The PDZ and LIM domain 5 (PDLIM5) gene may play a role in alcohol dependence (AD), bipolar disorder, and major depressive disorder; however, no study has identified shared genetic variants within PDLIM5 gene among AD, type 2 diabetes (T2D), and hypertension. This study investigated the association of 72 single nucleotide polymorphism (SNPs) with AD (1066 AD cases and 1278 controls) in the Study of Addiction - Genetics and Environment (SAGE) sample and 47 SNPs with T2D (878 cases and 2686 non-diabetic) and hypertension (825 cases and 2739 non-hypertensive) in the Marshfield sample. Multiple logistic regression models in PLINK software were used to examine the associations of genetic variants with AD, T2D, and hypertension and SNP x alcohol consumption interactions for T2D and hypertension. Twenty-five SNPs were associated with AD in the SAGE sample (p < 0.05); rs1048627 showed the strongest association with AD (p = 5.53 × 10−4). Of the 25 SNPs, 5 SNPs showed associations with both AD in the SAGE sample and T2D in the Marshfield sample (top SNP rs11097432 with p = 0.00107 for T2D and p = 0.0483 for AD) while 6 SNPs showed associations with both AD in the SAGE sample and hypertension in the Marshfield sample (top SNP rs12500426 with p = 0.0119 for hypertension and p = 1.51 × 10−3 for AD). SNP (rs6532496) showed significant interaction with alcohol consumption for hypertension. Our results showed that several genetic variants in PDLIM5 gene influence AD, T2D and hypertension. These findings offer the potential for new insights into the pathogenesis of AD, T2D, and hypertension.

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Vid klinisk enbaspolymorfi (SNP) analys inkluderas DNA-kontroller med kända genotyper i varje analysomgång för att säkerställa riktigheten vad gäller analysresultatet. DNA-kontrollerna har en central roll för resultatens trovärdighet vid genotypningen. Vissa kontrollprover som används är av sällsynt genotyp och kan vara mycket svåra att få tag på. Detta arbete har utförts för att undersöka om det går att erhålla DNA-material från sällsynta genotyper med hjälp av helgenomamplifiering och på så sätt säkerställa en tillgång till dessa. I arbetet testades helgenomamplifiering med hjälp av två olika kit. De helgenomamplifierade produkternas kvantitet och kvalitet analyserades och jämfördes med det ursprungliga DNA:t, med avsikt att redogöra för det mest fördelaktiga kitet för SNP-analys i kliniskt syfte. Båda helgenomamplifierings-kiten påvisade god förmåga att amplifiera genomiskt DNA med hög kvalité. Helgenomamplifierat DNA från det bästa kitet sekvenserades och här var skillnader mellan ursprungligt och helgenomamplifierat DNA marginella. Vid sekvensanalys av ett 464 baspar långt fragment av faktor II genen och 585 baspar långt fragment av ApoE genen på fem helgenomamplifierade DNA-prover påvisades endast en eventuell diskrepans. / Clinical single nucleotide polymorphisms (SNP) analysis includes DNA controls with known genotypes in each run to ensure the accuracy of the analysis results. DNA controls have a central role for the credibility of the results in the genotyping process. Some of the used control samples are rare and can be very difficult to obtain. This work was carried out to investigate whether it is possible to obtain DNA from samples with a rare genotype using whole genome amplification and as a result ensure access to these samples. In this work the whole genome amplification method was tested by two different kits. The quantity and quality of the whole genome amplification products were analyzed and compared with the original DNA, with the intention to describe the most advantageous kit for clinical SNP analysis. Both tested kits demonstrated a good ability to amplify genomic DNA with high quality. Whole genome amplified DNA from the best kit was sequenced and the difference between the original DNA and whole genome amplified DNA was negligible. Sequence analysis of 464 base pairs of the factor II gene and 585 base pairs of the ApoE gene in five whole genome amplified DNA samples indicated only one possible discrepancy.

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A narrow germplasm base and a complex allotetraploid genome have historically made the discovery of single nucleotide polymorphism (SNP) markers difficult in cotton (Gossypium hirsutum). We conducted a genome reduction experiment to identify SNPs from two accessions of G. hirsutum and two accessions of G. barbadense. Approximately 2 million sequence reads were assembled into contigs with an N50 of 508 bp and analyzed for SNPs. A total of 11,834 and 1,679 SNPs between the accessions G. hirsutum and G. barbadense, respectively, were identified with highly conservative parameters (a minimum read depth of 8x at each SNP and a 100% identity of all reads within an accession at the SNP). Additionally, 4,327 SNPs were identified between accessions of G. hirsutum in and assembly of Expressed Sequence Tags (ESTs). 320 and 252 KASPAR assays were designed for SNP mapping in non-genic and genic regions respectively. 187 markers in total (136 non-genic, 51 genic) were mapped using KBioscience KASPar genotyping assays in a segregating F2 population using the Fluidigm EP1 system. EST The target genome of EST markers was successfully predicted bioinformaticly diploid reference sequences. Examination of nucleotide substitutions and SNP frequencies further confirms validity of new markers. A genetic map was constructed using a large G. hirsutum segregating F2 population. Genetic maps generated by these newly identified markers will be used to locate quantitative, economically important regions within the cotton genome.

Following its introduction to North America in the late nineteenth century, Bromus tectorum L., an inbreeding invasive winter annual grass, has become dominant on millions of hectares of sagebrush steppe habitat throughout Intermountain Western North America. It appears that within the last 30-40 years, B. tectorum has expanded its range southward into the Mojave Desert and also into more climatically extreme salt desert environments. Previous research using microsatellite markers and experimental studies has suggested that lineages found in desert habitats are genetically distinct from those found in the sagebrush-steppe habitat and possess suites of traits that pre-adapt them to these environments. To provide additional support for our hypothesis that desert habitat-specific haplotypes dominate and are widely distributed across warm and salt desert habitats, we genotyped approximately 20 individuals from each of 39 B. tectorum populations from these habitats and adjacent sagebrush steppe habitats using 71 single nucleotide polymorphic (SNP) markers. Our data clearly demonstrate that populations throughout the Mojave Desert region, as well as in salt desert habitats further north, are dominated by a small number of closely related SNP haplotypes that belong to the desert clade. In contrast, populations from adjacent environments are largely dominated by haplotypes of the common clade, which is widely distributed throughout the North American sagebrush steppe. Populations across all habitats were usually dominated by 1-2 SNP haplotypes. This suggests that inbreeding B. tectorum lineages can often maintain their genetic integrity. It also explains the strong association between marker fingerprints and suites of adaptive traits in this species.

Many diseases of genetic origin originate from non-synonymous single nucleotide polymorphisms (nsSNPs). These cause changes in the final protein product encoded by a gene. Through large scale sequencing and population studies, there is growing availability of information of which variations are tolerated and which are not. Variant effect predictors use a wide range of information about such variations to predict their effect, often focusing on evolutionary information. Here, a novel amino acid substitution variant effect predictor is developed. The predictor is a deep convolutional neural network incorporating evolutionary information, sequence information, as well as structural information, to predict both the pathogenicity as well as the severity of amino acid substitutions. The model achieves state-of-the-art performance on benchmark datasets.

During the past three years, we assisted to the rise of a new pathogen that afflicted the world with a global pandemic. Working in an era of rapid change has posed important challenges and the focus of research has shifted more and more toward topics of greater social utility. However, this period has also brought a new role for wastewater highlighting how it can provide insight into the health of a community. This is the approach of Wastewater-based Epidemiology (WBE). The work presented here aimed to deepen this approach not only at the theoretical level but also contributing with an ongoing monitoring of about 30 months. The main objectives were (i) to collect information on the recently discovered SARS-CoV-2 virus, its biology, transmission mechanism, and role in wastewater treatment plants (WWTP); (ii) set up a surveillance system that would allow to monitor SARS-CoV-2 infections over time, obtaining early information on its spread among the population to support the Health Authority. Starting from a detailed study of the shedding mechanisms of SARS-CoV-2 in the feces of infected patients, we moved on to the evaluation of the viral concentrations in the sewage system and the wastewater entering the WWTP. The possibility of a faecal-oral transmission route of the virus was investigated by evaluating the data about viability and infectivity in wastewater. The natural processes of decay of the virus in wastewater and the reduction of its concentration in the different treatment stages of WWTPs were explored in literature and with experimental data. At the same time, we developed a SARS-CoV-2 surveillance system in wastewater by applying different detection methods. Some practical and scientific aspects of the analysis protocol have been studied in depth such as the choice of the type of sample, the storage temperatures, and the pre-heat treatments aimed at making the analysis safer for the operator. The choice of the concentration method was evaluated to comply with the low concentration of the viral titer and therefore the crucial importance of this phase of the protocol. During the monitoring campaign, we further investigated aspects related to data processing and developed normalization approaches. Samples from WWTPs in the province of Trento were analysed weekly and sampling frequencies and curve smoothing methods deriving from those data were evaluated. The trend curves thus obtained were compared with those deriving from clinical data provided by the local Health Authority and signals of early warnings of virus diffusion trends in the population were highlighted. With the alternation of the different variants of the virus and the evidence of their importance in the development of new waves of infection, a PCR based genotyping method has been devised to rapidly identify the already known variants. In conclusion, this research project addressed a broad spectrum of aspects related to the WBE approach in contrasting the COVID-19 emergency and confirmed that wastewater could be a valuable source of information and management support for this and other emerging pathogens or micropollutants.

Background: Development of brain metastases in advanced melanoma patients is a frequent event that limits patients’ quality of life and survival. Despite recent insights into melanoma genetics, systematic analyses of genetic alterations in melanoma brain metastasis formation are lacking. Moreover, whether brain metastases harbor distinct genetic alterations beyond those observed at different anatomic sites of the same patient remains unknown. Experimental Design and Results: In our study, 54 intracranial and 18 corresponding extracranial melanoma metastases were analyzed for mutations using targeted next generation sequencing of 29 recurrently mutated driver genes in melanoma. In 11 of 16 paired samples, we detected nucleotide modifications in brain metastases that were absent in matched metastases at extracranial sites. Moreover, we identified novel genetic variants in ARID1A, ARID2, SMARCA4 and BAP1, genes that have not been linked to brain metastases before; albeit most frequent mutations were found in ARID1A, ARID2 and BRAF. Conclusion: Our data provide new insights into the genetic landscape of intracranial melanoma metastases supporting a branched evolution model of metastasis formation.

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