitza x Piétrain cross and observed a coat color segregation pattern in the F2 generation that can be explained by virtue of two alleles at the MC1R locus and two alleles at the ASIP locus. Complete linkage of the black-and-tan phenotype to microsatellite alleles at the ASIP locus on SSC 17q21 was observed. Corroborated by the knowledge of similar mouse coat color mutants, it seems therefore conceivable that the black-and-tan pigmentation of Mangalitza pigs is caused by an ASIP allele a(t), which is recessive to the wild-type allele A. Toward positional cloning of the a(t) mutation, a 200-kb genomic BAC/PAC contig of this chromosomal region has been constructed and subsequently sequenced. Full-length ASIP cDNAs obtained by RACE differed in their 5' untranslated regions, whereas they shared a common open reading frame. Comparative sequencing of all ASIP exons and ASIP cDNAs between Mangalitza and Piétrain pigs did not reveal any differences associated with the coat color phenotype. Relative qRT-PCR analyses showed different dorsoventral skin expression intensities of the five ASIP transcripts in black-and-tan Mangalitza. The a(t) mutation is therefore probably a regulatory ASIP mutation that alters its dorsoventral expression pattern.

Animals↗

Novel mutations in the human sucrase-isomaltase gene (SI) that cause congenital carbohydrate malabsorption.

Disaccharide intolerance I or congenital sucrase-isomaltase deficiency (CSID) is a disorder leading to maldigestion of disaccharides, which is autosomal recessively inherited. Here we analyzed the sucrase-isomaltase (SI) gene from 11 patients of Hungarian origin with congenital sucrase-isomaltase deficiency. Variants in the SI gene had previously been described in CSID patients, which cause amino acid exchanges that affect the transport, the processing, or the function of the SI protein. None of our patients had known mutations for CSID. Our analyses revealed 43 SI variants in total, 15 within exons and one at a splice site. Eight of the exonic mutations lead to amino acid exchanges, causing hypomorph or null alleles. One new variation affects a splice site, which is also predicted to result in a null allele. All potential pathological alterations were present on one allele only. In six out of the 11 patients the phenotype of CSID could be explained by compound heterozygosity.

Biopsy↗

Black hair follicular dysplasia in Large Münsterländer dogs: clinical, histological and ultrastructural features.

Four Large Münsterländer cross-bred dogs affected with black hair follicular dysplasia (BHFD) and one unaffected control littermate were observed, and skin was sampled weekly over the first 19 weeks of life. Affected dogs were born with silvery grey hair, a consequence of melanin clumping in the hair shafts. Hair bulb melanocytes were densely pigmented, and contained abundant stage IV melanosomes but adjacent matrix keratinocytes lacked melanosomes. Melanin clumping was not prominent in epidermal melanocytes in the haired skin but occurred in the foot pads. Follicular changes progressed from bulbar clumping, clumping in the isthmus/infundibulum and finally to dysplastic hair shafts. Alopecia developed progressively in pigmented areas. Silver-grey hair, melanin clumping, accumulation of stage IV melanosomes within melanocytes and insufficient melanin transfer to adjacent keratinocytes are also classic features of human Griscelli syndrome. The underlying cause in Griscelli syndrome is a defect of melanocytic intracellular transport proteins leading to inadequate and disorganized melanosome transfer to keratinocytes with resultant melanin clumping. In view of the correlation in the phenotype, histology and ultrastructure between both disorders, a defect in intracellular melanosome transport is postulated as the pathogenic mechanism in BHFD.

Animals↗

Single linkage group per chromosome genetic linkage map for the horse, based on two three-generation, full-sibling, crossbred horse reference families.

A genetic linkage map of the horse consisting of 742 markers, which comprises a single linkage group for each of the autosomes and the X chromosome, is presented. The map has been generated from two three-generation full-sibling reference families, sired by the same stallion, in which there are 61 individuals in the F2 generation. Each linkage group has been assigned to a chromosome and oriented with reference to markers mapped by fluorescence in situ hybridization. The average interval between markers is 3.7 cM and the linkage groups collectively span 2772 cM. The 742 markers comprise 734 microsatellite and 8 gene-based markers. The utility of the microsatellite markers for comparative mapping has been significantly enhanced by comparing their flanking sequences with the human genome sequence; this enabled conserved segments between human and horse to be identified. The new map provides a valuable resource for genetically mapping traits of interest in the horse.

Animals↗

Bovine prion protein gene (PRNP) promoter polymorphisms modulate PRNP expression and may be responsible for differences in bovine spongiform encephalopathy susceptibility.

The susceptibility of humans to the variant Creutzfeldt-Jakob disease is greatly influenced by polymorphisms within the human prion protein gene (PRNP). Similar genetic differences exist in sheep, in which PRNP polymorphisms modify the susceptibility to scrapie. However, the known coding polymorphisms within the bovine PRNP gene have little or no effect on bovine spongiform encephalopathy (BSE) susceptibility in cattle. We have recently found a tentative association between PRNP promoter polymorphisms and BSE susceptibility in German cattle (Sander, P., Hamann, H., Pfeiffer, I., Wemheuer, W., Brenig, B., Groschup, M., Ziegler, U., Distl, O., and Leeb, T. (2004) Neurogenetics 5, 19-25). A plausible hypothesis explaining this observation could be that the bovine PRNP promoter polymorphisms cause changes in PRNP expression that might be responsible for differences in BSE incubation time and/or BSE susceptibility. To test this hypothesis, we performed a functional promoter analysis of the different bovine PRNP promoter alleles by reporter gene assays in vitro and by measuring PRNP mRNA levels in calves with different PRNP genotypes in vivo. Two variable sites, a 23-bp insertion/deletion (indel) polymorphism containing a RP58-binding site and a 12-bp indel polymorphism containing an SP1-binding site, were investigated. Band shift assays indicated differences in transcription factor binding to the different alleles at the two polymorphisms. Reporter gene assays demonstrated an interaction between the two postulated transcription factors and lower expression levels of the ins/ins allele compared with the del/del allele. The in vivo data revealed substantial individual variation of PRNP expression in different tissues. In intestinal lymph nodes, expression levels differed between the different PRNP genotypes.

Alleles↗

Polymorphisms within the canine MLPH gene are associated with dilute coat color in dogs.

BACKGROUND: Pinschers and other dogs with coat color dilution show a characteristic pigmentation phenotype. The fur colors are a lighter shade, e.g. silvery grey (blue) instead of black and a sandy color (Isabella fawn) instead of red or brown. In some dogs the coat color dilution is sometimes accompanied by hair loss and recurrent skin inflammation, the so called color dilution alopecia (CDA) or black hair follicular dysplasia (BHFD). In humans and mice a comparable pigmentation phenotype without any documented hair loss is caused by mutations within the melanophilin gene (MLPH). RESULTS: We sequenced the canine MLPH gene and performed a mutation analysis of the MLPH exons in 6 Doberman Pinschers and 5 German Pinschers. A total of 48 sequence variations was identified within and between the breeds. Three families of dogs showed co-segregation for at least one polymorphism in an MLPH exon and the dilute phenotype. No single polymorphism was identified in the coding sequences or at splice sites that is likely to be causative for the dilute phenotype of all dogs examined. In 18 German Pinschers a mutation in exon 7 (R199H) was consistently associated with the dilute phenotype. However, as this mutation was present in homozygous state in four dogs of other breeds with wildtype pigmentation, it seems unlikely that this mutation is truly causative for coat color dilution. In Doberman Pinschers as well as in Large Munsterlanders with BHFD, a set of single nucleotide polymorphisms (SNPs) around exon 2 was identified that show a highly significant association to the dilute phenotype. CONCLUSION: This study provides evidence that coat color dilution is caused by one or more mutations within or near the MLPH gene in several dog breeds. The data on polymorphisms that are strongly associated with the dilute phenotype will allow the genetic testing of Pinschers to facilitate the breeding of dogs with defined coat colors and to select against Large Munsterlanders carrying BHFD.

Animals↗

A duplication in the canine beta-galactosidase gene GLB1 causes exon skipping and GM1-gangliosidosis in Alaskan huskies.

GM(1)-gangliosidosis is a lysosomal storage disease that is inherited as an autosomal recessive disorder, predominantly caused by structural defects in the beta-galactosidase gene (GLB1). The molecular cause of GM(1)-gangliosidosis in Alaskan huskies was investigated and a novel 19-bp duplication in exon 15 of the GLB1 gene was identified. The duplication comprised positions +1688-+1706 of the GLB1 cDNA. It partially disrupted a potential exon splicing enhancer (ESE), leading to exon skipping in a fraction of the transcripts. Thus, the mutation caused the expression of two different mRNAs from the mutant allele. One transcript contained the complete exon 15 with the 19-bp duplication, while the other transcript lacked exon 15. In the transcript containing exon 15 with the 19-bp duplication a premature termination codon (PTC) appeared, but due to its localization in the last exon of canine GLB1, nonsense-mediated RNA decay (NMD) did not occur. As a consequence of these molecular events two different truncated GLB1 proteins are predicted to be expressed from the mutant GLB1 allele. In heterozygous carrier animals the wild-type allele produces sufficient amounts of the active enzyme to prevent clinical signs of disease. In affected homozygous dogs no functional GLB1 is synthesized and G(M1)-gangliosidosis occurs.

Alleles↗

Evolution of the spermadhesin gene family.

Spermadhesins belong to a novel family of secretory proteins of the male genital tract. They are major proteins of the seminal plasma and have been found peripherally associated to the sperm surface. So far, they have only been detected in ungulates, specifically in pig, cattle, and horse, respectively. Spermadhesins form a subgroup of the superfamily of proteins with a CUB-domain that has been found in a variety of developmentally regulated proteins. The structure and function of the spermadhesins have been investigated in the pig. They are multifunctional proteins showing a range of ligand-binding abilities, e.g. to carbohydrates, phospholipids, and protease inhibitors, suggesting that they may be involved in different steps of fertilization. We report here the genomic organization of the porcine spermadhesin gene cluster as well as a detailed comparative analysis with respect to other mammalian species. The porcine spermadhesin genes are located on SSC 14q28-q29 in a region syntenic to HSA 10q26. The pig contains five closely linked spermadhesin genes, whereas only two spermadhesin genes are present in the cattle genome. Inactive copies of spermadhesin genes are still detectable in the human, chimp, and dog genome while the corresponding region was lost from the rodent genomes of mouse and rat. Within the pig, the five spermadhesin genes contain both highly diverged and highly conserved regions. Interestingly, the pattern of divergence does not correlate with the position of the exons. Evolutionary analyses suggest that the pattern of diversity is shaped by ancestral variation, recombination, and new mutations.

Animals↗

Molecular characterization and chromosomal assignment of the bovine glycinamide ribonucleotide formyltransferase (GART) gene on cattle chromosome 1q12.1-q12.2.

The mammalian glycinamide ribonucleotide formyltransferase (GART) genes encode a trifunctional polypeptide involved in the de novo purine biosynthesis. We isolated a bacterial artificial chromosome (BAC) clone containing the bovine GART gene and determined the complete DNA sequence of the BAC clone. Cloning and characterization of the bovine GART gene revealed that the bovine gene consists of 23 exons spanning approximately 27 kb. RT-PCR amplification of bovine GART in different organs showed the expression of two GART transcripts in cattle similar to human and mouse. The GART transcripts encode two proteins of 1010 and 433 amino acids, respectively. Eleven single nucleotide polymorphisms (SNPs) were detected in a mutation scan of 24 unrelated animals of three different cattle breeds, including one SNP that affects the amino acid sequence of GART. The chromosomal localization of the gene was determined by fluorescence in situ hybridization. Comparative genome analysis between cattle, human and mouse indicates that the chromosomal location of the bovine GART gene is in agreement with a previously published mapping report.

Alternative Splicing↗

The role of stallion seminal proteins in fertilisation.

Seminal plasma proteins are secretory proteins originating mainly from the epididymis and the accessory sex glands. They are involved in the remodelling of the sperm surface which occurs during sperm transit through the male genital tract and continues later at ejaculation. During this process, collectively called post-testicular sperm maturation, the spermatozoa acquire the ability to fertilise an egg. Seminal plasma proteins have been shown to contribute to early and central steps of the fertilisation sequence, e.g. the establishment of the oviductal sperm reservoir, modulation of capacitation and gamete interaction. The major equine seminal plasma proteins belong to three protein classes, which contain widely occurring protein modules. Fn-2 type proteins are characterised by two or four tandemly arranged Fn-2 modules and have been implicated in the modulation of sperm capacitation. Multiple members of the cysteine-rich secretory proteins (CRISP) have been identified in the male genital tract of a number of species. CRISP proteins have been shown to be involved in various functions related to sperm-oocyte fusion, innate host defense function and ion channel blockage. Spermadhesins occur only in ungulate species. Their carbohydrate- and zona pellucida-binding properties would suggest a role of these proteins in gamete recognition. The major proteins of equine seminal plasma have been isolated and characterised regarding their expression along the male genital tract, protein structure and their functions.

Animals↗

Genetic markers for stallion fertility--lessons from humans and mice.

Our knowledge on the many aspects of mammalian reproduction in general and equine reproduction in particular has greatly increased during the last 15 years. Advances in the understanding of the physiology, cell biology, and biochemistry of reproduction have facilitated genetic analyses of fertility. Currently, there are more than 200 genes known that are involved in the production of fertile sperm cells. The completion of a number of mammalian genome projects will aid in the investigation of these genes in different species. Great progress has been made in the understanding of genetic aberrations that lead to male infertility. Additionally, the first genetic mechanisms are being discovered that contribute to the quantitative variation of fertility traits in fertile male animals. As artificial insemination (AI) represents a widespread technology in horse breeding, semen quality traits may eventually become an additional selection criterion for breeding stallions. Current research activities try to identify genetic markers that correlate to these semen quality traits. Here, we will review the current state of genetic research in male fertility and offer some perspectives for future research in horses.

Animals↗