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An increasing number of countries are investing efforts to exploit the human genome, in order to improve genetic diagnostics and to pave the way for the integration of precision medicine into health systems. The expected benefits include improved understanding of normal and pathological genomic variation, shorter time-to-diagnosis, cost-effective diagnostics, targeted prevention and treatment, and research advances. We review the 41 currently active individual national projects concerning their aims and scope, the number and age structure of included subjects, funding, data sharing goals and methods, and linkage with biobanks, medical data, and non-medical data (exposome). The main aims of ongoing projects were to determine normal genomic variation (90%), determine pathological genomic variation (rare disease, complex diseases, cancer, etc.) (71%), improve infrastructure (59%), and enable personalized medicine (37%). Numbers of subjects to be sequenced ranges substantially, from a hundred to over a million, representing in some cases a significant portion of the population. Approximately half of the projects report public funding, with the rest having various mixed or private funding arrangements. 90% of projects report data sharing (public, academic, and/or commercial with various levels of access) and plan on linking genomic data and medical data (78%), existing biobanks (44%), and/or non-medical data (24%) as the basis for enabling personal/precision medicine in the future. Our results show substantial diversity in the analysed categories of 41 ongoing national projects. The overview of current designs will hopefully inform national initiatives in designing new genomic projects and contribute to standardisation and international collaboration.

The G 4 C 2 hexanucleotide repeat expansion mutation (HREM) in C9ORF72 , represents the most common mutation associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Three main disease mechanisms have been proposed to date: C9ORF72 haploinsufficiency, RNA toxicity and accumulation of dipeptide repeat proteins. Pure GC content of the HREM potentially enables the formation of various non-B DNA structures such as G-quadruplexes and i-motifs. These structures are proposed to act as promoters and regulatory elements affecting replication, transcription and translation of the surrounding region. G-quadruplexes have already been shown on the G-rich sense DNA and RNA strands (G 4 C 2 ) n , the structure of the anti-sense (G 2 C 4 ) n strand remains unresolved. Similar C-rich sequences may, under acidic conditions, form i-motifs consisting of two parallel duplexes in a head to tail orientation held together by hemi-protonated C + -C pairs. We show that d(G 2 C 4 ) n repeats do form i-motif and protonated hairpins even under near-physiological conditions. Rather than forming a DNA duplex, i-motifs persist even in the presence of the sense strand. This preferential formation of G-quadruplex and i-motif/hairpin structures over duplex DNA, may explain HREM replicational and transcriptional instability. Furthermore, i-motifs/hairpins can represent a novel pharmacological target for C9ORF72 associated ALS and FTLD.

Parkinson’s disease (PD) is a neurological disorder characterized by rigidity, bradykinesia and tremor. Several genetic and environmental causes of PD are known, and there is emerging evidence of the possible contribution of the gut microbiome to the disease onset, severity, and response to therapy. While previous research has shown several differences in the microbiome of PD patients under therapy as opposed to healthy controls, few prospective studies have included drug-naïve patients. In order to evaluate the gut microbiome composition prior to therapy initiation, we collected and performed 16S rRNA gene sequencing of the stool samples from 49 drug-naïve PD patients and compared them to 34 diet and lifestyle-matched controls from the Croatian population (GiOPARK Project). While no significant alpha diversity difference was observed between the patients and controls, the differential relative abundance analysis showed an increase in Bacteroides fluxus , B. interstinalis , B. eggerthii , and Dielma fastidiosa in the drug-naïve PD patients compared to controls, while Alistipes , Barnesiella and Dialister spp . were decreased in patients compared to controls. Despite preserved overall diversity, these changes may indicate early microbial dysbiosis and represent a foundation for future studies exploring microbiome changes across disease progression and treatment.

BackgroundThe etiology of preterm birth (PTB) is heterogeneous and not yet well known. Maternal periodontal disease has been investigated for decades and is a known risk factor for adverse pregnancy outcomes. However, no particular bacterial species or higher taxonomic order has been found as causative of PTB, leading to studies of the whole oral microbiome. In order to determine if and how the composition of the oral microbiome is associated with PTB, we performed a large case–control study including women with term (TB) and PTB.MethodsWe compared oral microbiomes in PTB to TB, to examine differences in the microbial richness, diversity, and differential abundance of specific taxa. We obtained oral swab samples from 152 Caucasian pregnant women who were classified as either PTB (≤36 6/7 weeks, n  = 61) or TB (≥38 0/7 weeks, n  = 91) in exclusion of any other major medical or obstetric conditions. The oral microbiomes of these women were characterized by 16S ribosomal RNA (rRNA) gene sequencing of the V3–V4 region on the MiSeq platform.ResultsThe dominant microorganisms at the phylum level in all pregnant women regardless of birth week outcomes as belonging to Firmicutes, Proteobacteria, Bacteroidetes, Fusobacteria , and Actinobacteria . The phyla Firmicutes and Bacteroidetes were relatively more abundant in women with a PTB than in women with a TB, while Proteobacteria was less prevalent in women with a PTB. At the genus level, Veillonella , Prevotella , and Capnocytophaga were enriched in the PTB, and while many of the members of these genera could not be resolved to the species level, Veillonella massillensis was shown to be increased in the PTB group.ConclusionWe identified the genera Veillonella , Prevotella , and Capnocytophaga in the maternal oral microbiome as being associated with PTB independently of clinically apparent infection, uterine anomalies, and other pregnancy complications, including placenta previa, and placental abruption. The clarification of the role of those taxa in the etiology of PTB merits further research.

Parkinson’s disease (PD) guidelines lack clear criteria for genetic evaluation. We assessed the yield and rationale of genetic testing for PD in a routine clinical setting on a multicenter cohort of 149 early-onset and familial patients by exome sequencing and semi-quantitative multiplex ligation-dependent probe amplification of evidence-based PD-associated gene panel. We show that genetic testing for PD should be considered for both early-onset and familial patients alike, and a clinical yield of about 10% in the Caucasian population can be expected.

The DNA genome of a novel HPV genotype, HPV-125, isolated from a hand wart of an immuno-competent 19-year old male was fully cloned, sequenced and characterized. The full genome of HPV-125 is 7,809-bp in length with a GC content of 46.4%. By comparing the nucleotide sequence of the complete L1 gene, HPV-125 is phylogenetically placed within cutaneotrophic species 2 of Alphapapillomaviruses, and is most closely related to HPV-3 and HPV-28. HPV-125 has a typical genomic organization of Alphapapillomaviruses and contains genes coding for five early proteins, E6, E7, E1, E2 and E4 and two late capsid proteins, L1 and L2. The genome contains two non-coding regions: the first located between the L1 and E6 genes (nucleotide positions 7,137-7,809, length 673-bp) and the second between genes E2 and L2 (nucleotide positions 3,757-4,216, length 460-bp). The E6 protein of HPV-125 contains two regular zinc-binding domains at amino acid positions 29 and 102, whereas the E7 protein exhibits one such domain at position 50. HPV-125 lacks the regular pRb-binding core sequence within its E7 protein. In order to assess the tissue predilection and clinical significance of HPV-125, a quantitative type-specific real-time PCR was developed. The 95% limit-of-detection of the assay was 2.5 copies per reaction (range 1.7-5.7) and the intra- and inter-assay coefficients of variation were 0.47 and 2.00 for 100 copies per reaction, and 1.15 and 2.15 for 10 copies per reaction, respectively. Testing of a representative collection of HPV-associated mucosal and cutaneous benign and malignant neoplasms and hair follicles (a total of 601 samples) showed that HPV-125 is a relatively rare HPV genotype, with cutaneous tropism etiologically linked with sporadic cases of common warts.

DNA from two novel HPV genotypes, HPV-150 and HPV-151, isolated from hair follicles of immuno-competent individuals, was fully cloned, sequenced and characterized. The complete genomes of HPV-150 and HPV-151 are 7,436-bp and 7,386-bp in length, respectively. Both contain genes for at least six proteins, namely E6, E7, E1, E2, L2, L1, as well as a non-coding upstream regulatory region located between the L1 and E6 genes: spanning 416-bp in HPV-150 (genomic positions 7,371 to 350) and 322-bp in HPV-151 (genomic positions 7,213 to 148). HPV-150 and HPV-151 are phylogenetically placed within the Betapapillomavirus genus and are most closely related to HPV-96 and HPV-22, respectively. As in other members of this genus, the intergenic E2-L2 region is very short and does not encode for an E5 gene. Both genotypes contain typical zinc binding domains in their E6 and E7 proteins, but HPV-151 lacks the regular pRb-binding core sequence within its E7 protein. In order to assess the tissue predilection and clinical significance of the novel genotypes, quantitative type-specific real-time PCR assays were developed. The 95% detection limits of the HPV-150 and HPV-151 assays were 7.3 copies/reaction (range 5.6 to 11.4) and 3.4 copies/reaction (range 2.5 to 6.0), respectively. Testing of a representative collection of HPV-associated mucosal and cutaneous benign and malignant neoplasms and hair follicles (total of 540 samples) revealed that HPV-150 and HPV-151 are relatively rare genotypes with a cutaneous tropism. Both genotypes were found in sporadic cases of common warts and SCC and BCC of the skin as single or multiple infections usually with low viral loads. HPV-150 can establish persistent infection of hair follicles in immuno-competent individuals. A partial L1 sequence of a putative novel HPV genotype, related to HPV-150, was identified in a squamous cell carcinoma of the skin obtained from a 64-year old immuno-compromised male patient.

Amyotrophic lateral sclerosis is a late-onset disorder primarily affecting motor neurons and leading to progressive and lethal skeletal muscle atrophy. Small RNAs, including microRNAs (miRNAs), can serve as important regulators of gene expression and can act both globally and in a tissue-/cell-type-specific manner. In muscle, miRNAs called myomiRs govern important processes and are deregulated in various disorders. Several myomiRs have shown promise for therapeutic use in cellular and animal models of ALS; however, the exact miRNA species differentially expressed in muscle tissue of ALS patients remain unknown. Following small RNA-Seq, we compared the expression of small RNAs in muscle tissue of ALS patients and healthy age-matched controls. The identified snoRNAs, mtRNAs and other small RNAs provide possible molecular links between insulin signaling and ALS. Furthermore, the identified miRNAs are predicted to target proteins that are involved in both normal processes and various muscle disorders and indicate muscle tissue is undergoing active reinnervation/compensatory attempts thus providing targets for further research and therapy development in ALS.

Determining the genetic contribution of susceptibility to severe SARS-CoV-2 infection outcomes is important for public health measures and individualized treatment. Through intense research on this topic, several hundred genes have been implicated as possibly contributing to the severe infection phenotype(s); however, the findings are complex and appear to be population-dependent. We aimed to determine the contribution of human rare genetic variants associated with a severe outcome of SARS-CoV-2 infections and their burden in the Slovenian population. A panel of 517 genes associated with severe SARS-CoV-2 infection were obtained by combining an extensive review of the literature, target genes identified by the COVID-19 Host Genetic Initiative, and the curated Research COVID-19 associated genes from PanelApp, England Genomics. Whole genome sequencing was performed using PCR-free WGS on DNA from 60 patients hospitalized due to severe COVID-19 disease, and the identified rare genomic variants were analyzed and classified according to the ACMG criteria. Background prevalence in the general Slovenian population was determined by comparison with sequencing data from 8025 individuals included in the Slovenian genomic database (SGDB). Results show that several rare pathogenic/likely pathogenic genomic variants in genes CFTR, MASP2, MEFV, TNFRSF13B, and RNASEL likely contribute to the severe infection outcomes in our patient cohort. These results represent an insight into the Slovenian genomic diversity associated with a severe COVID-19 outcome.