Functional genetic variation of human miRNAs and phenotypic consequences

A large number of human protein-coding genes are finely regulated by one or more microRNAs. Members of this small noncoding RNA family have emerged as important post-transcriptional regulators of gene expression and are involved in a number of disease phenotypes. Variability in the human genome is extensive and includes the common and rare single nucleotide polymorphisms (SNPs) and copy number variations (CNVs). The functional significance of the genome's variability is under intense investigation. In this article we review the emerging literature on how human genomic variation influences the outcome of microRNA targeting and the associated phenotypic effects. Illustrative examples are discussed that demonstrate the biological importance of functional polymorphisms affecting miRNA-mediated gene regulatio

Welcome to PathoGenetics

Disease gene identification has made enormous strides in the past twenty years through functional, positional and candidate gene approaches, and more recently by the exploitation of genome-wide strategies. However, although pathogenic mutations in over 2000 genes have been identified as causative of human diseases, much less is known about the relationship between the molecular defects and mechanisms that lead to disease pathology and symptoms. Recent advances in diverse fields such as genomics, proteomics, cell biology, as well as studies on transgenic animals have greatly accelerated our understanding of the biochemical and cellular basis of many diseases but much still remains to be discovered. The current challenge is to understand the molecular and metabolic pathways by which a particular pathogenic variation leads to a specific phenotype. The study of abnormal conditions is of crucial importance for the understanding of normal physiology and often provides us with the rationale for the development of novel therapeutic strategies

Peutz-Jeghers LKB1 mutants fail to activate GSK-3β, preventing it from inhibiting Wnt signaling

Peutz-Jeghers syndrome (PJS) is caused by germline mutations in the LKB1 gene, which encodes a serine-threonine kinase that regulates cell proliferation and polarity. This autosomal dominant disorder is characterized by mucocutaneous melanin pigmentation, multiple gastrointestinal hamartomatous polyposis and an increased risk of developing various neoplasms. To understand the molecular pathogenesis of PJS phenotypes, we used microarrays to analyze gene expression profiles in proliferating HeLa cells transduced with lentiviral vectors expressing wild type or mutant LKB1 proteins. We show that gene expression is differentially affected by mutations that impair the kinase activity (K78I) or alter the cellular localization of the LKB1 protein. However, both mutations abrogate the ability of LKB1 to up-regulate the transcription of several genes involved in Wnt signaling, including DKK3, WNT5B and FZD2. In addition—and in contrast to the wild type protein—these LKB1 mutants fail to activate the GSK-3β kinase, which otherwise phosphorylates β-catenin. The increase in β-catenin phosphorylation that occurs upon expression of wild-type LKB1 results in transcriptional inhibition of a canonical Wnt reporter gene. This suggests that pathogenic LKB1 mutations that lead to activation of the Wnt/β-catenin pathway could contribute to the cancer predisposition of PJS patient

The subcellular localization of the ChoRE-binding protein, encoded by the Williams-Beuren syndrome critical region gene 14, is regulated by 14-3-3

The Williams-Beuren syndrome (WBS) is a contiguous gene syndrome caused by chromosomal rearrangements at chromosome band 7q11.23. Several endocrine phenotypes, in particular impaired glucose tolerance and silent diabetes, have been described for this clinically complex disorder. The WBSCR14 gene, one of the genes mapping to the WBS critical region, encodes a member of the basic-helix-loop-helix leucine zipper family of transcription factors, which dimerizes with the Max-like protein, Mlx. This heterodimeric complex binds and activates, in a glucose-dependent manner, carbohydrate response element (ChoRE) motifs in the promoter of lipogenic enzymes. We identified five novel WBSCR14-interacting proteins, four 14-3-3 isotypes and NIF3L1, which form a single polypeptide complex in mammalian cells. Phosphatase treatment abrogates the association between WBSCR14 and 14-3-3, as shown previously for multiple 14-3-3 interactors. WBSCR14 is exported actively from the nucleus through a CRM1-dependent mechanism. This translocation is contingent upon the ability to bind 14-3-3. Through this mechanism the 14-3-3 isotypes directly affect the WBSCR14:Mlx complexes, which activate the transcription of lipogenic gene

Three decades of the Human Genome Organization.

The Human Genome Organization (HUGO) was initially established in 1988 to help integrate international scientific genomic activity and to accelerate the diffusion of knowledge from the efforts of the human genome project. Its founding President was Victor McKusick. During the late 1980s and 1990s, HUGO organized lively gene mapping meetings to accurately place genes on the genome as chromosomes were being sequenced. With the completion of the Human Genome Project, HUGO went through some transitions and self-reflection. In 2020, HUGO (which hosts a large annual scientific meeting and comprises the renowned HUGO Gene Nomenclature Committee [HGNC], responsible for naming genes, and an outstanding Ethics Committee) was merged with the Human Genome Variation Society (HGVS; which defines the correct nomenclature for variation description) and the Human Variome Project (HVP; championed by the late Richard Cotton) into a single organization that is committed to assembling human genomic variation from all over the world. This consolidated effort, under a new Executive Board and seven focused committees, will facilitate efficient and effective communication and action to bring the benefits of increasing knowledge of genome diversity and biology to people all over the world

Characterization of mouse Dactylaplasia mutations: a model for human ectrodactyly SHFM3

SHFM3 is a limb malformation characterized by the absence of central digits. It has been shown that this condition is associated with tandem duplications of about 500 kb at 10q24. The Dactylaplasia mice display equivalent limb defects and the two corresponding alleles (Dac 1j and Dac 2j ) map in the region syntenic with the duplications in SHFM3. Dac 1j was shown to be associated with an insertion of an unspecified ETn-like mouse endogenous transposon upstream of the Fbxw4 gene. Dac 2j was also thought to be an insertion or a small inversion in intron 5 of Fbxw4, but the breakpoints and the exact molecular lesion have not yet been characterized. Here we report precise mapping and characterization of these alleles. We failed to identify any copy number differences within the SHFM3 orthologous genomic locus between Dac mutant and wild-type littermates, showing that the Dactylaplasia alleles are not associated with duplications of the region, in contrast with the described human SHFM3 cases. We further show that both Dac 1j and Dac 2j are caused by insertions of MusD retroelements that share 98% sequence identity. The differences between the nature of the human and mouse genomic abnormalities argue against models proposed so far that either envisioned SHFM3 as a local trisomy or Dac as a mutant allele of Fbxw4. Instead, both genetic conditions might lead to complex alterations of gene regulation mechanisms that would impair limb morphogenesis. Interestingly, the Dac 2j mutation occurs within a highly conserved element that may represent a regulatory sequence for a neighboring gen

Identifying protein-coding genes in genomic sequences

A review of the main computational pipelines used to generate the human reference protein-coding gene sets

TNPO3 protects HIV-1 replication from CPSF6-mediated capsid stabilization in the host cell cytoplasm

BACKGROUND: Despite intensive investigation the mechanism by which HIV-1 reaches the host cell nucleus is unknown. TNPO3, a karyopherin mediating nuclear entry of SR-proteins, was shown to be required for HIV-1 infectivity. Some investigators have reported that TNPO3 promotes HIV-1 nuclear import, as would be expected for a karyopherin. Yet, an equal number of investigators have failed to obtain evidence that supports this model. Here, a series of experiments were performed to better elucidate the mechanism by which TNPO3 promotes HIV-1 infectivity. RESULTS: To examine the role of TNPO3 in HIV-1 replication, the 2-LTR circles that are commonly used as a marker for HIV-1 nuclear entry were cloned after infection of TNPO3 knockdown cells. Potential explanation for the discrepancy in the literature concerning the effect of TNPO3 was provided by sequencing hundreds of these clones: a significant fraction resulted from autointegration into sites near the LTRs and therefore were not bona fide 2-LTR circles. In response to this finding, new techniques were developed to monitor HIV-1 cDNA, including qPCR reactions that distinguish 2-LTR circles from autointegrants, as well as massive parallel sequencing of HIV-1 cDNA. With these assays, TNPO3 knockdown was found to reduce the levels of 2-LTR circles. This finding was puzzling, though, since previous work has shown that the HIV-1 determinant for TNPO3-dependence is capsid (CA), an HIV-1 protein that forms a mega-dalton protein lattice in the cytoplasm. TNPO3 imports cellular splicing factors via their SR-domain. Attention was therefore directed towards CPSF6, an SR-protein that binds HIV-1 CA and inhibits HIV-1 nuclear import when the C-terminal SR-domain is deleted. The effect of 27 HIV-1 capsid mutants on sensitivity to TNPO3 knockdown was then found to correlate strongly with sensitivity to inhibition by a C-terminal deletion mutant of CPSF6 (R2 = 0.883, p \u3c 0.0001). TNPO3 knockdown was then shown to cause CPSF6 to accumulate in the cytoplasm. Mislocalization of CPSF6 to the cytoplasm, whether by TNPO3 knockdown, deletion of the CPSF6 nuclear localization signal, or by fusion of CPSF6 to a nuclear export signal, resulted in inhibition of HIV-1 replication. Additionally, targeting CPSF6 to the nucleus by fusion to a heterologous nuclear localization signal rescued HIV-1 from the inhibitory effects of TNPO3 knockdown. Finally, mislocalization of CPSF6 to the cytoplasm was associated with abnormal stabilization of the HIV-1 CA core. CONCLUSION: TNPO3 promotes HIV-1 infectivity indirectly, by shifting the CA-binding protein CPSF6 to the nucleus, thus preventing the excessive HIV-1 CA stability that would otherwise result from cytoplasmic accumulation of CPSF6