Impact of genetic structural variants in factor XI deficiency: identification, accurate characterization, and inferred mechanism by long-read sequencing.

BACKGROUND: Congenital factor XI (FXI) deficiency is a probably underestimated coagulopathy that confers antithrombotic protection. Characterization of genetic defects in F11 is mainly focused on the identification of single-nucleotide variants and small insertion/deletions because they represent up to 99% of the alterations accounting for factor deficiency, with only 3 gross gene defects of structural variants (SVs) having been described. OBJECTIVES: To identify and characterize the SVs affecting F11. METHODS: The study was performed in 93 unrelated subjects with FXI deficiency recruited in Spanish hospitals over a period of 25 years (1997-2022). F11 was analyzed by next-generation sequencing, multiplex ligand probe amplification, and long-read sequencing. RESULTS: Our study identified 30 different genetic variants. Interestingly, we found 3 SVs, all heterozygous: a complex duplication affecting exons 8 and 9, a tandem duplication of exon 14, and a large deletion affecting the whole gene. Nucleotide resolution obtained by long-read sequencing revealed Alu repetitive elements involved in all breakpoints. The large deletion was probably generated de novo in the paternal allele during gametogenesis, and despite affecting 30 additional genes, no syndromic features were described. CONCLUSION: SVs may account for a high proportion of F11 genetic defects implicated in the molecular pathology of congenital FXI deficiency. These SVs, likely caused by a nonallelic homologous recombination involving repetitive elements, are heterogeneous in both type and length and may be de novo. These data support the inclusion of methods to detect SVs in this disorder, with long-read-based methods being the most appropriate because they detect all SVs and achieve adequate nucleotide resolution.

Usefulness and Limitations of Multiple Ligation-Dependent Probe Amplification in Antithrombin Deficiency.

Multiplex ligation-dependent probe amplification (MLPA) identifies genetic structural variants in SERPINC1 in 5% of cases with antithrombin deficiency (ATD), the most severe congenital thrombophilia. Our aim was to unravel the utility and limitations of MLPA in a large cohort of unrelated patients with ATD (N = 341). MLPA identified 22 structural variants (SVs) causing ATD (6.5%). MLPA did not detect SVs affecting introns (four cases), and the diagnosis was inaccurate in two cases according to long-range PCR or nanopore sequencing. MLPA was used to detect possible hidden SVs in 61 cases with type I deficiency with single nucleotide variations (SNVs) or small insertion/deletion (INDEL). One case had a false deletion of exon 7, as the 29-bp deletion affected an MLPA probe. We evaluated 32 variants affecting MLPA probes: 27 SNVs and 5 small INDELs. In three cases, MLPA gave false-positive results, all diagnosed as deletions of the affected exon: a small INDEL complex, and two SNVs affecting MLPA probes. Our study confirms the utility of MLPA to detect SVs in ATD, but also shows some limitations in detecting intronic SVs. MLPA renders imprecise and false-positive results for genetic defects which affect MLPA probes. Our results encourage the validation of MLPA results.

Two SERPINC1 variants affecting N-glycosylation of Asn224 cause severe thrombophilia not detected by functional assays.

Antithrombin deficiency, the most severe congenital thrombophilia, might be underestimated, as some pathogenic variants are not detected by routine functional methods. We have identified 2 new SERPINC1 variants, p.Glu227Lys and p.Asn224His, in 4 unrelated thrombophilic patients with early and recurrent thrombosis that had normal antithrombin activity. In one case, the mutation was identified by whole genome sequencing, while in the 3 remaining cases, the mutation was identified by sequencing SERPINC1 based on a single functional positive finding supporting deficiency. The 2 variants shared a common functional defect, an impaired or null N-glycosylation of Asn224 according to a eukaryotic expression model. Carriers had normal anti-FXa or anti-FIIa activities but impaired anti-FVIIa activity and a detectable loss of inhibitory function when incubating the plasma for 1 hour at 41°C. Moreover, the ß glycoform of the variants, lacking 2 N-glycans, had reduced secretion, increased heparin affinity, no inhibitory activity, and a potential dominant-negative effect. These results explain the increased thrombin generation observed in carriers. Mutation experiments reflected the role that Lysine residues close to the N-glycosylation sequon have in impairing the efficacy of N-glycosylation. Our study shows new elements involved in the regulation of N-glycosylation, a key posttranslational modification that, according to our results, affects folding, secretion, and function, providing new evidence of the pathogenic consequence of an incorrect N-glycosylation of antithrombin. This study supports that antithrombin deficiency is underestimated and encourages the development of new functional and genetic tests to diagnose this severe thrombophilia.

Molecular Dissection of Structural Variations Involved in Antithrombin Deficiency.

Inherited antithrombin deficiency, the most severe form of thrombophilia, is predominantly caused by variants in SERPINC1. Few causal structural variants have been described, usually detected by multiplex ligation-dependent probe amplification or cytogenetic arrays, which only define the gain or loss and the approximate size and location. This study has done a complete dissection of the structural variants affecting SERPINC1 of 39 unrelated patients with antithrombin deficiency using multiplex ligation-dependent probe amplification, comparative genome hybridization array, long-range PCR, and whole genome nanopore sequencing. Structural variants, in all cases only affecting one allele, were deleterious and caused a severe type I deficiency. Most defects were deletions affecting exons of SERPINC1 (82.1%), but the whole cohort was heterogeneous, as tandem duplications, deletion of introns, or retrotransposon insertions were also detected. Their size was also variable, ranging from 193 bp to 8 Mb, and in 54% of the cases involved neighboring genes. All but two structural variants had repetitive elements and/or microhomologies in their breakpoints, suggesting a common mechanism of formation. This study also suggested regions recurrently involved in structural variants causing antithrombin deficiency and found three structural variants with a founder effect: the insertion of a retrotransposon, duplication of exon 6, and a 20-gene deletion. Finally, nanopore sequencing was determined to be the most appropriate method to identify and characterize all structural variants at nucleotide level, independently of their size or type.

Molecular and clinical characterization of transient antithrombin deficiency: A new concept in congenital thrombophilia.

Antithrombin deficiency, the most severe thrombophilia, might be underestimated, since it is only investigated in cases with consistent functional deficiency or family history. We have analyzed 444 consecutive, unrelated cases, from 1998 to 2021, with functional results supporting antithrombin deficiency in at least one sample. Plasma antithrombin was evaluated by functional and biochemical methods in at least two samples. SERPINC1 gene was analyzed by sequencing and MPLA. Hypoglycosylation was studied by electrophoresis and high-performance liquid chromatography (HPLC). In 260 of 305 cases (85.2%) with constitutive deficiency (activity < 80% in all samples), a SERPINC1 (N = 250), or N-glycosylation defect (N = 10) was observed, while 45 remained undetermined. The other 139 cases had normal antithrombin activity (≥ 80%) in at least one sample, what we called transient deficiency. Sixty-one of these cases (43.9%) had molecular defects: 48 had SERPINC1 variants, with two recurrent mutations (p.Ala416Ser[Cambridge II], N = 15; p.Val30Glu[Dublin], N = 12), and 13 hypoglycosylation. Thrombotic complications occurred in transient deficiency, but were less frequent, latter-onset, and had a higher proportion of arterial events than in constitutive deficiency. Two mechanisms explained transient deficiency: The limitation of functional methods to detect some variants and the influence of external factors on the pathogenic consequences of these mutations. Our study reveals a molecular defect in a significant proportion of cases with transient antithrombin deficiency, and changes the paradigm of thrombophilia, as the pathogenic effect of some mutations might depend on external factors and be present only at certain timepoints. Antithrombin deficiency is underestimated, and molecular screening might be appropriate in cases with fluctuating laboratory findings.

Cancer testis antigens in myelodysplastic syndromes revisited: a targeted RNA-seq approach.

Cancer-Testis antigens (CTA) are named after the tissues where they are mainly expressed: in germinal and in cancer cells, a process that mimics many gametogenesis features. Mapping accurately the CTA gene expression signature in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) is a prerequisite for downstream immune target-discovery projects. In this study, we take advantage of the use of azacitidine to treat high-risk MDS and CMML to draw the CTAs landscape, before and after treatment, using an ad hoc targeted RNA sequencing (RNA-seq) design for this group of low transcript genes. In 19 patients, 196 CTAs were detected at baseline. Azacitidine did not change the number of CTAs expressed, but it significantly increased or decreased expression in nine and five CTAs, respectively. TFDP3 and DDX53, emerged as the main candidates for immunotherapeutic targeting, as they showed three main features: i) a significant derepression on day +28 of cycle one in those patients who achieved complete remission with hypomethylating treatment (FC = 6, p = .008; FC = 2.1, p = .008, respectively), ii) similar dynamics at the protein level to what was observed at the RNA layer, and iii) to elicit significant specific cytotoxic immune responses detected by TFDP3 and DDX53 HLA-A*0201 tetramers. Our study addresses the unmet landscape of CTAs expression in MDS and CMML and revealed a previously unrecognized TFDP3 and DDX53 reactivation, detectable in plasma and able to elicit a specific immune response after one cycle of azacitidine.

Relación de los polimorfismos G-174C y G-572C de la interleucina-6 y C-511T y C+3953T de la interleucina-1 con la cardiopatía isquémica en Quito, Ecuador / Relationship of Interleukin-6 G-174C and G-572C and Interleukin-1 C-511T and C+3953T Polymorphisms with Ischemic Cardiomyopathy in Quito, Ecuador

RESUMEN: Introducción: En los últimos años se ha evidenciado que los procesos inflamatorios están directamente relacionados con la formación de la placa ateroesclerótica, causante de la cardiopatía isquémica (CI). Por esta razón, toda molécula relacionada con aquellos procesos es de vital importancia. Las interleucinas (IL) son citoquinas proinflamatorias y sus polimorfismos aparentemente incrementan el proceso inflamatorio. Los más asociados con la cardiopatía isquémica son algunos polimorfismos de las interleucinas 1 (IL-1) y 6 (IL-6). Objetivos: Establecer la relación de los polimorfismos G-174C y G-572C de la interleucina-6 y C-511T y C+3953T de la interleucina-1 con la cardiopatía isquémica. Material y métodos: Se desarrolló un estudio de tipo analítico retrospectivo, de 76 casos y 76 controles, de pacientes atendidos en el servicio de hemodinámica del Hospital Carlos Andrade Marín (HCAM), de Quito. La genotipificación se hizo mediante la reacción en cadena de la polimerasa con enzimas de restricción (PCR-RFLP). Resultados: De los cuatro polimorfismos estudiados, únicamente el IL-6 174 GG tuvo una asociación estadísticamente significativa con la cardiopatía isquémica. La regresión logística usada para determinar los predictores más importantes de cardiopatía isquémica mostró que el genotipo IL-6 174 GG (OR 4,065, p = < 0,001) se asoció con un incremento del riesgo de presentar cardiopatía isquémica de forma independiente. Conclusiones: El genotipo GG del polimorfismo IL-6 G-174C confiere un riesgo 4 veces superior de desarrollar cardiopatía isquémica, mientras que los tres polimorfismos restantes no confieren riesgos.

ABSTRACT: Background: It has been recently found that inflammatory processes are directly related to the development of atherosclerotic plaque, causing ischemic heart disease. For this reason, every molecule related to these processes is critically important. Interleukins (IL) are proinflammatory cytokines, and their polymorphisms seem to increase the inflammatory progress. IL-1 and IL-6 polymorphisms are the ones most significantly associated with ischemic heart disease. Objectives: The aim of this study was to establish the relationship of IL-6 G-174C and G-572 C and IL-1 C-511T and C+3953T polymorphisms with ischemic heart disease. Methods: A retrospective study of 76 cases and 76 controls was carried out in patients attending the hemodynamics service of Carlos Andrade Marín Hospital (HCAM) of Quito, Ecuador. Genotyping was done on the basis of polymerase chain reaction with restriction enzymes (PCR-RFLP). Results: Among the four polymorphisms studied, only IL-6 -174 GG was significantly associated with ischemic heart disease. The logistic regression analysis used to determine the most important predictors of ischemic heart disease showed that the IL-6 -174 GG genotype was associated with an increased risk of independently presenting ischemic heart disease (OR 4.065, p ≤ 0.001). Conclusions: The GG genotype of IL-6 G-174C polymorphism confers a fourfold higher risk of developing ischemic heart disease, while the remaining three polymorphisms do not pose risks in this human population.

Consumo de drogas en una población joven de una comuna rural: magnitud y caracteristicas del problema / Drug consumption in a young population of a rural community: extent and characteristics of the problem

Se realizó un estudio descriptivo con la finalidad de conocer la magnitud del consumo de drogas entre los adolescentes de la comuna de Coínco. Se entregó un cuestionario a 857 jóvenes, que consistió en 22 preguntas de tipo cerradas y que fue autoaplicado a la muestra en forma simultánea. Del análisis de los resultados de desprendió que la gran mayoría de los jóvenes se encontraba entre los 13 y 18 años de edad, siendo su actividad principal el estudio. El 4,8% de la muestra declaró haber consumido drogas, predominando en cuanto a tipo de drogas, los tranquilizantes del Sistema Nervioso Central y la marihuana. En general, la edad primer contacto con drogas fue antes de los 18 años, y en más de la mitad de los casos éste se ha mantenido con alguna frecuencia, predominando el consumo individual. La experiencia inicial fue por iniciativa propia, influencia de amigos o por tratamiento médico. El hallazgo de síntomas de dependencia síquica o física alcanzó al 25% de los consumidores. Finalmente, los investigadores postulan que existió cierto grado de ocultamiento de información por parte de la muestra, por temor a sanciones, lo que estaría disminuyendo la real magnitud del problema

Prevalencia y características del consumo de alcohol en escolares de la comuna de Pichidegua / Prevalence and characteristics of alcohol consumption in students of Pichidegua community

Se estudia la prevalencia y las características del consumo de alcohol en escolares de enseñanza básica de una comunidad rural, con el propósito de motivar este tipo de estudios para conocer mejor el problema a nivel regional y local y poder realizar programas educativos preventivos en conjunto con el profesorado. La encuesta fue aplicada a 1408 escolares del segundo ciclo de la enseñanza básica en un área rural y arrojó los siguientes resultados: el 81% de los encuestados tiene entre 10 y 14 años de edad; el 97% pertenece a hogares legalmente constituídos; el 50% de los padres tiene un trabajo estable; el 90% de las madres se desempeña como dueña de casa; el 90% de los encuestados reconoce tener un familiar en casa que bebe regularmente, en general el padre; el 67% reconoce que el padre se embriaga, un 36% como bebedor anormal y un 31% como bebedor moderado. El 78,3% de los escolares dijo haber ingerido bebidas alcohólicas alguna vez, y de éstos, un 43% antes de los 11 años de edad; el 26,2% reconoce haberse embriagado en alguna oportunidad, en su mayoría menores de 14 años. Un 23% consume bebidas alcohólicas en forma habitual, el 64% en circunstancias de tipo social, acompañados generalmente por familiares y de preferencia en el hogar. La imitación fue la principal razón para beber y en un 15% con estimulación directa de los padres. El 86,4% declara saber que el alcohol es perjudicial para la salud