Mutation affecting the proximal promoter of Endoglin as the origin of hereditary hemorrhagic telangiectasia type 1

[Background] Hereditary hemorrhagic telangiectasia (HHT) is a vascular multi-organ system disorder. Its diagnostic criteria include epistaxis, telangiectases in mucocutaneous sites, arteriovenous malformations (AVMs), and familial inheritance. HHT is transmitted as an autosomal dominant condition, caused in 85% of cases by mutations in either Endoglin (ENG) or Activin receptor-like kinase (ACVRL1/ACVRL1/ALK1) genes. Pathogenic mutations have been described in exons, splice junctions and, in a few cases with ENG mutations, in the proximal promoter, which creates a new ATG start site. However, no mutations affecting transcription regulation have been described to date in HHT, and this type of mutation is rarely identified in the literature on rare diseases.[Methods] Sequencing data from a family with HHT lead to single nucleotide change, c.-58G > A. The functionality and pathogenicity of this change was analyzed by in vitro mutagenesis, quantitative PCR and Gel shift assay. Student t test was used for statistical significance.[Results] A single nucleotide change, c.-58G > A, in the proximal ENG promoter co-segregated with HHT clinical features in an HHT family. This mutation was present in the proband and in 2 other symptomatic members, whereas 2 asymptomatic relatives did not harbor the mutation. Analysis of RNA from activated monocytes from the probands and the healthy brother revealed reduced ENG mRNA expression in the HHT patient (p = 0.005). Site-directed mutagenesis of the ENG promoter resulted in a three-fold decrease in luciferase activity of the mutant c.-58A allele compared to wild type (p = 0.005). Finally, gel shift assay identified a DNA-protein specific complex.[Conclusions] The novel ENG c.-58G > A substitution in the ENG promoter co-segregates with HHT symptoms in a family and appears to affect the transcriptional regulation of the gene, resulting in reduced ENG expression. ENG c.-58G > A may therefore be a pathogenic HHT mutation leading to haploinsufficiency of Endoglin and HHT symptoms. To the best of our knowledge, this is the first report of a pathogenic mutation in HHT involving the binding site for a transcription factor in the promoter of ENG.This study has been supported by grants from Ministerio de Economia y Competitividad of Spain (SAF2011-23475 and SAF2014-52374-R) to L.M. Botella and Centro de Investigación Biomedica en Red de Enfermedades Raras (CIBERER).Peer reviewe

Análisis de la topología del DNA durante la replicación

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias. Fecha de lectura: 16-05-2003The topology of replication intermediates (Rls) changes continuously as replication proceeds. But the dynamics of these changes remains to be fully understood. To shed some new light on this problem we used Escherichia coli plasmids as a model system. We constructed several plasmids bearing a unidirectional ColE 1 origin and a polar replication fork barrier located at different distances from the origin. Two dimensional agarose gel electrophoresis (2D gels) and electron microscopy allowed us to show that the negative supercoiling of partially replicated plasmids progressively diminishes as the replication fork advances. We found also that Chloroquine and Ethidium Bromide, two drugs that induce positive supercoiling of non-replicating molecules, are unable to introduce positive supercoiling in partially replicated plasmids. This occurs because in those plasmids with a replication fork, positive supercoiling is immediately adsorbed by a regression of the replication fork with the concomitant formation of a Holliday-like junction. Knotted bubbles form when topo IV knot the daughter duplexes behind the fork. We showed that knotted bubbles can form also during unimpaired DNA replication, although they become more evident in RIs containing a stalled fork. To learn more about the dynamics of knot formation as replication advances, we used 2D gels to identify knotted bubbles in molecules where the replication fork stalled at different stages of the process. The number and complexity of knotted bubbles rose as a function of bubble size, suggesting that during DNA replication knotting is inversely affected by precatenane density. Finally, we investigated the topological consequences of the collision of transcription and replication in plasmids where head-on collision between the replication fork and the RNA polymerase transcribing the tetracycline resistance gene was allowed or avoided. The results obtained indicate that this type of collision triggers knotting of the daughter duplexes behind the fork. We propose that too many knots would impair segregation and could be one of the main reasons for nature to avoid head-on collision of transcription and replication

DNA knotting caused by head-on collision of transcription and replication

26 p.-4 fig.Collision of transcription and replication is uncommon, but the reason for nature to avoid this type of collision is still poorly understood. In Escherichia coli pBR322 is unstable and rapidly lost without selective pressure. Stability can be rescued if transcription of the tetracycline-resistance gene (TetR), progressing against replication, is avoided. We investigated the topological consequences of the collision of transcription and replication in pBR322-derived plasmids where head-on collision between the replication fork and the RNA polymerase transcribing the TetR gene was allowed or avoided. The results obtained indicate that this type of collision triggers knotting of the daughter duplexes behind the fork. We propose this deleterious topological consequence could explain the instability of pBR322 and could be also one of the reasons for nature to avoid head-on collision of transcription and replication.This work was partially supported by grants PM97-0138 and PGC PB98-048 from the Spanish Comisión Interministerial de Ciencia y Tecnología (CICYT), 99/0850 from the Spanish Fondo de Investigación Sanitaria (FIS) and 08.6/0016/1997 from the Comunidad Autónoma de Madrid (CAM).Peer reviewe

Método para la predicción del riesgo de desarrollar la enfermedad de degeneración macular asociada a la edad en la población española

Método para predecir el riesgo de desarrollar la enfermedad de degeneración macular asociada a la edad en población caucásica española que se caracteriza porque se utilizan para el cálculo marcadores genéticos prevalentes en población afectada por dicha enfermedad en relación a población sana.Peer reviewedSegugen S.L., Universidad de Navarra, Consejo Superior de Investigaciones Científicas (España)B1 Patente sin examen previ

Relevance of complement factor H-related 1 (CFHR1) genotypes in age- related macular degeneration (AMD)

31 p.-4 fig.-4 tab.Purpose. Age-related macular degeneration (AMD) has a strong genetic component with a major locus at 1q31, including the complement factor H (CFH) gene. Detailed analyses of this locus have demonstrated the existence of one SNP haplotype block, carrying the CFH 402His allele, which confers increased risk for AMD, and two protective SNP haplotypes, one of them carrying a deletion of the CFHR1 and CFHR3 genes (ΔCFHR3-CFHR1). The purpose of these studies was to evaluate the contribution of newly described CFHR1 alleles to the association of the 1q31 locus with AMD. Methods. Two hundred fifty-nine patients and 191 age-matched controls of Spanish origin were included in a transversal case–control study using multivariate logistic regression analysis and ROC (receiver operating characteristic) statistics to generate and test models predictive of the development of AMD. Results. This study showed for the first time that a particular CFHR1 allotype, CFHR1*A, is strongly associated with AMD (odds ratio, 2.08; 95% confidence interval, 1.59–2.73; P < 0.0001) and illustrate a peculiar genotype–phenotype correlation between the CFHR1 alleles and different diseases that may have important implications for understanding the pathophysiology of AMD. It also shows that CFHR1*A is in strong linkage disequilibrium with the CFH 402His allele, which provides additional candidate variants within the major risk haplotype at 1q31, promoting its association with AMD. Further, using the Spanish population as a model, the results showed that analysis of the CFHR1 genotypes provide sufficient information to delineate the individual risk of developing AMD. Conclusions. The results support a relevant role of CFHR1 in the pathogenesis of AMDSupported by Grant SAF2008-00226 from the Spanish Ministerio de Ciencia e Innovación, the Ciber de Enfermedades Raras, and the Fundación Renal Iñigo Alvarez de Toledo (SRdeC); Grants RTICS RD07/0062, FIS PI 08/1705, and FIS 11/00898 from the Instituto de Salud Carlos III (AG-L); and Grant 35/2008 from the Comunidad Autónoma de Madrid, Dirección General de Innovación Tecnológica of the Consejeria Economía e Innovavión Tecnológica (JP-P)Peer reviewe

Detection of genetic rearrangements in the regulators of complement activation RCA cluster by high-throughput sequencing and MLPA

20 p.-6 fig.-3 tab.The regulators of complement activation (RCA) gene cluster in 1q31-1q32 includes most of the genes encoding complement regulatory proteins. Genetic variability in the RCA gene cluster frequently involve copy number variations (CNVs), a type of chromosome structural variation causing alterations in the number of copies of specific regions of DNA. CNVs in the RCA gene cluster often relate with gene rearrangements that result in the generation of novel genes, carrying internal duplications or deletions, and hybrid genes, resulting from the fusion or exchange of genetic material between two different genes. These gene rearrangements are strongly associated with a number of rare and common diseases characterized by complement dysregulation. Identification of CNVs in the RCA gene cluster is critical in the molecular diagnostic of these diseases. It can be done by bioinformatics analysis of DNA sequence data generated by massive parallel sequencing techniques (NGS, next generation sequencing) but often requires special techniques like multiplex ligation-dependent probe amplification (MLPA). This is because the currently used massive parallel DNA sequencing approaches do not easily identify all the structural variations in the RCA gene cluster. We will describe here how to use the MLPA assays and two computational tools to analyze NGS data, NextGENe and ONCOCNV, to detect CNVs and gene rearrangements in the RCA gene cluster.SRdeC is supported by the Spanish “Ministerio de Economía y Competitividad-FEDER” (SAF2015-66287R, PID2019-104912RB-I00, RTC-2016-4635-1 and the Autonomous Region of Madrid (S2017/BMD-3673). Secugen has received a soft loan from the Spanish “Ministerio de Economía y Competitividad” Retos Program RTC-2016-4635-1 cofinanced by FEDER funds.Peer reviewe

Aislamiento y caracterización de secuencias de AND contenidas en los cromosomas L de Sciara Coprophila

Trabajo presentado en el III Seminario de Citogenética, celebrado en Granada (España) del 30 de Junio al 3 de Julio de 2003

An retrospective analysis of genetic diagnosis in Atypical Hemolytic Uremic Syndrome (aHUS) and C3 glomerulopathy (C3G). Where we are now and where we are moving to.

1 p. Abstracts for the 17th IPNA Congress, Iguaçu, Brazil, September 2016Peer reviewe

Description of mpox reinfection by whole genome sequencing

Several possible mpox reinfections have been reported, however, the debate on whether these are confirmed reinfections remains open.A 30-year-old male living with HIV and a history of single-dose mpox vaccination, first diagnosed with mpox in September 2022, presented with genital ulcers in March 2023, testing positive for mpox virus. Real-time polymerase chain reaction revealed the presence of viral DNA with cycle threshold values of 24 and 25, respectively. Whole genome sequencing and phylogenetic approach allowed us to classify these viruses as Clade IIb lineage B.1 and Clade IIb lineage B.1.4, respectively. Twelve nucleotide differences were identified. The observed difference was higher than the estimate of mutations/genome/year described.These data confirm that mpox reinfection is possible and reinforces current vaccination campaigns

Characterization of a spontaneous, recessive, missense mutation arising in the Tecta gene

The TECTA gene encodes alpha-tectorin ( TECTA), a major noncollagenous component of the tectorial membrane (TM). In humans, mutations in TECTA lead to either dominant (DFNA8/A12) or recessive (DFNB21) forms of nonsyndromic hearing loss. All missense mutations in TECTA that have been reported thus far are associated with the dominant subtype, whereas those leading to recessive deafness are all inactivating mutations. In this paper, we characterize a spontaneous missense mutation (c.1046C9 &gt; A, p.A349D) arising in the mouse Tecta gene that is, unlike all previously reported missense mutations in TECTA, recessive. The morphological phenotype of the Tecta(A349D/A349D) mouse resembles but is not identical to that previously described for the Tecta(Delta ENT/Delta ENT) mouse. As in the Tecta(Delta ENT/Delta ENT) mouse, the TM is completely detached from the surface of the organ of Corti and spiral limbus, lacks a striated-sheet matrix, and is deficient in both beta-tectorin (Tectb) and otogelin. A significant amount of Tecta is, however, detected in the TM of the Tecta(A349D/A349D) mouse, and numerous, electron-dense matrix granules are seen interspersed among the disorganized collagen fibrils. Mutated Tecta(A349D) is therefore incorporated into the TM but presumably unable to interact with either Tectb or otogelin. The Tecta(A349D/A349D) mouse reveals that missense mutations in Tecta can be recessive and lead to TM detachment and suggests that should similar mutations arise in the human population, they would likely cause deafness