Functional noncoding variants within the TBX1 enhancer contribute to tetralogy of Fallot.

Complex Genomic Rearrangement Involving the TBX4 Promoter Manifesting With Variable Expressivity in a Five‐Generation Family

It is proposed that one or more of the variants within the TBX4 lung‐specific super‐enhancer or TAD may act in trans with the pathogenic CGR, modulating TBX4 expression from the intact allele.

Unravelling the role of IRX4 variants in non-syndromic and Down syndrome associated congenital heart disease

EP1343 - ECE_3558 - Rare 5′-UTR variants creating new upstream open reading frames provide a potential novel genetic mechanism in congenital hypogonadotropic hypogonadism

Congenital hypogonadotropic hypogonadism (CHH) is a genetically heterogeneous disorder with numerous genes implicated in its pathogenesis. To date, most studies have focused on the coding regions of the genome, leaving ~50% of the CHH genetically unsolved. While the impact of non-coding regions in disease pathogenesis has recently emerged, the role of non-coding variants affecting upstream open reading frames (uORFs) within the 5′ untranslated regions (5′-UTRs), which are known to suppress downstream translation, has not been thoroughly studied. In this study, we aimed: (i) to assess whether patients with CHH are enriched for rare 5′-UTR variants creating new uORFs, and thus having the potential to suppress downstream translation of known CHH genes compared to reproductively normal controls, (ii) to phenotypically characterize CHH individuals harboring such 5′-UTR variants. Whole exome sequencing data of 1403 CHH subjects and 7999 reproductively normal controls from the Mass General Brigham Biobank (MGBB) was analyzed using the GATK pipeline to identify rare 5′UTR variants [minor allele frequency (MAF) <0.1% in autosomal dominant genes and <1% in autosomal and X-linked recessive genes] in 82 known CHH genes. The variants' functional effect was assessed using the UTRannotator, a VEP plugin that identifies variants disrupting existing or creating new uORFs. We detected 50 5′-UTR variants (MAF<1%) with a predicted effect in 30 genes. Such variants were more common in CHH vs controls (1.3% vs 0.7%, P = .034). Variants creating new uORFs were also more common in CHH probands (CHH 0.8%—Controls 0.3%, P = .0159), and this pertained to autosomal dominant genes (P = .06) but not to autosomal and X-linked recessive (P = .114). Variants disrupting existing uORFs were equally prevalent in CHH and Controls (0.5% vs 0.4%, P = ns). Of the 11 CHH probands harboring variants creating new uORFs 6 were males and 5 females; 7 had anosmia (Kallmann syndrome) and 4 normosmic CHH; and 4 had additional non-reproductive characteristics (eg, clinodactyly, short metacarpal, learning disability/speech impairment, amblyopia, ptosis, strabismus). CHH cases are enriched for rare 5′UTR variants that create new uORFs in autosomal dominant genes compared to controls, underscoring the emerging role of non-coding variants in the genetic background of CHH.

P461 - LBA_ECE_1060 - Functional characterization of a non-canonical SDHD splice-site variant (c.314+4A > G) in a family with thyroid and carotid paragangliomas

Thyroid paragangliomas (TPGLs) are exceptionally rare (<0.1% of thyroid neoplasms, ~0.5% of all paragangliomas) and frequently occur within hereditary head and neck paraganglioma syndromes. While hereditary paragangliomas are frequently linked to SDHD germline variants, intronic splice-site alterations are scarcely reported in TPGLs. We describe the clinical and functional characterization of a TPGL associated with a novel intronic SDHD variant. We investigated a family of a 36-year-old woman with intrathyroidal TPGL and bilateral carotid body paragangliomas in her father and brother, all carrying a heterozygous intronic SDHD variant, c.314+4A > G (NM_003002.5). Germline testing was performed by next-generation sequencing, followed by in-depth RNA sequencing on whole blood to assess splicing and allele-specific expression. RNA-seq libraries were prepared with a directional protocol and sequenced on an Illumina NextSeq 2000 (~80 M 150-bp paired-end reads/sample), aligned with HISAT2, quantified with HTSeq, and analyzed with DESeq2; splice junctions were visualized by sashimi plots in IGV. The SDHD c.314+4A > G variant lies within the intron 3 donor site and is absent from gnomAD and the Leiden University Medical Center SDHD gene variant database; SpliceAI predicts splice disruption (Δ score 0.81). RNA-seq demonstrated two competing exon 3→4 junctions in both carriers: a canonical junction from the wild-type allele and a cryptic junction 4 bp downstream, attributable to the mutant allele. In the proband and her brother, aberrant junctions accounted for approximately half of all exon 3→4 splice events, consistent with a “leaky” splice-site defect and predicted frameshift with premature termination. Despite sharing the same variant, overall SDHD expression was lower in the proband than in her brother, consistent with allele-specific transcript imbalance and possible haploinsufficiency. Co-segregation of c.314+4A > G with thyroid and bilateral carotid body paragangliomas across three paternally related relatives supports its role in a hereditary SDHD-associated paraganglioma syndrome. This study provides direct RNA-level evidence that the intronic SDHD c.314+4A > G variant causes aberrant splicing and supports reclassification from VUS to likely pathogenic under ACMG/AMP criteria (PS3, PM2, PP3, segregation). These findings expand the spectrum of splice-disrupting SDHD c.314 variants implicated in hereditary head and neck paragangliomas, highlight TPGL as part of this spectrum, and underscore the value of RNA-based assays in interpreting non-canonical splice-site changes.

Whole-genome resequencing with multidimensional annotation revealed pathogenic networks in sirenomelia

The findings support a multilayered pathogenic model in which genetic variants collectively disrupt the core developmental pathways involved in posterior axis formation, vasculogenesis, and organogenesis and identify potential targets for future research and clinical applications.

Molecular Characterization of two NR2F2 Exons identifies a Rare Intronic Variant Enriched in Indonesian Congenital Heart Disease Patients

Background: Congenital heart disease is a globally prevalent congenital disorder that has a complicated genetic etiology that includes both coding and non-coding variants. Although there is much evidence of pathogenic coding variants in key transcription factors, the role of regulatory variants in the Indonesian population is less known. The NR2F2 plays an essential role in cardiac development. To determine potentially pathogenic variation, we performed a case-control genetic study in Indonesian patients with CHD to find variants in the NR2F2 gene.Methods: 27 ethnically matched controls and 35 CHD patients were examined, and genomic DNA was extracted from peripheral blood samples. Following targeted Sanger sequencing to identify potential pathogenic variants in exons 2 and 3 of the NR2F2 gene, extensive bioinformatic annotation was carried out using genomic visualization platforms.Results: This study identified a rare intronic variation of the NR2F2 gene, rs753513812 (c.970+11G>C; g.8677G>C), in eight of 35 CHD patients, and it was absent in all control individuals and the majority of CHD subtypes. The variation was significantly enriched in CHD patients. This finding suggests that CHD susceptibility may play a role through non coding regulatory variations. Among the 35 patients examined, no pathogenic or likely pathogenic variants were detected in exons 2 and 3 of the NR2F2 gene.Conclusion: These findings imply that pathogenic coding variants within these exons are unlikely to be a frequent genetic cause of CHD in Indonesian patients. Also, the study highlights the need for population-specific genetic investigations and their contribution to human cardiac development.