Irene Madrigal

The mechanisms underlying the development and severity of pulmonary hypertension (PH) in chronic obstructive pulmonary disease (COPD) and interstitial lung disease (ILD) remain poorly understood. To investigate the contribution of genetic factors, we performed targeted sequencing of PH-related genes in 63 patients with COPD, 51 with ILD, and 38 with idiopathic pulmonary arterial hypertension (IPAH), stratifying COPD and ILD patients according to PH severity, assessed by right heart catheterisation. Patients with ILD-associated PH harboured fewer genetic variants than those without PH, whereas an opposite trend was observed in COPD-associated PH. Variants of uncertain significance (VUS) in PAH-associated genes (BMPR2, BMPR1B) were identified in two out of 28 patients with severe COPD-PH and in one with nonsevere ILD-PH (SMAD9). VUS in extracellular matrix-related genes (THBS1, LOXL2) were also identified in four patients with severe COPD-PH, and in inflammatory-related genes (TNFAIP6) in two patients with ILD-PH. Pathogenic/likely pathogenic variants in PAH-associated genes were exclusive to IPAH. We conclude that there is limited genetic overlap between COPD-PH and ILD-PH, with a stronger genetic contribution to PH in COPD, particularly in its severe form, where a subset of patients carries VUS in PAH-associated genes or genes involved in vascular remodelling.

Whole exome sequencing (WES) is a standard, relevant diagnostic strategy in medical genetics. However, commercial WES library preparation kits vary significantly in design and covered regions. Very few studies have assessed DNA tissue of origin’s impact on WES performance, which relates to preanalytical factors that may compromise DNA quality. We evaluated three WES commercial solutions using eight DNA samples extracted from peripheral blood, amniotic fluid, fetal tissue, and fibroblasts. A second set of 24 previously sequenced samples was resequenced using the highest-performing kit to validate results. A comprehensive bioinformatics pipeline was applied to evaluate key metrics such as coverage uniformity, duplication rates, or estimated library size, among others. Twist Exome 2.0 capture kit demonstrated the best overall performance, achieving more uniform coverage, lower duplication rates (8% vs. 16–17%), and a larger estimated library size (243 M vs. 102–129 M). Additionally, this kit achieved 93% of regions covered at the standard threshold of 38x (compared to ~80% in the other two solutions). Notably, Twist Exome 2.0 capture consistently delivered superior results for DNA extracted from non-peripheral blood tissues. This study suggests that Twist Exome 2.0 provides robust performance and is particularly well-suited for samples where DNA quality may be compromised due to preanalytical limitations.

TRIM28 is an epigenetic co-repressor protein that silences transposable elements (TEs). Although loss-of-function studies in mice led to neurodevelopmental defects, a functional link between TRIM28 and human neurodevelopment has yet to be established. In this study, we describe two patients with neurodevelopmental delay who carry de novo TRIM28 missense variants. Using CRISPR-edited induced pluripotent stem cell lines and differentiated neural organoids, we demonstrate that these variants result in the loss of the histone mark H3K9me3 over TEs. This releases the regulatory potential of TEs resulting in altered expression of nearby genes. These findings could be replicated using CRISPRi-based TRIM28 silencing, which suggests that the two variants result in a loss of function. Our results highlight the critical role of TRIM28 in regulating TEs during human brain development, establishing a link between TRIM28 variants and neurodevelopmental delay. One Sentence Summary: TRIM28 variants disrupt epigenetic control of transposable elements in the developing human brain, linking them to neurodevelopmental delay.

Siddiqi syndrome is a rare autosomal recessive deafness-dystonia disorder caused by pathogenic variants in the FITM2 gene. To date, only 5 unrelated families have been reported in the literature carrying loss-of-function variants in FIMT2 gene. In this report, we describe a 29-year-old woman with compound heterozygous novel variants identified by trio-based exome sequencing. She carries the paternally inherited delins variant c.158_161delinsTCAT, p.(Arg53_Asn54delinsLeuIle) and the maternally inherited frameshift variant c.567del, p.(Thr190ProfsTer9) in FITM2 gene. The patient exhibits the main features of the disease, including deafness, intellectual disability, regression of motor skills and poor overall growth. Additionally, she presents with spastic paraplegia which supports recent phenotypic expansion. We describe for the first time, novel brain magnetic resonance imaging signal alterations, not previously associated with this disorder. These neuroimaging findings may provide new insights into the neurological manifestations of Siddiqi syndrome. This case expands the phenotypic and molecular spectrum of FITM2 associated disease and emphasizes the adult-features of this syndrome. Graphical abstract

Programs for Undiagnosed Rare Diseases (URD) with anonymized data sharing are contributing to the earlier genetic diagnosis of patients and to the identification and characterization of novel genetic disorders. Recently, de novo pathogenic variants in two non-coding spliceosomal small nuclear RNAs (snRNAs), RNU4-2 and RNU2-2 , key regulators of gene expression during neurodevelopment, have been linked to neurodevelopmental disorders (NDD). With the aim to identify individuals with causal genetic variants in known and novel snRNA genes, we analyzed WGS and clinical data of 1,708 probands with undiagnosed NDD enrolled in the Spanish URD Programs. Selected individuals underwent in-depth re-phenotyping including facial mask analyses (Face2Gene), and blood-DNA methylation profiling was obtained (Infinium methylation Epicv2 microarrays). We identified 38 patients (2.22%) with de novo pathogenic variants in snRNA genes, 28 in RNU4-2 (1.64%), 6 in RNU2-2 (0.35%), and 4 in RNU5B-1 (0,23%). A clinically recognizable NDD (ReNU syndrome) is caused by RNU4-2 variants, with dysmorphic features making a disease-associated facial mask, RNU2-2 associates an early onset epileptic encephalopathy, and RNU5-B1 a less defined NDD with overlapping features. A DNA methylomic episignature was also identified for RNU4-2 , which does not overlap with the other conditions. We have also defined autosomal recessive inheritance in 8 additional patients (0.47%) with NDD and biallelic variants in three snRNA genes (3 in RNU4-2 , 3 in RNU2-2 , and 2 in RNU4-1 ). Our study has facilitated the quick diagnosis, management, and genetic counselling of 2.69% previously undiagnosed patients with NDD, further reinforcing the value and utility of the URD Programs. GRAPHICAL

The implementation of non-invasive prenatal testing (NIPT) in maternal plasma, based on cell-free DNA (cfDNA) analysis, has progressed over the last two decades and is now integrated into the Spanish National Health System. However, there remains significant heterogeneity in its indications, technical methodologies, and reporting standards, reflecting international variability. This guide, developed by experts from the Spanish Association of Prenatal Diagnosis (AEDP) and the Spanish Association of Human Genetics (AEGH), provides recommendations to standardize NIPT application. It addresses key aspects such as technical and analytical requirements, integration with invasive diagnostic methods, pre- and post-test genetic counseling, and legal considerations. Additionally, the guide discusses the detection of common aneuploidies, the limitations in identifying structural chromosomal abnormalities and rare variants, and the impact of biological and clinical factors on test performance. By establishing a minimum framework based on scientific evidence, this document aims to optimize NIPT implementation in a cost-effective manner, ensuring clinical validity and informed decision-making for both healthcare professionals and pregnant women.

Introduction: Circulating cell-free DNA (cfDNA) consists of extracellular DNA fragments that circulate in the bloodstream and derived from apoptotic cells such as hematopoietic cells or placental trophoblast cells during pregnancy. Contents: cfDNA screening has been included in prenatal screening programs for the detection of chromosomal abnormalities. Unlike other invasive techniques, such as amniocentesis or chorionic villus sampling, cfDNA screening only requires a maternal plasma test. The use of advanced technologies for cfDNA testing, including DNA sequencing and SNP arrays, enables the detection of pregnancies at risk for trisomy 21, 18 or 13. Summary: This test has demonstrated a high accuracy and reliability, with detection rates exceeding 99 % for trisomy 21, and a very low rate of false-positive and false-negative results. In some countries, cfDNA screening has already been integrated in combined or universal prenatal screening programs. Outlook: As new technologies emerge and become widely available, more accurate prenatal tests will be developed for other genetic abnormalities.

Resumen: Introducción: El ADN libre circulante (ADN-lc) son fragmentos de ADN extracelulares que circulan libremente por la sangre y que se originan a partir de la apoptosis de diversos tipos celulares, como por ejemplo células hematopoyéticas o, en gestaciones, de células trofoblásticas provenientes de la placenta. Contenido: El estudio del ADN-lc se ha incluido como prueba de cribado prenatal para la detección de anomalías cromosómicas y, a diferencia de otras técnicas invasivas como la amniocentesis o la biopsia de vellosidades coriónicas, se realiza mediante un análisis de sangre materna. Gracias a la utilización de tecnologías avanzadas en el estudio del ADN-lc, como la secuenciación de ADN o los arrays de SNPs, es posible identificar gestaciones con riesgo de trisomía 21, trisomía 18 o trisomía 13. Resumen: Este test ha demostrado una alta precisión y fiabilidad, con tasas de detección superiores al 99 % para trisomía 21 y una muy baja tasa de falsos positivos y falsos negativos. En algunos países ya se ha implementado como una herramienta de cribado combinado o cribado universal. Perspectiva: A medida que la tecnología avanza y se vuelve más accesible, se espera que se puedan obtener pruebas aún más precisas para otras anomalías genéticas en el diagnóstico prenatal.

Background/Objectives: Cell-free DNA (cfDNA) is a non-invasive prenatal test used to screen for common trisomies (target cfDNA) that can be expanded to assess all autosomal chromosomes (genome-wide cfDNA). As cfDNA testing gains popularity, it is crucial to examine the factors influencing the decision-making process of pregnant individuals when choosing between these two approaches. Methods: In this prospective cohort study, 190 individuals undergoing cfDNA testing for aneuploidy screening, according to the current screening protocol, were allowed to make their own choice between target and genome-wide cfDNA testing. They were asked to complete a first survey at 11–13 weeks, designed to explore their characteristics, preferences, and satisfaction with the prenatal genetic counseling session, as well as a Decisional Conflict Scale. A postnatal survey was administered three months after delivery, including the Decisional Regret Scale and two open questions. Results: 84% of participants opted for genome-wide cfDNA. However, 17% found the decision challenging, and 14% felt that the results might increase anxiety. No significant differences in participant characteristics were found when comparing decisions between genome-wide and target cfDNA. However, significant differences were observed regarding ethnicity (p = p = 0.029), previous cfDNA experience (p = 0.004), and having sufficient information when comparing termination options (p = 0.002). After delivery, only 4% would have changed their decision. Conclusions: Individuals, regardless of their characteristics, prefer genome-wide cfDNA; however, the complexity of the results necessitates enhanced genetic education for prenatal care clinicians.