The status of the human gene catalogue.

Scientists have been trying to identify every gene in the human genome since the initial draft was published in 2001. In the years since, much progress has been made in identifying protein-coding genes, currently estimated to number fewer than 20,000, with an ever-expanding number of distinct protein-coding isoforms. Here we review the status of the human gene catalogue and the efforts to complete it in recent years. Beside the ongoing annotation of protein-coding genes, their isoforms and pseudogenes, the invention of high-throughput RNA sequencing and other technological breakthroughs have led to a rapid growth in the number of reported non-coding RNA genes. For most of these non-coding RNAs, the functional relevance is currently unclear; we look at recent advances that offer paths forward to identifying their functions and towards eventually completing the human gene catalogue. Finally, we examine the need for a universal annotation standard that includes all medically significant genes and maintains their relationships with different reference genomes for the use of the human gene catalogue in clinical settings.

Systematic assessment of long-read RNA-seq methods for transcript identification and quantification.

The Long-read RNA-Seq Genome Annotation Assessment Project (LRGASP) Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. The consortium generated over 427 million long-read sequences from cDNA and direct RNA datasets, encompassing human, mouse, and manatee species, using different protocols and sequencing platforms. These data were utilized by developers to address challenges in transcript isoform detection and quantification, as well as de novo transcript isoform identification. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. When aiming to detect rare and novel transcripts or when using reference-free approaches, incorporating additional orthogonal data and replicate samples are advised. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

CapTrap-Seq: A platform-agnostic and quantitative approach for high-fidelity full-length RNA transcript sequencing.

Long-read RNA sequencing is essential to produce accurate and exhaustive annotation of eukaryotic genomes. Despite advancements in throughput and accuracy, achieving reliable end-to-end identification of RNA transcripts remains a challenge for long-read sequencing methods. To address this limitation, we developed CapTrap-seq, a cDNA library preparation method, which combines the Cap-trapping strategy with oligo(dT) priming to detect 5'capped, full-length transcripts, together with the data processing pipeline LyRic. We benchmarked CapTrap-seq and other popular RNA-seq library preparation protocols in a number of human tissues using both ONT and PacBio sequencing. To assess the accuracy of the transcript models produced, we introduced a capping strategy for synthetic RNA spike-in sequences that mimics the natural 5'cap formation in RNA spike-in molecules. We found that the vast majority (up to 90%) of transcript models that LyRic derives from CapTrap-seq reads are full-length. This makes it possible to produce highly accurate annotations with minimal human intervention.

The status of the human gene catalogue.

Scientists have been trying to identify all of the genes in the human genome since the initial draft of the genome was published in 2001. Over the intervening years, much progress has been made in identifying protein-coding genes, and the estimated number has shrunk to fewer than 20,000, although the number of distinct protein-coding isoforms has expanded dramatically. The invention of high-throughput RNA sequencing and other technological breakthroughs have led to an explosion in the number of reported non-coding RNA genes, although most of them do not yet have any known function. A combination of recent advances offers a path forward to identifying these functions and towards eventually completing the human gene catalogue. However, much work remains to be done before we have a universal annotation standard that includes all medically significant genes, maintains their relationships with different reference genomes, and describes clinically relevant genetic variants.

GENCODE: reference annotation for the human and mouse genomes in 2023.

GENCODE produces high quality gene and transcript annotation for the human and mouse genomes. All GENCODE annotation is supported by experimental data and serves as a reference for genome biology and clinical genomics. The GENCODE consortium generates targeted experimental data, develops bioinformatic tools and carries out analyses that, along with externally produced data and methods, support the identification and annotation of transcript structures and the determination of their function. Here, we present an update on the annotation of human and mouse genes, including developments in the tools, data, analyses and major collaborations which underpin this progress. For example, we report the creation of a set of non-canonical ORFs identified in GENCODE transcripts, the LRGASP collaboration to assess the use of long transcriptomic data to build transcript models, the progress in collaborations with RefSeq and UniProt to increase convergence in the annotation of human and mouse protein-coding genes, the propagation of GENCODE across the human pan-genome and the development of new tools to support annotation of regulatory features by GENCODE. Our annotation is accessible via Ensembl, the UCSC Genome Browser and https://www.gencodegenes.org.

Profiling subcellular localization of nuclear-encoded mitochondrial gene products in zebrafish.

Most mitochondrial proteins are encoded by nuclear genes, synthetized in the cytosol and targeted into the organelle. To characterize the spatial organization of mitochondrial gene products in zebrafish (Danio rerio), we sequenced RNA from different cellular fractions. Our results confirmed the presence of nuclear-encoded mRNAs in the mitochondrial fraction, which in unperturbed conditions, are mainly transcripts encoding large proteins with specific properties, like transmembrane domains. To further explore the principles of mitochondrial protein compartmentalization in zebrafish, we quantified the transcriptomic changes for each subcellular fraction triggered by the chchd4a -/- mutation, causing the disorders in the mitochondrial protein import. Our results indicate that the proteostatic stress further restricts the population of transcripts on the mitochondrial surface, allowing only the largest and the most evolutionary conserved proteins to be synthetized there. We also show that many nuclear-encoded mitochondrial transcripts translated by the cytosolic ribosomes stay resistant to the global translation shutdown. Thus, vertebrates, in contrast to yeast, are not likely to use localized translation to facilitate synthesis of mitochondrial proteins under proteostatic stress conditions.

RATTLE: reference-free reconstruction and quantification of transcriptomes from Nanopore sequencing.

Nanopore sequencing enables the efficient and unbiased measurement of transcriptomes. Current methods for transcript identification and quantification rely on mapping reads to a reference genome, which precludes the study of species with a partial or missing reference or the identification of disease-specific transcripts not readily identifiable from a reference. We present RATTLE, a tool to perform reference-free reconstruction and quantification of transcripts using only Nanopore reads. Using simulated data and experimental data from isoform spike-ins, human tissues, and cell lines, we show that RATTLE accurately determines transcript sequences and their abundances, and shows good scalability with the number of transcripts.

GENCODE 2021.

The GENCODE project annotates human and mouse genes and transcripts supported by experimental data with high accuracy, providing a foundational resource that supports genome biology and clinical genomics. GENCODE annotation processes make use of primary data and bioinformatic tools and analysis generated both within the consortium and externally to support the creation of transcript structures and the determination of their function. Here, we present improvements to our annotation infrastructure, bioinformatics tools, and analysis, and the advances they support in the annotation of the human and mouse genomes including: the completion of first pass manual annotation for the mouse reference genome; targeted improvements to the annotation of genes associated with SARS-CoV-2 infection; collaborative projects to achieve convergence across reference annotation databases for the annotation of human and mouse protein-coding genes; and the first GENCODE manually supervised automated annotation of lncRNAs. Our annotation is accessible via Ensembl, the UCSC Genome Browser and https://www.gencodegenes.org.

Annotation of Full-Length Long Noncoding RNAs with Capture Long-Read Sequencing (CLS).

Metazoan genomes produce thousands of long-noncoding RNAs (lncRNAs), of which just a small fraction have been well characterized. Understanding their biological functions requires accurate annotations, or maps of the precise location and structure of genes and transcripts in the genome. Current lncRNA annotations are limited by compromises between quality and size, with many gene models being fragmentary or uncatalogued. To overcome this, the GENCODE consortium has developed RNA capture long-read sequencing (CLS), an approach combining targeted RNA capture with third-generation long-read sequencing. CLS provides accurate annotations at high-throughput rates. It eliminates the need for noisy transcriptome assembly from short reads, and requires minimal manual curation. The full-length transcript models produced are of quality comparable to present-day manually curated annotations. Here we describe a detailed CLS protocol, from probe design through long-read sequencing to creation of final annotations.

From research to rapid response: mass COVID-19 testing by volunteers at the Centre for Genomic Regulation.

The COVID-19 pandemic has posed and is continuously posing enormous societal and health challenges worldwide. The research community has mobilized to develop novel projects to find a cure or a vaccine, as well as to contribute to mass testing, which has been a critical measure to contain the infection in several countries. Through this article, we share our experiences and learnings as a group of volunteers at the Centre for Genomic Regulation (CRG) in Barcelona, Spain. As members of the ORFEU project, an initiative by the Government of Catalonia to achieve mass testing of people at risk and contain the epidemic in Spain, we share our motivations, challenges and the key lessons learnt, which we feel will help better prepare the global society to address similar situations in the future.

GENCODE reference annotation for the human and mouse genomes.

The accurate identification and description of the genes in the human and mouse genomes is a fundamental requirement for high quality analysis of data informing both genome biology and clinical genomics. Over the last 15 years, the GENCODE consortium has been producing reference quality gene annotations to provide this foundational resource. The GENCODE consortium includes both experimental and computational biology groups who work together to improve and extend the GENCODE gene annotation. Specifically, we generate primary data, create bioinformatics tools and provide analysis to support the work of expert manual gene annotators and automated gene annotation pipelines. In addition, manual and computational annotation workflows use any and all publicly available data and analysis, along with the research literature to identify and characterise gene loci to the highest standard. GENCODE gene annotations are accessible via the Ensembl and UCSC Genome Browsers, the Ensembl FTP site, Ensembl Biomart, Ensembl Perl and REST APIs as well as https://www.gencodegenes.org.

LRP5 gene polymorphism and cortical bone.

BACKGROUND AND AIMS: There is evidence that distinct genetic polymorphisms of LRP5 are associated with low Bone Mineral Density (BMD) and the risk of fracture. However, relationships between LRP5 polymorphisms and micro- and macro architectural bone characteristics assessed by pQCT have not been studied. The aim of the present study was to investigate the association of Ala1330Val and Val667Met polymorphisms in LRP5 gene with volumetric BMD (vBMD) and macro-architectural bone parameters in a population-based sample of men and women. METHODS: We studied 959 participants of the InCHIANTI study (451 men and 508 women, age range: 21-94 yrs). Trabecular vBMD (vBMDt, mg/cm3), cortical vBMD (vBMDc, mg/cm3), cortical bone area (CBA, mm2) and cortical thickness (Ct.Th, mm) at the level of the tibia were assessed by peripheral quantitative computed tomography (pQCT). Ala1330Val and Val667Met genotypes were determined on genomic DNA by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: In age-adjusted analyses both LRP 1330-valine and LRP 667-metionin variants were associated with lower vBMDt in men (p<0.05), and lower vBMDt (p<0.05), Ct.Th (p<0.05) and CBA (p<0.05) in women. After adjusting for multiple confounders, only the association of LRP5 1330-valine and 667-metionin with CBA remained statistically significant (p=0.04 and p=0.01, respectively) in women. CONCLUSION: These findings suggest that both Ala1330Val and Val667Met LRP5 polymorphisms may affect the determination of geometric bone parameters in women.

Inhibition of in vitro growth and arrest in the G0/G1 phase of HCT8 line human colon cancer cells by kaempferide triglycoside from Dianthus caryophyllus.

The effects of phytoestrogens have been studied in the hypothalamic-pituitary-gonadal axis and in various non-gonadal targets. Epidemiologic and experimental evidence indicates a protective effect of phytoestrogens also in colorectal cancer. The mechanism through which estrogenic molecules control colorectal cancer tumorigenesis could possibly involve estrogen receptor beta, the predominantly expressed estrogen receptor subtype in colon mucosa.To validate this hypothesis, we therefore used an engineered human colon cancer cell line induced to overexpress estrogen receptor beta, beside its native cell line, expressing very low levels of ERbeta and not expressing ERalpha; as a phytoestrogenic molecule, we used kaempferide triglycoside, a glycosylated flavonol from a Dianthus caryophyllus cultivar. The inhibitory properties of this molecule toward vegetal cell growth have been previously demonstrated: however, no data on its activity on animal cell or information about the mechanism of this activity are available. Kaempferide triglycoside proved to inhibit the proliferation of native and estrogen receptor beta overexpressing colon cancer cells through a mechanism not mediated by ligand binding dependent estrogen receptor activation. It affected HCT8 cell cycle progression by increasing the G(0)/G(1) cell fraction and in estrogen receptor beta overexpressing cells increased two antioxidant enzymes. Interestingly, the biological effects of this kaempferide triglycoside were strengthened by the presence of high levels of estrogen receptor beta.Pleiotropic molecular effects of phytoestrogens may explain their protective activity against colorectal cancer and may represent an interesting area for future investigation with potential clinical applications.

In vitro differentiation of human mesenchymal stem cells on Ti6Al4V surfaces.

Long-term stability of arthroplasty prosthesis depends on the integration between the bone tissue and the implanted biomaterials, which requires the contribution of osteoblastic precursors and their continuous differentiation into the osteoblastic phenotype. Classically, these interactions are tested in vitro using mesenchymal stem cells (MSCs) isolated and ex vivo expanded from bone marrow aspirates. Human adipose tissue-derived stromal cells (AMSCs) may be a more convenient source of MSCs, according to their abundance and accessibility, but no data are available on their in vitro interactions with hard biomaterials. The aim of this work is to compare the osteogenic potential of human AMSCs and bone marrow-derived MSCs (BMMSCs) and to evaluate their response to Ti6Al4V alloy in terms of adhesion, proliferation and differentiation features, using the human osteosarcoma cell line SaOS-2 for comparison. The overall results showed that AMSCs have the same ability to produce bone matrix as BMMSCs and that Ti6Al4V surfaces exhibit an osteoinductive action on AMSCs, promoting their differentiation into functional osteoblasts and increasing bone formation. In conclusion, adipose tissue is a promising autologous source of osteoblastic cells with important clinical implications for bone tissue engineering.

Clodronate acts on human osteoclastic cell proliferation, differentiation and function in a bioreversible manner.

Background. Clodronate is used in high bone resorption diseases. Its action was defined as "cytotoxic" based on the induced cellular ATP loss, without any experimental verification of reversibility. In the present report the reversibility of clodronate action was tested on cultured human osteoclastic cell cultures. As "in vitro" bioeffects of clodronate are reversible, this compound should not be defined as "cytotoxic".Introduction. Bisphosphonates are pyrophosphate analogs able to inhibit osteoclast-mediated bone resorption widely used in the treatment of diseases with high bone turnover. Several evidences have shown that bisphosphonates can be divided into two groups with distinct molecular mechanisms of action depending on the nature of the R(2)side chain. The nitrogen-containing bisphosphonates act on osteoclasts by preventing protein prenylation, while non-nitrogen-containing bisphosphonates, like clodronate, are metabolized intracellularly to a ß-γ-methylene analog of ATP that induces inhibition of the ADP/ATP translocase.Materials and Methods. In order to evaluate clodronate effects on osteoclastic cells and the bioreversibility of its action, we have used a human preosteoclastic (FLG 29.1) cell line and primary cultures of human osteoclast-like (HOC) cells. Functional and differentiative modifications were evaluated with immunocytochemical tartrate-resistant acid phosphatase activity (TRAcP) assay and with rapid quantitative detection of the complex "matrix metalloproteinase 9/tissue inhibitor of metalloproteinase" (MMP9/TIMP1) by RT-PCR analysis based on "TaqMan" technology. The apoptosis phenomenon were detected by DNA ladder analysis and quantified by counting apoptotic cells with Transmission Electron Microscopy (TEM) analysis. Adenosine-5'-[ ß - γ -dichloromethylene] triphosphate (AppCCl(2)p) was detected and identified in cell extract by HPLC-ESI-MS-MS Mass Spectrometry. Intracellular ATP modulation in the presence of clodronate was evaluated by luciferin-luciferase assay. The Mann-Whitney "U" test was conducted for statistical analysis.Results. We found that clodronate inhibited both proliferation and differentiative features of cells of the osteoclastic lineage. Furthermore, treatment of both cell types with clodronate caused apoptosis, generation of measurable levels of AppCCl(2)p, and reduction of intracellular ATP levels. Addition of ATP to the culture medium caused an inhibition of the biological actions of clodronate on the human osteoclastic cell lineage.Conclusions. These data indicate that intracellular accumulation of the metabolite AppCCl(2)p is the likely route by which clodronate inhibits osteoclastic function and this effect is reversed by ATP.

The role of osteoprotegerin (OPG) and estrogen receptor (ER-α) gene polymorphisms in rheumatoid arthritis.

Objective. Osteoclast activation at the cartilage pannus junction is an essential step in the destruction of bone matrix in patients affected by rheumatoid arthritis (RA). Receptor activator of NFkappaB ligand (RANK-L) is responsible for osteoclast differentiation and activation. Osteoprotegerin (OPG) is an alternative, high-affinity soluble receptor for RANK-L which significantly inhibits osteoclastogenesis. Estrogens and the specific receptors α and ß (ER-α and ER-α) are known to play an important role in the pathophysiology of osteoarthritis (OA). Scope of the present study is to investigate the role of ER-α and OPG gene polymorphisms in a group of women affected by RA.Materials and Methods. 139 consecutive RA patients (115 females and 34 males; median age 65.8 years) were selected. Bone mineral density (BMD) was measured by dual energy x-ray absorptinometry at the lumbar spine (LS-BMD) and femoral neck (FN-BMD) and the presence of bone erosions was evaluated by conventional X-ray. ER-α gene polymorphisms were determined by PvuII and XbaI restriction endonuclease digestion of polymerase chain reaction (PCR) products. By convention, the presence of the endonuclease restriction site was indicated with lowercase (p and x) letters while the absence of the restriction site was indicated with uppercase letters (P and X). OPG gene polymorphism was determined by RsaI restriction endonuclease digestion of PCR products and the presence and absence of restriction fragment was identified as TT and CC respectively.Results. Pearson's χ(2)analysis for the ER-α gene polymorphism showed a prevalence of Pp genotype (58%) (p=0.04) and Xx (54%) (p=0.04) in the total population, without differences between males and females. We did not observed any significant differences between ER-α genotypes and LS-BMD. However subjects with xx or pp genotype had a lower LS-BMD in comparison with the opposite genotype.For OPG gene polymorphism, non significant differences in the distribution of the genotypes were observed between males and females. In addition, we did not observed significant differences on LS-BMD between the genotypes.Finally, we observed that patients with ER-α pp genotype was significant more represented in patients with hand erosions (p = 0.05). No significant correlation was observed for ER-α XbaI genotype, however a trend characterized by a correlation between xx and hand erosions was observed (p = 0.13). For OPG gene polymorphism, we found a statistical correlation between C allele of OPG and hands bone erosions (p = 0.02).Conclusion. We found a significant association between ER-α and OPG gene polymorphisms and the presence of bone erosions in RA patients. These preliminary data suggest a role of ER-α and OPG gene polymorphisms in bone turnover and disease progression.

Genetics and pharmacogenetics of estrogen response.

Estrogens are a steroid hormone group distributed widely in animals and human beings. Estrogens diffuse across cell phospholipidic membranes and interact with estrogen receptors. Their highest concentration is found in target tissues with reproductive function (breast, ovary, vagina and uterus). High estrogen levels are usually associated with tumor onset and progression, while loss of estrogen or its receptor(s) contributes to development and/or progression of various diseases (osteoporosis, neurodegenerative disease and cardiovascular disease). Despite the numerous efforts to highlight estrogen's mechanism of action, recent discoveries showed an unexpected degree of complexity of estrogenic response.

Multiple endocrine neoplasia type 2.

Multiple Endocrine Neoplasia Type 2 (MEN2) is a rare hereditary complex disorder characterized by the presence of medullary thyroid carcinoma (MTC), unilateral or bilateral pheochromocytoma (PHEO) and other hyperplasia and/or neoplasia of different endocrine tissues within a single patient. MEN2 has been reported in approximately 500 to 1000 families worldwide and the prevalence has been estimated at approximately 1:30,000. Two different forms, sporadic and familial, have been described for MEN2. Sporadic form is represented by a case with two of the principal MEN2-related endocrine tumors. The familial form, which is more frequent and with an autosomal pattern of inheritance, consists of a MEN2 case with at least one first degree relative showing one of the characteristic endocrine tumors. Familial medullary thyroid carcinoma (FMTC) is a subtype of MEN2 in which the affected individuals develop only medullary thyroid carcinoma, without other clinical manifestations of MEN2. Predisposition to MEN2 is caused by germline activating mutations of the c-RET proto-oncogene on chromosome 10q11.2. The RET gene encodes a single-pass transmembrane tyrosine kinase that is the receptor for glial-derived neurotrophic growth factors. The combination of clinical and genetic investigations, together with the improved understanding of the molecular and clinical genetics of the syndrome, helps the diagnosis and treatment of patients. Currently, DNA testing makes possible the early detection of asymptomatic gene carriers, allowing to identify and treat the neoplastic lesions at an earlier stage. In particular, the identification of a strong genotype-phenotype correlation in MEN2 syndrome may enable a more individualized treatment for the patients, improving their quality of life. At present, surgical treatment offers the only chance of cure and therefore, early clinical and genetic detection and prophylactic surgery in subjects at risk are the main therapeutic goal.

Multiple endocrine neoplasia type 1.

Multiple Endocrine Neoplasia type 1 (MEN1) is a rare autosomal dominant hereditary cancer syndrome presented mostly by tumours of the parathyroids, endocrine pancreas and anterior pituitary, and characterised by a very high penetrance and an equal sex distribution. It occurs in approximately one in 30,000 individuals. Two different forms, sporadic and familial, have been described. The sporadic form presents with two of the three principal MEN1-related endocrine tumours (parathyroid adenomas, entero-pancreatic tumours and pituitary tumours) within a single patient, while the familial form consists of a MEN1 case with at least one first degree relative showing one of the endocrine characterising tumours. Other endocrine and non-endocrine lesions, such as adrenal cortical tumours, carcinoids of the bronchi, gastrointestinal tract and thymus, lipomas, angiofibromas, collagenomas have been described. The responsible gene, MEN1, maps on chromosome 11q13 and encodes a 610 aminoacid nuclear protein, menin, with no sequence homology to other known human proteins. MEN1 syndrome is caused by inactivating mutations of the MEN1 tumour suppressor gene. This gene is probably involved in the regulation of several cell functions such as DNA replication and repair and transcriptional machinery. The combination of clinical and genetic investigations, together with the improving of molecular genetics knowledge of the syndrome, helps in the clinical management of patients. Treatment consists of surgery and/or drug therapy, often in association with radiotherapy or chemotherapy. Currently, DNA testing allows the early identification of germline mutations in asymptomatic gene carriers, to whom routine surveillance (regular biochemical and/or radiological screenings to detect the development of MEN1-associated tumours and lesions) is recommended.