Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Footrot is a contagious hoof disease of ruminants. It is endemic in New Zealand and throughout sheep and goat farming regions of the world. The disease results from a mixed bacterial infection, but the essential agent is Dichelobacter nodosus, a Gram-negative, anaerobic bacterium that possesses type-IV fimbriae on its surface. Genetic variation in the fimbriae of D. nodosus was investigated in this study. Using the polymerase chain reaction (PCR), the variable region of the gene encoding the fimbrial subunit (fimA) was amplified from bacterial DNA extracted from footrot lesions. Different fimA amplimers were differentiated by single-strand conformation polymorphism (SSCP) analysis. In conjunction with DNA sequencing, 15 unique sequences of D. nodosus fimA were obtained from 14 footrot samples taken from 6 farming regions throughout New Zealand. When these sequences were compared to fimA of known serogroups, it revealed that there were at least 15 D. nodosus strains, representing 8 serogroups, present on New Zealand farms. The predominant serogroup was B which contained 6 strains, followed by serogroups F, H and G. No strains from serogroups D and I were detected in this investigation. Twelve out of the 15 New Zealand D. nodosus strains had fimbriae different to those previously reported and the presence of multiple strains on a single hoof was common (86% of samples). The fimA sequences from the 12 D. nodosus strains incorporated into the footrot vaccine currently available in New Zealand were determined. A primer set targeting the relatively conserved fimA regions and based on the published sequence of serogroup M Nepalese isolates (designated M-Nep), failed to amplify fimA from the vaccine serotype M strain (designated as M-SPAHL). When the downstream primer was substituted with a primer that was specific for other serogroups of D. nodosus, the fimA gene was successfully amplified. Cloning followed by DNA sequencing, revealed that M-SPAHL fimA was different to M-Nep fimA. The predicted amino acid sequence of M-SPAHL fimA did not show homology to any known serogroups or serotypes. The most similar sequence was from serotype F1, and not M-Nep. The sequence difference between M-SPAHL and M-Nep was larger than that expected within a serogroup. The consequences of serological relatedness and sequence dissimilarity are discussed. Only eight of the 15 New Zealand field strains had fimbriae identical to those of the vaccine strains, while the remaining seven strains possessed different fimbriae. In addition, the vaccine contained two more D. nodosus strains, representing two sera groups, that were not found on the New Zealand farms investigated in this study. This may, to some extent, explain why the current footrot vaccine is at times less efficient in New Zealand. Another 17 footrot samples were screened for new or additional D. nodosus strains. Two PCR amplimers (designated X and Y) derived from footrot samples generated SSCP patterns different to those of previously identified strains. DNA sequencing revealed that these two fragments possessed novel sequences. The upstream of X (nt 1-183) was identical to serotype M1 while its downstream (nt 223-414) was identical to serotype F1; the upstream of Y (nt 1-116) was identical to serotype E1 whereas its downstream (nt 148-423) was identical to serotype F1. A 14-mer sequence consisting of two partially overlapping Chi-like sequences, 5'-GCTGGTGCTGGTGA-3', was also found in these fragments. Two primer sets with the downstream primer specific for serotype Fl and the upstream primer specific for serotype M1 or E1, produced PCR products of the expected sizes from the footrot samples from which fragments X and Y were isolated, respectively. These primer sets did not appear to amplify artificially mixed genomic DNA from serotypes M1 and F1 or E1 and F1. However, when the reactions were re-amplified, PCR recombination artefacts were observed, suggesting that PCR recombination does occur, but at a low frequency. It therefore seems more likely that fragments X and Y reflect genuine fimA sequences of D. nodosus which have resulted from in vivo DNA recombination, than from a PCR recombination artifact. The genetic capability for recombination at the fimbrial subunit locus may therefore endow D. nodosus with the ability to alter its antigenic appearance. D. nodosus strains present in footrot lesions can be genotyped using a PCR-SSCP/sequencing technique. However, this typing technique requires cloning and screening of D. nodosus fimA sequences, which is both laborious and costly. A rapid molecular typing system for D. nodosus was therefore developed in this study. A close examination of available D. nodosus fimA sequences revealed regions that appear to be specific for serogroups and serotypes. These regions were used to design a panel of sequence-specific oligonucleotide probes (SSOPs), and a rapid and accurate D. nodosus typing system using PCR and reverse dot-blot hybridisation (PCR/oligotyping) was subsequently developed. The variable region of D. nodosus fimA, amplified and labelled with digoxigenin (DIG) in a single multiplex PCR amplification, was hybridised to a panel of group- and type-specific, poly-dT tailed oligonucleotides that were immobilised on a nylon membrane strip. A mixture of positive control poly-dT tailed oligonucleotides was also included on the membrane. After hybridisation the membrane was washed to a defined specificity, and DIG-labelled fragments that had hybridised were detected. The specificity of the oligonucleotides was verified by the lack of cross-reactivity with D. nodosus fimA sequences that had a single base difference. DNA from 14 footrot samples previously genotyped by PCR-SSCP/sequencing, was assayed using the PCR/oligotyping technique. All types of D. nodosus which had been detected previously with a PCR-SSCP/sequencing method were detected by this procedure. However, for three of the 14 footrot samples, PCR/oligotyping detected additional types of D. nodosus. Further PCR amplification using type-specific primers, confirmed that these types were present in the original footrot samples. These results indicate that PCR/oligotyping is a specific, accurate, and useful tool for typing footrot samples. In combination with a rapid DNA extraction protocol, D. nodosus present in a footrot sample can be accurately genotyped in less than two days. Individual animals from the same farm, or the same paddock, were often infected by different strains of D. nodosus. This suggests a host role in mediating footrot infection, or that the interaction between the pathogen and the host is important. In order to better understand the interaction between the bacterium and the host, two polymorphic ovine class II MHC genes DQA1 and DQA2, which have been previously shown to be important in footrot infection, were also investigated in this study. PCR-SSCP/sequencing analysis of the DQA1 locus revealed ten unique ovine DQA1 sequences, with five of them being newly identified. This increases the number of known ovine DQA1 alleles from 8 to 13 (including a null allele), implying a high level of polymorphism at the ovine DQA1 locus. D. nodosus present on 20 footrot infected sheep from the same flock were genotyped, together with the ovine DQA1 and DQA2 genotypes of their hosts. Preliminary results showed that sheep with the same DQA1 and DQA2 genotypes tended to be infected by similar types of D. nodosus. Different types of D. nodosus were generally found on sheep with different genotypes at either the DQA1 or the DQA2 locus. This suggests the diversity in D. nodosus infection may be associated with the heterogeneity in the host MHC. However, as only a small number of animals from the same sire were analysed, further investigation is needed to gain a better understanding of the interaction between D. nodosus and the host MHC.

A candidate gene approach was used to identify potential genetic markers associated with wool quality traits including mean fibre diameter (MFD), fibre diameter standard deviation (FDSD), coefficient of variation of fibre diameter (CVD), prickle factor, curvature, yellowness, brightness, staple strength, staple length, yield, greasy fleece weight (GFW) and clean fleece weight (CFW). Inheritance of potential genetic markers was studied in two half-sib Merino families and assessed for association with the wool quality traits. The sire for one of the half-sib families is referred to as MV144-58-00, and wool measurements from its progeny were taken at 12 (n = 131), 24 (n =128) and 36 (n = 37) months of age. The sire for the second half-sib family is referred to as Stoneyhurst, and wool measurements from its progeny (n = 35) were taken at 12 months of age. Genes that code for the keratin intermediate-filament proteins (KRTs) (KRT1.2, KRT2.10) and the keratin intermediate-filament-associated proteins (KAPs) (KAPl.1, KAPl.3, KAP3.2, KAP6.1, KAP 7, KAP8) were targeted for this investigation, along with the beta 3-adrenergic receptor (ADRB3) gene and microsatellites BfMS and OarFCB193. Polymerase chain reaction (PCR) was used to amplify specific DNA fragments from each locus and PCR- single strand conformational polymorphism (PCR-SSCP) analysis was used to detect polymorphism within the half-sib families for all the loci, except for the KAP1.1 gene, where length polymorphism was detected using agarose gel electrophoresis. Only the loci that were heterozygous for the sire (KAP1.1, KAP1.3, KRT1.2, ADRB3, KAP8) and hence were informative, were genotyped in the progeny. The total number of alleles observed at the KAP1.1, KAP1.3, KRT1.2, KAP8 and the ADRB3 loci were four, ten, six, five and six, respectively. Analysis of each of the informative loci revealed allelic associations with various wool traits. In the MV144-58-00 (genotypes KAP1.1 AB; KAP1.3 BD; KRT1.2 AB; ADRB3 CE) half-sib, inheritance of the KAP1.1 A allele was associated with a higher yield at 24 months of age (P = 0.037). This trend also observed at 36 months of age (P = 0.078). At 12 months of age, the KAP1.1 A allele tended to be associated with increased staple length (P = 0.08). At 36 months of age, the inheritance of the KAP1.1 B allele tended towards being associated with whiter wool (P = 0.080). The MV144-58-00 KAP1.3 D allele tended to be associated with increased yield at 24 and 36 months of age (P = 0.091 and 0.059, respectively), and with lower FDSD at 12 months of age (P = 0.055). The sire KAP1.3 B allele was associated with whiter wool colour at 36 months of age (P = 0.045). The inheritance of the MV144-58-00 KR T1.2 B allele was associated with or tended to be associated with a smaller FDSD (P = 0.040), an increase in staple strength (P = 0.025) and an increase in GFW (P = 0.069) at 12 months of age. At 24 months of age, the KR T1.2 B allele tended to be associated with increased yield (P = 0.057). At 36 months of age, the KRTl.2 A allele was associated with whiter wool (P = 0.019) and tended to be associated with increased crimp within the wool fibre (P = 0.089). In the Stoneyhurst (genotypes KAP1.1 BC; KAP1.3 CJ; KRT1.2 DE; ADRB3 CE) half-sib, inheritance of the KAP1.1 B allele was associated with longer staple length (P = 0.018) and a decrease in wool brightness (P = 0.039). In contrast, KAP1.1 C allele was associated with lowest staple length (P = 0.018) and brighter wool colour (P = 0.039). Associations observed with the inheritance of Stoneyhurst KAP 1.1 alleles were similar to the inheritance ofKAPl.3 alleles. Stoneyhurst KAP1.3 J allele was associated with longer staple length (P = 0.017) and a decrease in wool brightness (P = 0.010). In contrast, KAP1.3 C allele was associated with lowest staple length (P = 0.017) and brighter wool colour (P = 0.010). The Stoneyhurst KRT12 D allele was associated with longer staple length and a decrease in wool brightness (P = 0.033). In contrast, KRT1.2 E allele was associated with lowest staple length (P = 0.033) and brighter wool colour (P = 0.022). Sire alleles at the ADRB3 gene locus were associated with variation in staple strength (P = 0.025) for MV144-58-00's progeny, and with variation in yield (P = 0.023) for Stoneyhurst's progeny. The results obtained in this thesis are consistent with KAP1.1, KAP1.3 and KRT1.2 being clustered on one chromosome because both sires in this study passed on two major KAP1.1-KAP1.3-KRT1.2 haplotypes to their progeny, and the associations with wool traits were very similar for all the three loci. The major sire derived KAP1.1 – KAP1.3 - KRT1.2 haplotypes observed within the MV144-58-00 half-sib were: BBA (frequency of 43.4%; n = 43) and ADB (frequency of 44.4%; n = 44). Other minor haplotypes observed were: ADA (frequency of 4.0%; n = 4); BDA (frequency of 2.0%; n = 2); BBB (frequency of 3.0%; n = 3) and BDB (frequency of 3.0%; n = 3). In the Stoneyhurst half-sib, major sire-derived KAP 1.1 - KAP 1.3 - KR Tl.2 haplotypes observed were CCE (frequency of 53.1 %; n = 17) and BJD (frequency of 40.6%; n = 13). The minor haplotype BJE (frequency of 6.3%; n = 2) was also observed. Statistical analyses within the MVI44-58-00 half-sib showed that KAP1.1 AKAP1.3 D - KRT1.2 B haplotype was associated with increased yield (P = 0.023) and tended towards whiter wool colour (P = 0.059), smaller FDSD (P = 0.081) and stronger staple strength (P = 0.092). In the Stoneyhurst half-sib, the KAP1.1 B - KAP1.3 J - KRT1.2 D haplotype was associated with longer staple length (P = 0.010), while the KAP1.1 C - KAP1.3 C - KRT1.2 E haplotype showed a strong trend with increased wool brightness (P = 0.096). Result from this study indicated that the keratin genes on chromosome 11 are recombining relatively frequently at recombination "hotspots". A high rate of recombination among loci that impact on wool traits would make breeding for consistent wool quality very difficult. The results presented in this thesis suggest that genes coding for the KRTs and KAPs have the potential to impact on wool quality. KAP1.1, KAP1.3 and KRT1.2 could potentially be exploited in gene marker-assisted selection programmes within the wool industry to select for animals with increased staple length, 'increased staple strength, higher yield and brighter wool. This study was however limited to two half-sib families, and further investigation is required.

Déterminer le régime alimentaire des gastéropodes terrestres est important étant donné leurs impacts écologiques et économiques. L’observation en nature est difficile, car les gastéropodes sont principalement actifs la nuit et sont de petite taille. De plus, l’analyse visuelle des contenus digestifs est compliquée car les gastéropodes broient leurs aliments, ne permettant pas une identification précise. L’objectif est de produire une méthode fiable et peu onéreuse pour déterminer le régime alimentaire des gastéropodes en milieu naturel. Le modèle, Arion fuscus, a été échantillonné au printemps dans quatre érablières anciennes du sud du Québec (Canada). Une approche basée sur l’ADN a été utilisée avec amplification par PCR d’un segment d’un gène chloroplastique. La diversité a été déterminée par électrophorèse sur gel non-dénaturant suivie d’un séquençage des variants d’intérêts pour identifier les plantes consommées. Pour les 52 limaces analysées, les PCR ont fonctionné avec succès et les extractions d’ADN de mêmes individus traités indépendamment ont montré les mêmes résultats sur les gels SSCP. Des résultats fiables ont été produits en quelques semaines et le nombre d’échantillons à séquencer a été limité, réduisant ainsi les coûts. Les résultats montrent une faible diversité végétale consommée (6 haplotypes) par A. fuscus; 78,8% des limaces ont uniquement consommé la même espèce de plante. Cette méthode peut permettre de meilleurs suivis des espèces consommées par les gastéropodes dans un contexte d’écologie de la conservation ou de contrôle des espèces nuisibles. / The diet of terrestrial gastropods is difficult to assess in nature because of their often-nocturnal activity and their small size. Moreover, visual analyzes of digestive contents are problematic because gastropods grind food into small particles, making visual identification unexploitable. Determining the gastropods ‘diet is nevertheless highly relevant because gastropods may have strong ecological and economic impacts. On the one hand, several species are listed endangered or critically endangered. On the opposite, numerous species are considered agricultural pests. The objective is to produce an accurate and inexpensive method to determine the diet of gastropods. More specifically, by using an approach based on PCR-amplified DNA followed by SSCP gels, it was possible to determine taxa diversity before Sanger sequencing, and thus reduce costs. We used Arion fuscus from four old maple forests sites in southern Quebec (Canada) as model to test the method. The 52 individuals’ digestive contents analyzed resulted in a successful PCR amplification and independent DNA extractions from a given individual gave the same SSCP pattern. This method produced reliable results in a few weeks and at a low cost, making it possible to sequence only a few samples to know the plant species consumed by all individuals in the study. Results revealed a low diversity of plants consumed (6 haplotypes). 78.8% of the slugs harvested in spring consumed one and the same species of plant. This method enables the monitoring of plant species consumed by gastropods in conservation and agricultural pest control.

Une identification précise des espèces exotiques est essentielle afin de déterminer la nature et l’ampleur des impacts que ces espèces auront sur leurs nouveaux habitats. Le complexe d’Arion subfuscus s. l., originaire d’Europe, fait partie des limaces les plus abondantes dans le nord-est de l’Amérique du Nord et plusieurs impacts dus à leur présence ont été rapportés. Cependant, l’identité des espèces introduites demeure inconnue dans la plupart des régions. L’objectif de ce projet est donc de déterminer la répartition récente, la diversité taxonomique et l’origine des membres du complexe d’A. subfuscus s. l. au Québec en se basant sur leur identité mitochondriale (16S rDNA). Un total de 526 spécimens provenant de 68 sites à travers le Québec et un site en Nouvelle-Écosse ont été analysés à l’aide de la technique des SSCP et leurs séquences ont été déterminées. Huit haplotypes de deux espèces allopatriques, A fuscus et A. subfuscus s. s. (lignées S1 et S2) ont été détectés. Les résultats confirment que des limaces provenant de régions distinctes d’Europe ont été introduites à de multiples reprises. Une comparaison avec des données historiques de répartition a révélé une expansion fulgurante de la répartition depuis les 50 dernières années. Arion fuscus est la principale espèce envahissante qui a été détectée dans toutes les régions échantillonnées, ce qui contraste avec les études antérieures réalisées ailleurs en Amérique du Nord. Le rôle potentiel des échanges commerciaux internationaux dans l’histoire d’introduction des espèces exotiques est discuté. / Accurate identification of exotic species is required to assess the magnitude and nature of consequences on their new habitats. The Arion subfuscus s. l. species complex comprised slugs of European origins that are amongst the most abundant slug species in northeastern North America and various impacts of their presence are reported. However, the identities of the species introduced remain unknown in most regions. This study aims at determining the current distribution, taxonomic identity and the origins of the members of the A. subfuscus s. l. complex in Quebec (Canada) based on mitochondrial 16S rDNA. A total of 526 specimens from 68 locations throughout Quebec and one site in Nova Scotia were SSCP analysed and their sequences were determined. Eight haplotypes of the allopatric A. fuscus and A. subfuscus s. s. (lineages S1 and S2) were detected. Results confirmed that slugs from distinct European regions were introduced multiple times. Comparison with previous survey revealed an impressive expansion of the distribution during the last 50 years. Arion fuscus is the major invasive species found throughout Quebec, contrasting with previous North American studies. The potential role of international trade in the introduction history of exotic species is discussed.

The overall goal of this research was to determine what, if any, role grapevine rhizosphere bacteria play in the differing susceptibilities of New Zealand grown rootstocks to Cylindrocarpon black foot disease. The size and diversity of bacterial populations associated with the rhizospheres of grapevine rootstocks: 101-14, 5C, Schwarzmann and Riparia Gloire were evaluated. Dilution plating showed that total bacterial (P=0.012, P=0.005 for NA and KB, respectively) and fluorescent Pseudomonad (P=0.035) rhizosphere counts differed between rhizosphere and bulk soils but did not correlate with the differing susceptibilities of the rootstock varieties to black foot. No varietal differences were found for spore forming bacteria (P=0.201). SSCP banding patterns showed that species diversity was similar for most rootstocks, but that there were some differences in the composition of bacterial populations, probably attributable to vigour. Some functional characteristics of the bacteria isolated from the rhizospheres of the most and least susceptible rootstock varieties were assessed to investigate their potential to suppress the pathogen. In dual culture, bacteria from Riparia Gloire, 101-14 and the control soil all had little ability to antagonise Cylindrocarpon destructans. However, they differed in their degrees of activity for glucanase (P=0.000), protease (P=0.001) and siderophores (P=0.000). In all tests, bacterial isolates from the rhizosphere of 101-14 had the largest number of active isolates (P≤0.002); however, those from Riparia Gloire had the greatest degree of positive responses for the glucanase and siderophore assays. Bacterial isolates from the control soil produced few glucanases and no siderophores, but had the highest degree of protease activity. Bands excised and sequenced from SSCP gels frequently matched to other ‘uncultured bacteria’ in GenBank, as well as to other bacterial phyla, classes and genera commonly isolated from soil and sediment samples. These included members of the Firmicutes, Proteobacteria (α, δ, γ), Verrucomicrobia, Acidobacteria and Chromatiales. The pathogenicity of C. destructans and Fusarium oxysporum was investigated by inoculating soil containing wounded ungrafted rootstocks of 101-14, 5C, Schwarzmann and Riparia Gloire. Results indicated that F. oxysporum might be a more aggressive pathogen than C. destructans. Inoculation with F. oxysporum or C. destructans increased disease severity, P=0.018 and P=0.056, respectively at 0 cm. Rootstock variety influenced disease severity caused by C. destructans (P<0.001) and F. oxysporum (P=0.090), with rootstocks 101-14 and 5C being most susceptible to C. destructans, and Riparia Gloire and Schwarzmann most susceptible to F. oxysporum. There was also an indication that inoculation with one pathogen increased plant susceptibility to the other, with increased F. oxysporum infection in the C. destructans inoculated treatments of Riparia Gloire and Schwarzmann (P<0.05). The effect of carbohydrate stress (leaf trimming) and inoculation on C. destructans disease severity, incidence, and rootstock rhizosphere bacterial populations was evaluated by inoculating the soil containing one year old plants of Sauvignon Blanc scion wood grafted to rootstocks 101-14 and Schwarzmann. Disease severity and incidence was similar for both Schwarzmann (8.4% and 29.3%, respectively) and 101-14 (14.9% and 31.0%, respectively). When data for the moderate and no stress treatments were combined, because their effects were similar, the disease severity was significantly higher for the highly stressed plants(P=0.043). Stress did not influence disease incidence (P=0.551). Infection occurred in the non-inoculated plants, but disease severity was higher in the plants inoculated with C. destructans than those that were not. Root dry weight of highly stressed plants was lower than in both the moderately stressed (P=0.000) and unstressed plants (P=0.003). An interaction between inoculation and stress (P=0.031) showed that inoculated and highly stressed plants had the lowest root dry weight but there was no effect of rootstocks (P=0.062). There was no significant effect of carbohydrate stress (P=0.259) or inoculation (P=0.885) on shoot dry weight. SSCP banding patterns showed that bacterial diversity was generally similar between rootstocks, but stress and inoculation altered rhizosphere bacterial communities. This study has demonstrated that functionality of grapevine rhizosphere bacteria do differ between grapevine rootstock varieties that have different susceptibilities to black foot disease, but that this role needs to be further investigated if more accurate and practically relevant conclusions are to be drawn.

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

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Cardiotrophin-1 ist ein Zytokin der Familie Interleukin-6-Familie, zu der auch IL-11, CNTF, OSM und LIF gehören. Diese Substanzen wirken über die gemeinsame Rezeptoruntereinheit gp130. CT-1 induziert die Hypertrophie von Kardiomyozyten und inhibiert die Apoptose kardialer und Zellen. In verschiedenen Tiermodellen der Herzinsuffizienz konnte eine gesteigerte myokardiale CT-1 Expression beobachtet werden. Kardiomyopathien sind wiederum kardiale Erkrankungen, die mit einer Hypertrophie und Apoptose einhergehen und zu einer Herzinsuffizienz führen können. Man geht davon aus, dass 25-50 Prozent der familiär sind. Hierbei handelt es sich um eine monogenetische Erkrankung, die überwiegend autosomal-dominant vererbt werden. Daneben konnten aber auch modifizierende Polymorphismen in neurohumoralen Faktoren identifiziert werden. Basierend auf diesen Ergebnissen war das Ziel dieser Arbeit die Analyse der möglichen Beteiligung genetischer Varianten der kodierenden sowie der regulatorischen Region an der Pathogenese der Hypertrophen bzw. Dilatativen Kardiomyopathie. Zusätzlich sollte die mRNA-Expression von CT-1 in Myokardbiopsien von Patienten mit Herzinsuffizienz quantifiziert werden. Hierfür musste zunächst die Sequenzen der 5´-flankierenden Region identifiziert und bezüglich ihrer regulatorischen Eigenschaften analysiert werden. Es konnten 1,1 kb der 5´-flankierenden Region sequenziert werden. Die anschließende Luciferase-Reportergen-Analyse wies regulatorische Aktivitäten für den gesamten Bereich nach. Diese Region enthält zahlreiche cis-aktive DANN-Sequenzen aber keine TATA-Box. Für die Mutationssuche wurden 64 Patienten mit DCM, 53 Patienten mit HCM sowie 100 Kontrollpersonen mittels PCR-SSCP-Analyse untersucht. Es konnte eine kodierende Variante A92T bei jeweils einem DCM- bzw. HCM-Patienten identifiziert werden. Diese Substitution liegt in einem Bereich, der zwischen verschiedenen Spezies (Ratte, Maus, Mensch) konserviert ist. Diese Mutation könnte eine Veränderung der Sekundärstruktur bewirken und liegt in einem möglichen funktionellen Bereich. Die Promotorregion wies eine Basenpaarsubstitution bei -130 (G/T) sowie eine Deletion der Basen CTTT zwischen -992 und -995 auf. Der Polymorphismus an Position -130 fand sich tendenziell häufiger bei Patienten mit Dilatativer Kardiomyopathie. Die CTTT-Deletion konnte nur bei einer Patientin mit HCM nachgewiesen werden. Für die Quantifizierung der CT-1 mRNA wurden rechtsventrikuläre Endomyokardbiopsien von 6 Patienten mit eingeschränkter LVEF (CHI), 5 Patienten nach Herztransplantation (TX) sowie 3 Kontrollpatienten (KO) eingesetzt. Es konnte ein relativer Anstieg der CT-1 Expression um 82% bei den Patienten mit eingeschränkter LVEF festgestellt werden. Interessanterweise besteht eine enge Korrelation zur Schwere der eingeschränkten Herzfunktion sowie zur Zunahme der Hypertrophie. / Cardiotrophin-1 is a cytokine, which belongs to the interleukin-6 family, which includes IL-11, CNTF, OSM and LIF. These factors act via the receptor subunit gp130. CT-1 induces the hypertrophy of cardiomyocytes and inhibits the apoptosis of cardiac cells. Studies in animal models of congestive heart failure showed an enhanced expression of CT-1 in the myocardium. Cardiomyopathies are cardiac diesorders, which are charakterized by hypertrophy and apoptosis and which can terminate with congestive heart failure. About 25-50 percent of all cases are familial. It is a monogenetic mendelian disorder with an autosomal-dominant inheritance in most cases. Beside this, modifying polymorphisms in neurohunoral factors could be identified. Based on these facts, the aim of this study was to identify genetic variants within the coding and regulatory region of the CT-1 gene, which could influence the pathogenesis of hypertrophic or dilated cardiomyopathy. Additionally, the mRNA-expression of CT-1 in myocardial biopsies of heart failure patients should be quantified. First, it was necessary to sequence the 5´-untranslated region and to analyse its regulatory function. We could sequence 1.1 kb of the 5´-UTR. The luciferase reportergene assay showed a significant promoter activity for the whole region. The region contains various cis-active DNA sequences but no TATA-box.The TRANSFAC-analysis identified different binding sites for transcription factors but no TATA-box. The genetic material of 64 DCM and 53 HCM patients and 100 controls was screened for mutaions by using a PCR-based SSCP-analysis. A coding variant A92T could be identified for a patient with DCM and for an HCM patient. This mutation lies within a region which is conserved between different species (rat, mouse, human). This variant could disturb the secondary structure and lies in a probable functional region. Within the promoter we could identify a basepair substitution at position -130 (G/T) and a 4-basepair deletion between -992 and -995 (CTTTdel). The polymorphism at -130 showed a tendency for a higher occurrence in DCM patients. One HCM patient was heterozygous for the CTTT-deletion. To quantify the CT-1 mRNA we used endomyocardial biopsies of 6 patients with reduced LVEF (CHI), 5 patients after heart transplantation (TX) and 3 controls (KO). We performed a semiquantitative analysis by using HPLC and an external standard (PDH mRNA). We found an increased expression of CT-1 by 82% for patients with heart failure. Interestingly, we saw a tight correlation with to the reduction in LV function and to the degree of hypertrophy.

The aetiological diagnosis of recessive non-syndromic hearing loss poses a challenge owing to marked heterogeneity and the lack of identifying clinical features. The finding that up to 50% of recessive non-syndromal genetic hearing loss among Caucasians was due to mutations in GJB2, the gene encoding Connexin 26 (Cx26) was a breakthrough, whose value as a diagnostic tool has been limited by the significant variation in the prevalence of deafness genes and loci among population groups. The significant association of the GJB6-D13S1830 deletion among individuals with one mutant GJB2 allele highlighted the need to explore population specific genetic mutations for NSHL. Although data from Sub-Saharan Africa is limited, reported studies found a high prevalence of R143W GJB2 mutation among Ghanaian, the 35delG mutation in 5 out of 139 Sudanese and a low prevalence of GJB2 variations among 385 Kenyan deaf children. The mutation spectrum of Waardenburg Syndrome (WS) in Africans has not been documented. During a visit to a School for the Deaf in the Limpopo Province of South Africa in 1997, it was noted that a high number of students came from Nzhelele sub-district. All had childhood onset hearing loss with no associated anomalies or disorders. The question arose as to whether there was a high-risk area for deafness in the Limpopo Province and what the aetiology of this hearing loss was.The main aim of this study was to investigate the role of GJB2, the GJB6-D13S1830 deletion, and the four common mitochondrial mutations, A1555G, A3243G, A7511C and A7445G, in the African hearing-impaired population of Limpopo province in South Africa, and to identify the mutation spectrum of the deafness genes found. The type and degree of hearing loss in this hearing impaired population would also be assessed. Secondly, this study sought to identify the mutations in a sibling pair with 2 clinical WS and to use the findings in a future study to establish the mutation spectrum of WS in the African population of the Limpopo province and of South Africa in general. The study was designed as a two phase study, in which phase 1 was used for hypothesis formulation and phase 2 was for hypothesis testing. While phase 1 was a descriptive retrospective case study, phase 2 was a combination of sample survey and prospective descriptive case study. In phase 1, demographic data of 361 students in two schools of the deaf in the Limpopo province was analyzed for evidence of areas of high risk populations for deafness in the province. In phase 2, a group of 182 individuals with genetic non-syndromic hearing loss (NSHL) and two siblings with clinical WS from two schools for the Deaf in the Limpopo Province of South Africa were investigated. A thorough clinical examination, audiological evaluation and urinalysis were done. Mutational screening was carried out in all 184 subjects using genomic DNA using single-strand conformation polymorphism (SSCP), multiplex polymerase chain reaction (PCR), and direct sequencing for GJB2, and Restriction Fragment-Length Polymorphism (PCR–RFLP) analysis for GJB6, and SSCP, hetero-duplex analysis, and direct sequencing of the first 8 exons of PAX3 and all of MITF for Waarenburg syndrome. Data analysis was by geographical mapping, frequency tables, tests of association with calculation of odds ratios, and binary logistic regression analysis using STATA and GIS mapping systems. The results indicate that there seem to be areas of genuine populations at risk for hearing loss in the Limpopo province of South Africa, namely Mutale and parts of Makhado and Thulamela municipalities. In Thulamela (NP343) wards 11-15, 26-30 and 31-35, and in Mutale (NP 344) wards 6-10, together accounted for 67 (18%) of participants in phase 1, and 33 (18%) of the participants in phase 2 of the study. Mutale municipality in the Vhembe 3 district gave with a projected prevalence of at least 13.14 deaf children per 100,000 African population attending the local school for the deaf. The observed hearing loss is a genetic, non-syndromic form, which is mainly severe and severe to profound, although without any clear defining configuration or shape. It is a stable, non-progressive and prelingual form of hearing loss, implying that this may be a recessive form of deafness. No identifiable environmental confounding factors or associations were identified. The deafness is not linked the common known auditory gene mutations in GJB2, the GJB6-D13S1830 deletion, or the common mitochondrial mutations A1555G, A3243G, A7511C and A7445G. Severe and profound levels of hearing loss were found in 22.8% and 75% of the cohort respectively, with the majority exhibiting flat (70.1%) or sloping (23.4%) audiograms that were commonly symmetrical (81.5%). However, as indicated, there was no clear pattern in the audiological findings overall. None of the 184 hearing impaired individuals exhibited any of the reported disease causing mutations of GJB2, including 35delG. There was, however, a high prevalence of two variants, the C>T variant at position g.3318-15 and the C>T variant at position g.3318-34, occurring in 21.4% and 46.2% of the deaf cohort respectively. The same variants were found to occur in 35% and 42.6% of a normal hearing control group (n = 63) respectively, indicating that these variations are polymorphisms. In three subjects (1.63% of the cohort), a T>A homozygous variation at position g.3318-6 was detected. Its significance in the causation of NSSNHL is yet to be determined. The GJB6-D13S1830 deletion was not detected in any of the participants. None of the four mitochondrial mutations screened for were found. 4 These results indicate that GJB2 is not a significant deafness gene in the African population of the Limpopo Province of South Africa and that significant genes for non-syndromic recessive hearing loss in this population are yet to be found. The geographical clustering of deafness found in this study, combined with the lack of identifiable common associated clinical features among the subjects of this study (excluding the WS sibling pair), suggests that these subjects have a genetic recessive non-syndromal type of hearing loss. In the context of historical and cultural evidence of consanguinity in this population, a founder effect cannot be ruled out. A rare mutation, R223X, previously identified only once out of 470 WS patients, was identified in the PAX3 gene among the WS sibling pair. A novel silent change GGG>GGT at amino acid 293, was also identified. These identical findings document, for the first time, a molecular defect in WS in an African sibling pair, and confirm WS Type I in this family, which could be found in other WS type I South Africans in the Limpopo Province of South Africa. The current study demonstrated that parents of genetically hearing impaired children in these areas are able to detect hearing loss at an early age, with over 60% suspecting their children’s hearing loss below 6 months of age. A child-centered management model encompassing all the areas relevant to childhood deafness/hearing impairment, which takes into consideration the prevailing logistical and financial constraints of the available healthcare system, is proposed. The implementation of this model requires a paradigm shift from the current fragmented model of service delivery to a cohesive patient-centered approach, based on concrete data from appropriate community based research, in which all the relevant parties communicate and share resources. 5 It would achieve the goals of early detection and intervention, as well as inclusive education for all. The relevant health and education policies are already in place and the posts funded. Equitable implementation of these policies would require appropriate community based research, as well as improved communication and consultation between the various stakeholders to ensure an efficient and affordable quality healthcare service for all hearing impaired South Africans.