Síndrome de bardet-biedl y síndrome de alströmcaracterización molecular y propuesta de nuevos mecanismos

Resumo

Bardet-Biedl syndrome (BBS) and Alström syndrome (ALMS) are two rare, multisystem genetic disorders belonging to the group of ciliopathies, which encompasses different diseases frequently caused by defects in primary cilia structure and/or function. These organelles are central hubs for coordination of multiple cellular signaling pathways, functioning as sensorial antennae. Regarding the phenotype displayed by these patients, an extensive overlapping is generally observed for both syndromes especially in early stages, which usually leads to misdiagnosis. Thus, BBS has as primary diagnostic features retinal dystrophy, obesity, polydactyly, renal abnormalities, urogenital anomalies and cognitive impairment. On its part, ALMS patients usually develop retinal dystrophy with nystagmus and photophobia, obesity typically accompanied by severe insulin resistance, hyperinsulinemia and impaired glucose tolerance, sensorineural hearing loss, dilated cardiomyopathy, and renal, pulmonary and hepatic injury with extensive multiorgan fibrosis. BBS is one of the most genetically heterogeneous ciliopathies, with 21 BBS genes involved to date, which explain about 70-80% of clinically-diagnosed patients, whereas only one gene has been linked to ALMS phenotypes (ALMS1). Both syndromes are generally inherited in an autosomal recessive pattern, although a more complex model of oligogenic inheritance has been proposed for some BBS families to explain the high interfamilial and intrafamilial clinical variability that is frequently reported. The biological functions of BBS proteins are mainly related to ciliary membrane elongation and ciliary trafficking. Thus, eight BBS proteins form a multimeric complex called BBSome, which is involved in the selective entry of cargoes to the cilium and the bidirectional transport along the ciliary axoneme. The remaining BBS proteins participate in BBSome assembly (specifically, by means of the CCT/TRiC/BBS-chaperonin complex, formed by BBS6, BBS10 and BBS12 proteins together with six chaperonins of the CCT/TRiC family), its localization at the ciliary base (ARL6/BBS3) or in different stages of BBSome transport. ALMS1 protein, on the other hand, has been associated with diverse biological processes, including regulation of endosome trafficking and recycling of membrane receptors, cell cycle control and ciliary maintenance and function. However, the precise molecular model that reconciles the multiple functions of this protein is pending to be uncovered. Despite the outstanding progress made in characterizing the contribution of BBS genes to design diagnostic strategies and also in identifying the underlying molecular mechanisms, they are still insufficient to offer real therapeutic options for these patients. In addition, the progressive implementation of massive sequencing technologies within diagnostic laboratories is bringing to light a large amount of variants identified in only one individual, whose biological role is usually unknown but they could not be ignored under the assumption of an oligogenic inheritance model influenced by the total mutational load. This is the context wherein arise the need to delve into some issues related to the optimization of diagnostic algorithms and mainly to the research about new molecular mechanisms potentially involved in BBS and ALMS, which have been addressed in this project and will be explained in greater detail below. We first aimed to analyze the contribution of BBS10 and BBS12 genes in a group of 55 patients (53 families) with BBS suspicion but a negative result (one or no pathogenic variants identified) for a previous mutational screening with the Asper Biotech BBS-ALMS1 genotyping microarray. We made this selection because BBS12 screening had not been previously performed in our BBS cohort, and also the exonic structure of both genes makes them ideal for their inclusion in a diagnostic algorithm. After performing direct sequencing in the candidate families, we found two pathogenic variants in seven probands for BBS12 and other two patients for BBS10 gene. Thus, BBS10 accounted for 8.3%, whereas BBS12 contributed 10.4% to the total load (considering the 96 families that form the whole cohort). Based on these results, BBS12 gene is the second gene most frequently involved in Bardet-Biedl syndrome in our cohort, just behind the BBS1 gene. The low contribution of BBS10 in this group of patients is a striking result reported for the first time, since this gene have an average contribution of at least 20% in the remaining worldwide cohorts. This has also major implications in the improvement of our own diagnostic strategies for guiding the molecular analysis of suspected BBS patients. So that we propose the inclusion of direct sequencing of BBS12 gene in the second step of our algorithm, together with BBS1 and BBS10 genes, once the presence of the predominant variants p.Met390Arg and p.Cys91Leufs*5 had been excluded. Remarkably, we identified ten different pathogenic variants in ten families, three of them novel (which were submitted to the European Nucleotide Archive) and also with 70% of these variants being private. The data abovementioned suggest that our cohort, consisting of 86% families of Spanish background, is genetically more heterogeneous than other reported, which has important consequences for designing specific diagnostic strategies aimed at Spanish population. In addition, we identified up to 16 variants of unknown significance (VUS) in both genes, considering as such those variants that are not causal but whose impact on phenotype could not be discarded under the assumption of an oligogenic inheritance model. We performed bioinformatics predictions on potential impact at splicing level as a first approach to characterize these variants, obtaining positive results for four BBS12 changes. Moreover, we intended to establish a genotype-phenotype correlation for patients with two pathogenic variants in BBS10 and BBS12 genes for the first time in the Spanish BBS cohort, through the compilation of the available phenotype data in each case. We finally obtained comprehensive clinical information for 15 families, although the small sample size and the few data for some categories prevented us from performing statistical analyses. However, we found interesting trends that is noteworthy to mention. Thus, the clinical spectrum displayed by those patients with two causal variants in these two BBS genes is more severe and early-onset than showed by patients with BBS1 changes, since there is higher prevalence of all primary features. It is particularly noticeable the frequency of urogenital anomalies in BBS10 patients (83%) and the more severe cognitive impairment associated with BBS12 variants (found in 75% of patients with available data), usually accompanied by psychomotor delay. These findings provided the first evidence of severe phenotypes in BBS12 patients comparable to those previously reported for BBS10 gene. In fact, BBS12 changes produced the most severe clinical spectrum in our cohort, with high prevalence of overlapping features with ALMS and McKusick-Kauffman syndrome, which has direct impact on genetic counseling and patients´ management. In addition to analyzing the genetic contribution of two BBS genes in the Spanish cohort, we aimed to assess the potential impact on splicing of some variants in several BBS/ALMS1 genes which had been previously identified in some families of our cohort. In this sense, there is a growing requirement for validating functional assays that allow us to properly interpret the biological consequences and potential impact on phenotype of the different variants identified in patients. This is particularly important in oligogenic diseases such as BBS, where it has been demonstrated that common variants can be deleterious and/or interact with pathogenic changes in an epistatic way to modulate the phenotype. For this purpose, we selected five changes: c.266A>G/p.(Tyr89Cys) and c.823C>T/p.(Arg275*) in BBS2, c.4G>T/p.(Gly2*) in ARL6/BBS3, c.77-6A>G in BBS4 and c.11641C>T/p.(His3882Tyr) in ALMS1gene, which were previously predicted as putative splicing variants after reaching a minimum of two positive predictions with the four bioinformatics tools used. We then performed the functional validation based on minigene assay, using the pSPL3 reporting vector and COS-7 cells as in vitro model. As a result, we did not observe alterations in mRNA processing due to the single substitutions tested, since wild-type and mutant constructions produced the same splicing pattern. Interestingly, three variants (two in BBS2 gene and also the missense change in ALMS1) generated several bands apart from the canonical transcript, which presumably correspond to splicing isoforms from alternative splicing events using variable splice sites, and/or heteroduplex formations involving different isoforms. Moreover, the results obtained for the two nonsense variants showed that nonsense-mediated decay appears not to be triggered by these transcripts containing premature termination codons. Therefore, if these variants were translated, they would produce truncated proteins with negative impact on normal cell activity. This is the first study which validates the impact on splicing of these particular five variants found in BBS/ALMS1 genes, trying to classify them based on their biological effect as suggested by the current guidelines. Also, it contributes to the few studies reported that assess the functional consequences of variants identified in BBS and ALMS patients. The evidences obtained here, combined with genotypic, phenotypic and population data, allowed us to draw some conclusions regarding the causal or modifier effect of the five variants analyzed. On the other hand, one of the main objectives of this project was to research on new mechanisms potentially involved in the pathogenesis and phenotype modulation of BBS and ALMS, especially focused on this last syndrome, since there are currently no molecular models that can explain the diverse functions carried out by ALMS1 protein. In this regard, we first aimed to test if epigenetic regulation through a methylation mechanism could be taking place in ALMS syndrome, an unexplored field to our knowledge. We hypothesized that epigenetic modulation could be influencing ALMS phenotypes since the high variability reported for many families cannot be explained by an oligogenic inheritance pattern, as it has been suggested for BBS. With this aim, we first characterized the promoter region of ALMS1 gene to search for CpG islands, X-box motifs (highly conserved DNA sequences known to be recognized by RFX transcription factors (TFs), which are specifically involved in transcriptional regulation of ciliary genes) and any other binding sequences. We identified a CpG island with 67 CpG sites potentially methylated and also harboring up to 20 different binding motifs for transcription factors that could be potentially regulating ALMS1 expression; none of them was compatible with mammal consensus sequences for X-box motifs. Among them, E2F1 and Eomesodermin could be good candidates for transcriptional regulation of ALMS1 gene, since they promote the expression of genes related to cell cycle control and cardiac differentiation, respectively, and there are solid evidences pointing the role of ALMS1 in both processes. In addition, DNA binding sequences for these two factors are located within the CpG island found in ALMS1 promoter, which increases the interest of studying their role in transcription maybe through a methylation mechanism. After that, we designed and optimized a nested quantitative methylation-specific PCR (qMSP) assay to evaluate the methylation status of the island previously identified, including DNA samples from peripheral blood of seven ALMS patients and also two DNA samples from serum-starved cultures of hTERT RPE-1 cells and primary fibroblasts (from a healthy donor). As a result, we did not detect methylation-specific amplification for the regions analyzed in the selected samples; thereby suggesting that ALMS1 promoter methylation is not an epigenetic mechanism that regulates the expression of this gene, at least under our experimental approach. In this sense, the identification of four additional CpG islands in the first 5´half of ALMS1 gene-body might point out that methylation is not acting in ALMS1 promoter region, but is related to alternative patterns in different genomic regions. As last approach in researching on new mechanisms underlying ALMS syndrome, we considered the possible functional relationship between ALMS and TGF-β/BMP signaling for the first time. Thus, several phenotypes displayed by ALMS patients such as multiorgan fibrosis and cardiomyopathy have been linked to defects in this pathway, which has a central role in regulating cross-talk with other signaling cascades to produce context-dependent biological outcomes critical for development and homeostasis. In order to address this hypothesis, we established a knockdown model for ALMS1 using hTERT RPE-1 cells by transient post-transcriptional silencing mediated by small interference RNA, reaching a 67% average reduction of gene expression at mRNA level. Once validated, we analyzed the impact of ALMS1 depletion on ALMS1 protein localization, ciliogenesis, cilia morphology and centrosomal integrity through immunofluorescence experiments from fixed cells. We then evaluated the effect of ALMS1 knockdown on TGF-β/BMP activation by the independent stimulation of each individual pathway (TGF-β and BMP) at different time points (0, 10, 30 and 90 minutes). For that, we quantified the levels of several phosphorilated proteins by inmunoblot: the specific R-SMAD protein (pSMAD2 for TGF-β and pSMAD1/5 for BMP) to measure canonical activation, and pERK1/2 to assess the noncanonical one. Regarding the effect of ALMS1 depletion on primary cilia, we first determined that the localization pattern of ALMS1 protein is not altered in silenced cells, since this protein is exclusively associated with centrosome/basal body and retained this pattern in silenced cells. In addition, ALMS1 knockdown did not affect the cell ability to produce primary cilia. However, several remarkably results were observed: cells with reduced expression of ALMS1 gene formed longer cilia than control cells (mean±SD=3.28±0.18 µm compared with 2.83±0.06 µm of controls), after measuring at least 50 cilia for each condition (control or silenced cells) and from three independent biological replicates. Notably, this difference in length reached statistically significance (p-value=0.041). Furthermore, we assessed the presence of aberrant morphologies of primary cilia after immunofluorescence experiments, considering as “aberrant” those cilia with evident segmentation, swelled or constricted axonemes, clearly anomalous forms and cilia bending greater than 45°. Thus, depleted cells were observed to produce a higher frequency of aberrant forms (mean±SD=44.1±5.6% compared with 25.5±9.6% of controls), being this difference statistically significant (p-value=0.001). An increased ciliary length has profound implications in cell cycle control and also in several cilia-related processes such as intraflagellar transport or cargo positioning along the cilium; hence, maintaining normal ciliary length is essential for proper signaling of those pathways that operate through the primary cilium. All these observations therefore suggest that ALMS1 regulates primary cilia assembly, although more experimental evidences are required. Moreover, silencing of ALMS1 gene in hTERT RPE-1 cells occasionally led to centrosome splitting, that is, the separation of both centrioles in the centrosome complex. This phenomenon was observed only in 9/161 cells, but they did not reach the minimum distance of 2 µm for being formally considered as splitting centrosomes. Consequently, we cannot consider that ALMS1 knockdown produce centrosomal/basal body anomalies, although the localization pattern of ALMS1 protein anticipated more anomalies at this level that could be linked with the cell cycle defects reported in cells derived from patients. Concerning TGF-β/BMP signaling activation in our knockdown model, remarkable findings were also noted. Thus, we could determine that ALMS1 depletion downregulated both TGF-β and BMP pathways in response to independent stimulation, affecting both the canonical and noncanonical cascades. Therefore, ALMS1 could also be a key regulator of balancing between canonical and noncanonical signaling in response to TGF-β/BMP stimulation, which will finally determine the biological output of this pathway. In addition, ALMS1 depletion was observed to alter the dynamics of TGF-β/BMP activation, especially noted after TGF-β stimulation. Thus, the reduction of ALMS1 expression led to smaller magnitude responses with a time delay in peaking, from ten to thirty minutes in the case of canonical signaling associated to both TGF-β and BMP pathways. Interestingly, this effect was also observed for noncanonical signaling after stimulation of silenced cells, but only for TGF-β pathway. These data suggest that ALMS1 could be modulating the temporal pattern of signaling, and that this regulation could affect the two main pathways, TGF-β and BMP, in different ways. Finally, we intended to propose a model which reconciles the different biological functions attributed to ALMS1 protein, taking into account all the evidences obtained in this study from ALMS1 silencing in hTERT RPE-1 cells and also from the promoter characterization of this gene, together with the literature reports. Thus, since two different isoforms have been described by other authors, one restricted to centrosomes and other located in the cleavage furrow of dividing cells, we hypothesized that ALMS1 has two well-defined functions according to these two isoforms. Considering this model, the centrosomal isoform would be related to ciliary assembly regulation and cell cycle control, whereas the second one would assume extra-ciliary roles, as endosome recycling, possibly under TGF-β/BMP signaling control. In this sense, we identified DNA binding sequences for SMAD3 and SMAD4 factors in ALMS1 promoter, which reinforces the possibly involvement of this pathway in developing ALMS phenotype. Although we cannot conclude anything about the precise molecular mechanisms that are operating, there are several steps in which ALMS1 functions could better fit based on current evidence reported by other authors, such as the translocation to the nucleus of SMAD complexes (since ALMS1 protein harbors nuclear localization signals) or the regulation of receptor availability through the endosome recycling pathway.