Opitz syndrome: improving clinical interpretation of intronic variants in MID1 gene.

BACKGROUND: Loss-of-function variants in MID1 are the most common cause of Opitz G/BBB syndrome (OS). The interpretation of intronic variants affecting the splicing is a rising issue in OS. METHODS: Exon sequencing of a 2-year-old boy with OS showed that he was a carrier of the de novo c.1286-10G>T variant in MID1. In silico predictions and minigene assays explored the effect of the variant on splicing. The minigene approach was also applied to two previously identified MID1 c.864+1G>T and c.1285+1G>T variants. RESULTS: Minigene assay demonstrated that the c.1286-10G>T variant generated the inclusion of eight nucleotides that predicted generation of a frameshift. The c.864+1G>T and c.1285+1G>T variants resulted in an in-frame deletion predicted to generate a shorter MID1 protein. In hemizygous males, this allowed reclassification of all the identified variants from "of unknown significance" to "likely pathogenic." CONCLUSIONS: Minigene assay supports functional effects from MID1 intronic variants. This paves the way to the introduction of similar second-tier investigations in the molecular diagnostics workflow of OS. IMPACT: Causative intronic variants in MID1 are rarely investigated in Opitz syndrome. MID1 is not expressed in blood and mRNA studies are hardly accessible in routine diagnostics. Minigene assay is an alternative for assessing the effect of intronic variants on splicing. This is the first study characterizing the molecular consequences of three MID1 variants for diagnostic purposes and demonstrating the efficacy of minigene assays in supporting their clinical interpretation. Review of the criteria according to the American College of Medical Genetics reassessed all variants as likely pathogenic.

TRIM E3 Ubiquitin Ligases in Rare Genetic Disorders.

The TRIM family comprises proteins characterized by the presence of the tripartite motif composed of a RING domain, one or two B-box domains and a coiled-coil region. The TRIM shared domain structure underscores a common biochemical function as E3 ligase within the ubiquitination cascade. The TRIM proteins represent one of the largest E3 ligase families counting in human more than 70 members. These proteins are implicated in a plethora of cellular processes such as apoptosis, cell cycle regulation, muscular physiology, and innate immune response. Consistently, their alteration results in several pathological conditions emphasizing their medical relevance. Here, the genetic and pathogenetic mechanisms of rare disorders directly caused by mutations in TRIM genes will be reviewed. These diseases fall into different pathological areas, from malformation birth defects due to developmental abnormalities, to neurological disorders and progressive teenage neuromuscular disorders. In many instances, TRIM E3 ligases act on several substrates thus exerting pleiotropic activities: the need of unraveling disease-specific TRIM pathways for a precise targeting therapy avoiding dramatic side effects will be discussed.

The E3 ubiquitin ligase MID1/TRIM18 promotes atypical ubiquitination of the BRCA2-associated factor 35, BRAF35.

MID1/TRIM18 is a member of the TRIM family of ubiquitin E3 ligases characterized by the presence of a conserved RING-containing N-terminal tripartite motif. Mutations in the MID1 gene have been associated with the X-linked form of Opitz Syndrome, a developmental disorder characterized by midline defects and intellectual disability. The effect of MID1 E3 ligase activity within the cell and the role in the pathogenesis of the disease is still not completely unraveled. Here, we report BRAF35, a non-canonical HMG nuclear factor, as a novel MID1 substrate. MID1 is implicated in BRAF35 ubiquitination promoting atypical poly-ubiquitination via K6-, K27- and K29-linkages. We observed a partial co-localization of the two proteins within cytoplasmic bodies. We found that MID1 depletion alters BRAF35 localization in these structures and increases BRAF35 stability affecting its cytoplasmic abundance. Our data reveal a novel role for MID1 and for atypical ubiquitination in balancing BRAF35 presence, and likely its activity, within nuclear and cytoplasmic compartments.

The combination of transcriptomics and informatics identifies pathways targeted by miR-204 during neurogenesis and axon guidance.

Vertebrate organogenesis is critically sensitive to gene dosage and even subtle variations in the expression levels of key genes may result in a variety of tissue anomalies. MicroRNAs (miRNAs) are fundamental regulators of gene expression and their role in vertebrate tissue patterning is just beginning to be elucidated. To gain further insight into this issue, we analysed the transcriptomic consequences of manipulating the expression of miR-204 in the Medaka fish model system. We used RNA-Seq and an innovative bioinformatics approach, which combines conventional differential expression analysis with the behavior expected by miR-204 targets after its overexpression and knockdown. With this approach combined with a correlative analysis of the putative targets, we identified a wider set of miR-204 target genes belonging to different pathways. Together, these approaches confirmed that miR-204 has a key role in eye development and further highlighted its putative function in neural differentiation processes, including axon guidance as supported by in vivo functional studies. Together, our results demonstrate the advantage of integrating next-generation sequencing and bioinformatics approaches to investigate miRNA biology and provide new important information on the role of miRNAs in the control of axon guidance and more broadly in nervous system development.

Midline 1 directs lytic granule exocytosis and cytotoxicity of mouse killer T cells.

Midline 1 (MID1) is a microtubule-associated ubiquitin ligase that regulates protein phosphatase 2A activity. Loss-of-function mutations in MID1 lead to the X-linked Opitz G/BBB syndrome characterized by defective midline development during embryogenesis. Here, we show that MID1 is strongly upregulated in murine cytotoxic lymphocytes (CTLs), and that it controls TCR signaling, centrosome trafficking, and exocytosis of lytic granules. In accordance, we find that the killing capacity of MID1(-/-) CTLs is impaired. Transfection of MID1 into MID1(-/-) CTLs completely rescued lytic granule exocytosis, and vice versa, knockdown of MID1 inhibited exocytosis of lytic granules in WT CTLs, cementing a central role for MID1 in the regulation of granule exocytosis. Thus, MID1 orchestrates multiple events in CTL responses, adding a novel level of regulation to CTL activation and cytotoxicity.

TRIM family: Pleiotropy and diversification through homomultimer and heteromultimer formation.

The TRIM family is composed of multidomain ubiquitin E3 ligases characterized by the presence of the N-terminal tripartite motif (RING, B-boxes, and coiled coil). TRIM proteins transfer the ubiquitin moiety to specific substrates but are also involved in ubiquitin-like modifications, in particular SUMOylation and ISGylation. The TRIM family members are involved in a plethora of biological and physiological processes and, when altered, are implicated in many pathological conditions. Growing evidence indicates the pleiotropic effect of several TRIM genes, each of which might be connected to very diverse cellular processes. As a way to reconcile a single family member with several functions, we propose that structural features, that is, their ability to homo- and hetero-di(multi)merize, can increase and diversify TRIM ubiquitin E3 ligase capability.

Functional interactions between ubiquitin E2 enzymes and TRIM proteins.

The TRIM (tripartite motif) family of proteins is characterized by the presence of the tripartite motif module, composed of a RING domain, one or two B-box domains and a coiled-coil region. TRIM proteins are involved in many cellular processes and represent the largest subfamily of RING-containing putative ubiquitin E3 ligases. Whereas their role as E3 ubiquitin ligases has been presumed, and in several cases established, little is known about their specific interactions with the ubiquitin-conjugating E2 enzymes or UBE2s. In the present paper, we report a thorough screening of interactions between the TRIM and UBE2 families. We found a general preference of the TRIM proteins for the D and E classes of UBE2 enzymes, but we also revealed very specific interactions between TRIM9 and UBE2G2, and TRIM32 and UBE2V1/2. Furthermore, we demonstrated that the TRIM E3 activity is only manifest with the UBE2 with which they interact. For most specific interactions, we could also observe subcellular co-localization of the TRIM involved and its cognate UBE2 enzyme, suggesting that the specific selection of TRIM-UBE2 pairs has physiological relevance. Our findings represent the basis for future studies on the specific reactions catalysed by the TRIM E3 ligases to determine the fate of their targets.

Genomics and evolution of the TRIM gene family.

The TRIM family comprises proteins characterized by the presence of thetripartite motifthat is composed of a RING domain, one or two B-box domains and a Coiled-coil region. These proteins are implicated in a plethora of cellular processes such as apoptosis, cell cycle regulation, muscular physiology and innate immune response. Consistently, their alteration results in several pathological conditions emphasizing their medical relevance. The TRIM members domain structure underscores a common biochemical function as E3 ligases within the ubiquitylation cascade, which is then translated into diverse biological processes. The TRIM proteins represent one of the largest families in mammals counting in human almost 70 members. TRIM proteins are metazoan-specific and have been now identified in several species although the great increase in their number was generated in vertebrate species. The important expansion of the number of TRIM genes underlie the success of the tripartite module in ubiquitylation process. Furthermore, their massive diversification among species was achieved through fast evolution of the TRIM genes implicated in pathogen response.

TRIM proteins in development.

TRIM proteins play important roles in several patho-physiological processes. Their common activity within the ubiquitylation pathway makes them amenable to a number of diverse biological roles. Many of the TRIM genes are highly and sometimes specifically expressed during embryogenesis, it is therefore not surprising that several of them might be involved in developmental processes. Here, we primarily discuss the developmental implications of two subgroups of TRIM proteins that conserved domain composition and functions from their invertebrate ancestors. The two groups are: the TRIM-NHL proteins implicated in miRNA processing regulation and the TRIM-FN3 proteins involved in ventral midline development.

Cellular Function of TRIM E3 Ubiquitin Ligases in Health and Disease.

The field of the Tripartite Motif (TRIM) family has progressively attracted increasing interest during the last two decades [...].

Microtubular TRIM36 E3 Ubiquitin Ligase in Embryonic Development and Spermatogenesis.

TRIM36 is a member of the tripartite motif (TRIM) family of RING-containing proteins, also known as Haprin, which was first discovered for its abundance in testis and found to be implicated in the spermatozoa acrosome reaction. TRIM36 is a microtubule-associated E3 ubiquitin ligase that plays a role in cytoskeletal organization, and according to data gathered in different species, coordinates growth speed and stability, acting on the microtubules' plus end, and impacting on cell cycle progression. TRIM36 is also crucial for early developmental processes, in Xenopus, where it is needed for dorso-ventral axis formation, but also in humans as bi-allelic mutations in the TRIM36 gene cause a form of severe neural tube closure defect, called anencephaly. Here, we review TRIM36-related mechanisms implicated in such composite physiological and pathological processes.

SPECC1L Mutations Are Not Common in Sporadic Cases of Opitz G/BBB Syndrome.

Opitz G/BBB syndrome (OS) is a rare genetic developmental condition characterized by congenital defects along the midline of the body. The main clinical signs are represented by hypertelorism, laryngo-tracheo-esophageal defects and hypospadias. The X-linked form of the disease is associated with mutations in the MID1 gene located in Xp22 whereas mutations in the SPECC1L gene in 22q11 have been linked to few cases of the autosomal dominant form of this disorder, as well as to other genetic syndromes. In this study, we have undertaken a mutation screening of the SPECC1L gene in samples of sporadic OS cases in which mutations in the MID1 gene were excluded. The heterozygous missense variants identified are already reported in variant databases raising the issue of their pathogenetic meaning. Recently, it was reported that some clinical manifestations peculiar to OS signs are not observed in patients carrying mutations in the SPECC1L gene, leading to the proposal of the designation of 'SPECC1L syndrome' to refer to this disorder. Our study confirms that patients with diagnosis of OS, mainly characterized by the presence of hypospadias and laryngo-tracheo-esophageal defects, do not carry pathogenic SPECC1L mutations. In addition, SPECC1L syndrome-associated mutations are clustered in two specific domains of the protein, whereas the missense variants detected in our work lies elsewhere and the impact of these variants in the function of this protein is difficult to ascertain with the current knowledge and will require further investigations. Nonetheless, our study provides further insight into the SPECC1L syndrome classification.

Lack of Mid1, the mouse ortholog of the Opitz syndrome gene, causes abnormal development of the anterior cerebellar vermis.

Opitz G/BBB syndrome (OS) is a genetic disorder characterized by midline developmental defects. Male patients with the X-linked form of OS, caused by loss-of-function mutations in the MID1 gene, show high variability of the clinical signs. MID1 encodes a ubiquitin ligase that controls phosphatase 2A, but its role in the pathogenesis of the disease is still unclear. Here, we report a mouse line carrying a nonfunctional ortholog of the human MID1 gene, Mid1. Mid1-null mice show the brain anatomical defect observed in patients (i.e., hypoplasia of the anterior portion of the medial cerebellum, the vermis). We found that the presence of this defect correlates with motor coordination and procedural and nonassociative learning impairments. The defect is limited to the most anterior lobes of the vermis, the region of the developing cerebellum adjacent to the dorsal midbrain. Analyses at midgestation reveal that lack of Mid1 causes the shortening of the posterior dorsal midbrain, the rostralization of the midbrain/cerebellum boundary, and the downregulation of a key player in the development of this region, Fgf17. Thus, lack of Mid1 causes a misspecification of the midbrain/cerebellar boundary that results in an abnormal development of the most anterior cerebellar lobes. This animal model provides a tool for additional in vivo studies of the physiological and pathological role of the Mid1 gene and a system to investigate the development and function of anterior cerebellar domains.

TRIM32 and Malin in Neurological and Neuromuscular Rare Diseases.

Tripartite motif (TRIM) proteins are RING E3 ubiquitin ligases defined by a shared domain structure. Several of them are implicated in rare genetic diseases, and mutations in TRIM32 and TRIM-like malin are associated with Limb-Girdle Muscular Dystrophy R8 and Lafora disease, respectively. These two proteins are evolutionary related, share a common ancestor, and both display NHL repeats at their C-terminus. Here, we revmniew the function of these two related E3 ubiquitin ligases discussing their intrinsic and possible common pathophysiological pathways.

An online tool for fetal fraction prediction based on direct size distribution analysis of maternal cell-free DNA.

The discovery of circulating fetal DNA in the plasma of pregnant women has greatly promoted advances in noninvasive prenatal testing. Screening performance is enhanced with higher fetal fraction and analysis of samples whose fetal DNA fraction is lower than 4% are unreliable. Although current approaches to fetal fraction measurement are accurate, most of them are expensive and time consuming. Here we present a simple and cost-effective solution that provides a quick and reasonably accurate fetal fraction by directly evaluating the size distribution of circulating DNA fragments in the extracted maternal cell-free DNA. The presented approach could be useful in the presequencing stage of noninvasive prenatal testing to evaluate whether the sample is suitable for the test or a repeat blood draw is recommended.

The MID1 gene product in physiology and disease.

MID1 is an E3 ubiquitin ligase of the Tripartite Motif (TRIM) subfamily of RING-containing proteins, hence also known as TRIM18. MID1 is a microtubule-binding protein found in complex with the catalytic subunit of PP2A (PP2Ac) and its regulatory subunit alpha 4 (α4). To date, several substrates and interactors of MID1 have been described, providing evidence for the involvement of MID1 in a plethora of essential biological processes, especially during embryonic development. Mutations in the MID1 gene are responsible of the X-linked form of Opitz syndrome (XLOS), a multiple congenital disease characterised by defects in the development of midline structures during embryogenesis. Here, we review MID1-related physiological mechanisms as well as the pathological implication of the MID1 gene in XLOS and in other clinical conditions.

Emerging Roles of the TRIM E3 Ubiquitin Ligases MID1 and MID2 in Cytokinesis.

Ubiquitination is a post-translational modification that consists of ubiquitin attachment to target proteins through sequential steps catalysed by activating (E1), conjugating (E2), and ligase (E3) enzymes. Protein ubiquitination is crucial for the regulation of many cellular processes not only by promoting proteasomal degradation of substrates but also re-localisation of cellular factors and modulation of protein activity. Great importance in orchestrating ubiquitination relies on E3 ligases as these proteins recognise the substrate that needs to be modified at the right time and place. Here we focus on two members of the TRIpartite Motif (TRIM) family of RING E3 ligases, MID1, and MID2. We discuss the recent findings on these developmental disease-related proteins analysing the link between their activity on essential factors and the regulation of cytokinesis highlighting the possible consequence of alteration of this process in pathological conditions.

Analysis of the Zn-Binding Domains of TRIM32, the E3 Ubiquitin Ligase Mutated in Limb Girdle Muscular Dystrophy 2H.

Members of the tripartite motif family of E3 ubiquitin ligases are characterized by the presence of a conserved N-terminal module composed of a RING domain followed by one or two B-box domains, a coiled-coil and a variable C-terminal region. The RING and B-box are both Zn-binding domains but, while the RING is found in a large number of proteins, the B-box is exclusive to the tripartite motif (TRIM) family members in metazoans. Whereas the RING has been extensively characterized and shown to possess intrinsic E3 ligase catalytic activity, much less is known about the role of the B-box domains. In this study, we adopted an in vitro approach using recombinant point- and deletion-mutants to characterize the contribution of the TRIM32 Zn-binding domains to the activity of this E3 ligase that is altered in a genetic form of muscular dystrophy. We found that the RING domain is crucial for E3 ligase activity and E2 specificity, whereas a complete B-box domain is involved in chain assembly rate modulation. Further, in vitro, the RING domain is necessary to modulate TRIM32 oligomerization, whereas, in cells, both the RING and B-box cooperate to specify TRIM32 subcellular localization, which if altered may impact the pathogenesis of diseases.

Cellular TRIM33 restrains HIV-1 infection by targeting viral integrase for proteasomal degradation.

Productive HIV-1 replication requires viral integrase (IN), which catalyzes integration of the viral genome into the host cell DNA. IN, however, is short lived and is rapidly degraded by the host ubiquitin-proteasome system. To identify the cellular factors responsible for HIV-1 IN degradation, we performed a targeted RNAi screen using a library of siRNAs against all components of the ubiquitin-conjugation machinery using high-content microscopy. Here we report that the E3 RING ligase TRIM33 is a major determinant of HIV-1 IN stability. CD4-positive cells with TRIM33 knock down show increased HIV-1 replication and proviral DNA formation, while those overexpressing the factor display opposite effects. Knock down of TRIM33 reverts the phenotype of an HIV-1 molecular clone carrying substitution of IN serine 57 to alanine, a mutation known to impair viral DNA integration. Thus, TRIM33 acts as a cellular factor restricting HIV-1 infection by preventing provirus formation.