Genetic analysis of genodermatoses

The skin is the largest organ of the body. It provides a barrier that protects the body from harmful environmental factors as well as from a loss of fluids. Impaired skin integrity can lead to a disruption of the skin barrier and to disease. Genodermatoses are a heterogenous group of mostly rare single gene disorders affecting the skin and its appendages. After decades of genetic and due to recent advances in technology, the genetic cause of numerous genodermatoses was uncovered. Based on the function of the involved genes and the affected skin compartments, monogenic skin disorders can be grouped into ichthyoses, epidermolysis and blistering disorders, pigmentation disorders, disorders of ectodermal appendages, vascular disorders, connective tissue defects, dermal mosaics, and genodermatoses with tumor predisposition. Many human genodermatoses have a very similar counterpart in animals. Spontaneous mutants in purebred animals such as dogs that have a unique population structure and physical similarity to humans, are therefore valuable models for human genodermatoses. In this thesis, I took part in the analysis of 12 canine, equine and feline phenotypes with manifestations in skin or its appendages applying different genetic mapping techniques and whole genome sequencing. In cats, a frameshift variant in the COL5A1 gene was identified in a single case with Ehlers-Danlos syndrome. In a female cat with inflammatory linear epidermal lesions, a missense variant in the X-chromosomal NSDHL gene was found, explaining the observed cutaneous mosaicism. Interestingly, I identified a large deletion in the same gene, NSDHL, underlying a related congenital cornification disorder in a Labrador Retriever and her equally affected crossbred daughter. In a single canine case of ichthyosis, whole genome sequencing revealed a de novo variant in the ASPRV1 gene, probably disturbing filaggrin processing during cornification. A deregulation in cornification of nasal keratinocytes was also suspected in a litter of Greyhounds with nasal parakeratosis, in which I identified a splice defect in the SUV39H2 gene. Pigmentation and hair texture are diverse in dogs. During a study in Chow Chows with coat colour dilution I identified a variant in the MLPH gene encoding melanophilin, and found that this variant segregated in other dog breeds as well. In dogs with oculocutaneous albinism, variants in OCA2 and SLC45A2 encoding transporters in membranes of melanosomes were identified. In dogs with a previously unknown cause for their curly coat, a candidate gene approach led to the identification of a second KRT71 allele for curls, which has a potential predisposing role for hair loss that warrants further investigation. Hairless skin patches on the head and back was also a prominent feature in male Dachshund puppies with X-linked hypohidrotic ectodermal dysplasia, where a single base deletion in the EDA gene was found using targeted Sanger sequencing. Lethal acrodermatitis in Bull terriers is a disorder causing early death of affected animals. All cases in our cohort were homozygous for a splice defect in MKLN1, a gene not yet described in human genodermatoses. Finally, in horses from the Akhal-Teke breed, I identified a nonsense variant in the ST14 gene most likely causing the lethal naked foal syndrome in a monogenic autosomal recessive mode. The identification of these candidate causative variants enables genetic testing, controlled breeding and in the long term eradication of the corresponding disorders from the animal population. The majority of identified variants was located in genes already known from human disorders. However, ASPRV1 and MKLN1 variants have never been reported as cause for human genodermatoses. This thesis therefore demonstrates that genetic anlysis of spontaneous animal mutants offers the chance to gain new biological knowledge and candidate genes for rare human genodermatoses

The lifespan of web references : an example in graduate papers at the Department of information sciences in Zagreb

An increasing amount of students and scholars use web references as a prime source in their papers. The main concern is that those references have a short lifespan. In retrospect to that, the aim of this article is to show how many of the web references, gathered in a corpus of 1947 web references within 362 graduate papers at the Department of Information Sciences at the Faculty of Humanities and Social Sciences in Zagreb in the period from 2003 to 2010, are no longer active, i.e. accessible. It was also substantial to know how many of the web references have disappeared in the period between two researches. The main hypothesis is that there has been an increase in the number of inactive web references within the aforementioned corpus. It is expected that the highest number of inactive web references is present in the graduate papers that have been written during the first half of the analysed period, i.e. more time has passed since those references have been accessed. As well as the availability of the web references, certain bibliographic data were also analysed, where it is predicted that according to the type of web references web pages are more likely to be inactive than any other types such as scientific articles, etc. In response to that, it is expected that the web sites of inactive web references are non-scientific in snature. The analysis of the presence of authors and publishers among inactive web references is also included

Web References in Graduate Papers at the Department of Information Sciences at the Faculty of Humanities and Social Sciences in Zagreb

The aim of this article is to give insight into how graduate students at the Department of Information Sciences at the Faculty of Humanities and Social Sciences of the University of Zagreb tend to collect their references online. The research is based on the hypothesis, which was drawn up regarding the continuous spread of the Internet use, that the usage of web references in graduate papers has substantially grown in the period from 2004 to 2010. The period in question was chosen according to the availability of digitalised graduate papers in the Digital Repository of the Faculty Library. For this purpose a bibliometric method was used on the sample of over 360 graduate papers in the given period which include graduate papers from Archive Documentation, Library Science, General Information Science and Museology. A detailed analysis of the web references cited in the theses, together with the total number of references, is presented in this article. The analysis includes distinction based on authors and type of online references. One of the aims of the analysis was to investigate which author is most frequently cited even though web references are not always signed by an author. The different types of online references that are used, such as online books, articles, reference materials and others, were taken into consideration as well. The analysis of the collected data proved the hypothesis that the number of web references increased over the period; however, there is still a great deal of offline based references used in the graduate papers

A single base deletion in the SLC45A2 gene in a Bullmastiff with oculocutaneous albinism.

Oculocutaneous albinism type 4 (OCA4) in humans and similar phenotypes in many animal species are caused by variants in the SLC45A2 gene, encoding a putative sugar transporter. In dog, two independent SLC45A2 variants are known that cause oculocutaneous albinism in Doberman Pinschers and several small dog breeds respectively. For the present study, we investigated a Bullmastiff with oculocutaneous albinism. The affected dog was highly inbred and resulted from the mating of a sire to its own grandmother. We obtained whole genome sequence data from the affected dog and searched specifically for variants in candidate genes known to cause albinism. We detected a single base deletion in exon 6 of the SLC45A2 gene (NM_001037947.1:c.1287delC) that has not been reported thus far. This deletion is predicted to result in an early premature stop codon. It was confirmed by Sanger sequencing and perfectly co-segregated with the phenotype in the available family members. We genotyped 174 unrelated dogs from diverse breeds, all of which were homozygous wildtype. We therefore suggest that SLC45A2:c.1287delC causes the observed oculocutaneous albinism in the affected Bullmastiff

Compound heterozygosity for TNXB genetic variants in a mixed-breed dog with Ehlers-Danlos syndrome.

The Ehlers-Danlos syndromes (EDSs) are a heterogeneous group of inherited connective tissue disorders characterized by skin hyperextensibility, joint hypermobility and tissue fragility. Inherited disorders similar to human EDS have been reported in different mammalian species. In the present study, we investigated a female mixed-breed dog with clinical signs of EDS. Whole-genome sequencing of the affected dog revealed two missense variants in the TNXB gene, encoding the extracellular matrix protein tenascin XB. In humans, TNXB genetic variants cause classical-like EDS or the milder hypermobile EDS. The affected dog was heterozygous at both identified variants. Each variant allele was transmitted from one of the case's parents, consistent with compound heterozygosity. Although one of the variant alleles, XM_003431680.3:c.2012G>A, p.(Ser671Asn), was private to the family of the affected dog and absent from whole-genome sequencing data of 599 control dogs, the second variant allele, XM_003431680.3:c.2900G>A, p.(Gly967Asp), is present at a low frequency in the Chihuahua and Poodle population. Given that TNXB is a functional candidate gene for EDS, we suggest that compound heterozygosity for the identified TNXB variants may have caused the EDS-like phenotype in the affected dog. Chihuahuas and Poodles should be monitored for EDS cases, which might confirm the hypothesized pathogenic effect of the segregating TNXB variant

A second KRT71 allele in curly coated dogs.

Major characteristics of coat variation in dogs can be explained by variants in only a few genes. Until now, only one missense variant in the KRT71 gene, p.Arg151Trp, has been reported to cause curly hair in dogs. However, this variant does not explain the curly coat in all breeds as the mutant Trp allele, for example, is absent in Curly Coated Retrievers. We sequenced the genome of a Curly Coated Retriever at 22× coverage and searched for variants in the KRT71 gene. Only one protein-changing variant was present in a homozygous state in the Curly Coated Retriever and absent or present in a heterozygous state in 221 control dogs from different dog breeds. This variant, NM_001197029.1:c.1266_1273delinsACA, was an indel variant in exon 7 that caused a frameshift and an altered and probably extended C-terminus of the KRT71 protein NP_001183958.1:p.(Ser422ArgfsTer?). Using Sanger sequencing, we found that the variant was fixed in a cohort of 125 Curly Coated Retrievers and segregating in five of 14 additionally tested breeds with a curly or wavy coat. KRT71 variants cause curly hair in humans, mice, rats, cats and dogs. Specific KRT71 variants were further shown to cause alopecia. Based on this knowledge from other species and the predicted molecular consequence of the newly identified canine KRT71 variant, it is a compelling candidate causing a second curly hair allele in dogs. It might cause a slightly different coat phenotype than the previously published p.Arg151Trp variant and could potentially be associated with follicular dysplasia in dogs

Whole Genome Sequencing of Giant Schnauzer Dogs with Progressive Retinal Atrophy Establishes NECAP1 as a Novel Candidate Gene for Retinal Degeneration

Canine progressive retinal atrophies (PRA) are genetically heterogeneous diseases characterized by retinal degeneration and subsequent blindness. PRAs are untreatable and affect multiple dog breeds, significantly impacting welfare. Three out of seven Giant Schnauzer (GS) littermates presented with PRA around four years of age. We sought to identify the causal variant to improve our understanding of the aetiology of this form of PRA and to enable development of a DNA test. Whole genome sequencing of two PRA-affected full-siblings and both unaffected parents was performed. Variants were filtered based on those segregating appropriately for an autosomal recessive disorder and predicted to be deleterious. Successive filtering against 568 canine genomes identified a single nucleotide variant in the gene encoding NECAP endocytosis associated 1 (NECAP1): c.544G>A (p.Gly182Arg). Five thousand one hundred and thirty canids of 175 breeds, 10 cross-breeds and 3 wolves were genotyped for c.544G>A. Only the three PRA-affected GS were homozygous (allele frequency in GS, excluding proband family = 0.015). In addition, we identified heterozygotes belonging to Spitz and Dachshund varieties, demonstrating c.544G>A segregates in other breeds of German origin. This study, in parallel with the known retinal expression and role of NECAP1 in clathrin mediated endocytosis (CME) in synapses, presents NECAP1 as a novel candidate gene for retinal degeneration in dogs and other species

Identification of Two Independent COL5A1 Variants in Dogs with Ehlers-Danlos Syndrome.

The Ehlers-Danlos syndromes (EDS) are a heterogeneous group of heritable disorders affecting connective tissues. The mutations causing the various forms of EDS in humans are well characterized, but the genetic mutations causing EDS-like clinical pathology in dogs are not known, thus hampering accurate clinical diagnosis. Clinical analysis of two independent cases of skin hyperextensibility and fragility, one with pronounced joint hypermobility was suggestive of EDS. Whole-genome sequencing revealed de novo mutations of COL5A1 in both cases, confirming the diagnosis of the classical form of EDS. The heterozygous COL5A1 p.Gly1013ValfsTer260 mutation characterized in case 1 introduced a premature termination codon and would be expected to result in α1(V) mRNA nonsense-mediated mRNA decay and collagen V haploinsufficiency. While mRNA was not available from this dog, ultrastructural analysis of the dermis demonstrated variability in collagen fibril diameter and the presence of collagen aggregates, termed 'collagen cauliflowers', consistent with COL5A1 mutations underlying classical EDS. In the second case, DNA sequencing demonstrated a p.Gly1571Arg missense variant in the COL5A1 gene. While samples were not available for further analysis, such a glycine substitution would be expected to destabilize the strict molecular structure of the collagen V triple helix and thus affect protein stability and/or integration of the mutant collagen into the collagen V/collagen I heterotypic dermal fibrils. This is the first report of genetic variants in the COL5A1 gene causing the clinical presentation of EDS in dogs. These data provided further evidence of the important role of collagen V in dermal collagen fibrillogenesis. Importantly, from the clinical perspective, we showed the utility of DNA sequencing, combined with the established clinical criteria, in the accurate diagnosis of EDS in dogs

MKLN1 splicing defect in dogs with lethal acrodermatitis

Lethal acrodermatitis (LAD) is a genodermatosis with monogenic autosomal recessive inheritance in Bull Terriers and Miniature Bull Terriers. The LAD phenotype is characterized by poor growth, immune deficiency, and skin lesions, especially at the paws. Utilizing a combination of genome wide association study and haplotype analysis, we mapped the LAD locus to a critical interval of similar to 1.11 Mb on chromosome 14. Whole genome sequencing of an LAD affected dog revealed a splice region variant in the MKLN1 gene that was not present in 191 control genomes (chr14:5,731,405T>G or MKLN/:c.400+3A>C). This variant showed perfect association in a larger combined Bull Terrier/Miniature Bull Terrier cohort of 46 cases and 294 controls. The variant was absent from 462 genetically diverse control dogs of 62 other dog breeds. RT-PCR analysis of skin RNA from an affected and a control dog demonstrated skipping of exon 4 in the MKLN1 transcripts of the LAD affected dog, which leads to a shift in the MKLN1 reading frame. MKLN1 encodes the widely expressed intracellular protein muskelin 1, for which diverse functions in cell adhesion, morphology, spreading, and intracellular transport processes are discussed. While the pathogenesis of LAD remains unclear, our data facilitate genetic testing of Bull Terriers and Miniature Bull Terriers to prevent the unintentional production of LAD affected dogs. This study may provide a starting point to further clarify the elusive physiological role of muskelin 1 in vivo.Peer reviewe

Životni vijek mrežnih izvora: primjer mrežnih izvora u diplomskim radovima informacijskih znanosti na Filozofskom fakultetu u Zagrebu

Istraživanjem je utvrđeno povećanje broja nevažećih mrežnih izvora. Najveći naglasak stavljen je na problem nedostupnosti znanstveno relevantnih izvora gdje se poziva na poboljšanje standarda za citiranje i uvođenje obvezujućih pravila kod navođenja izvora kako bi se eventualno produljio njihov vijek. Drugi problem odnosio se na nemogućnost točne procjene životnog vijeka mrežnih izvora zbog nepoznavanja točnog datuma njezinog nestanka gdje je kao jedna od mogućnosti predloženo češće i potpunije indeksiranje stranica. Kao dodatni problemi koji utječu na skraćivanje životnog vijeka mrežnih stranica spomenuti su loše izabrani formati za očuvanje zapisa gdje se predlaže njihova detaljna provjera kako ne bi došlo do gubitka zapisa. Dobivanjem uvida u sve moguće prijedloge za produžetak životnog vijeka mrežnih izvora i razloge njihova propadanja rezultira većom razinom vjerodostojnosti korištenih izvora što izravno djeluje na vjerodostojnost rada u kojem su korišteni