A rare splice-site variant in TNNT2: the need for ancestral diversity in genomic reference data sets

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Inherited cardiomyopathies, including hypertrophic (HCM), dilated (DCM), and restrictive (RCM) cardiomyopathies affect ∼1 in 200–500 in the population. Genetic testing is standard practice and typically identifies causal variants in approximately half of cases. Cardiac troponin T (TNNT2) is an integral protein in the cardiac sarcomere and is definitively associated with HCM and DCM. The underrepresentation of diverse ancestries in genomic data sets complicates variant interpretation, especially for non-European populations, where genetic testing has a lower diagnostic yield.1 Among rare monogenic diseases, allele frequency in population reference databases contributes to understanding whether a variant is potentially pathogenic. While global efforts like gnomAD v4.0 have improved ancestral diversity, individuals with Oceanian ancestry, i.e. a geographical region on the Pacific Ocean comprising Australasia, Melanesia, Micronesia, and Polynesia, remain underrepresented. We report a variant, TNNT2; NM_001001430.3: c.571-1G>A (rs483352835), in two unrelated probands with Oceanian ancestry and cardiac phenotypes, seen at a specialized genetic heart disease clinic in Sydney, Australia, who consented to research-based whole-genome sequencing (RPA Hospital Sydney Local Health District Human Research Ethics Committee X15-0089). Variant classification was performed by research genetic counsellors using the MYH7-modified ACMG/AMP criteria and discussed at a dedicated cardiac genetic multidisciplinary team meeting. There were 16 ClinVar entries for this variant (ClinVar variation ID: 132940), updated between December 2014 and January 2024. Fourteen entries classify this variant as being of uncertain significance due to haploinsufficiency not being established as a disease mechanism for TNNT2. One proposed this variant as likely pathogenic in 2010, primarily due to the absence of TNNT2 splice variants among available controls. The final entry gives no classification. Of the 16 submitters, 13 are diagnostic genetic laboratories. Data were collected from ClinVar, literature, and specialized cardiovascular centres, identifying 26 unrelated probands with the variant (excluding our two cases). Of these, 24 had a known cardiac phenotype: 15 with HCM (2 SCD), 5 with DCM (2 diagnosed before age 3), 3 with sudden unexplained death, and 1 with premature ventricular contractions and syncope. Four probands had family histories of SCD. In one HCM case, co-segregation with disease was reported in affected relatives. Notably, 15% of probands had additional pathogenic variants explaining their cardiac phenotype. Ancestry was known for 22 probands, 21 of whom had Oceanian ancestry, including Māori, Samoan, Tongan, Polynesian, Pacific Islander, Aboriginal Australian, Torres Strait Islander, and Hawaiian backgrounds (Figure 1A). (A) Probands with the TNNT2 c.571-1G>A (rs483352835) splice-site variant by self-reported ancestry and disease phenotype. (Bi) NCBI RefSeq transcripts for the TNNT2 gene. Exons are numbered as per NM_001001430.3. (Bii) SpliceAI predicts weakening of the canonical acceptor splice site in Exon 12 and strengthening of a cryptic acceptor splice site three nucleotides downstream, resulting in deletion of Gln191. There is no significant change in SpliceAI score for the Exon 12 canonical donor splice site. (Biii) The cryptic acceptor is the annotated acceptor splice site for NM_001001432.3. (C) Frequency of variant TNNT2; NM_001001430.2: c.571-1G>A (rs483352835) in genomic reference databases. (D) Geographical Map of Oceania (Polynesia, Micronesia, and Melanesia) with population-specific TNNT2 c.571-1G>A (rs483352835) allele frequencies. This work is licensed under Attribution-ShareAlike 3.0 Unported (CC BY-SA 3.0). The original can be found at: https://commons.wikimedia.org/wiki/File:Oceania_UN_Geoscheme_-_Map_with_Zones.svg#/media/File:Oceania_UN_Geoscheme_Regions. HCM, hypertrophic cardiomyopathy; DCM, dilated cardiomyopathy; RCM, restrictive cardiomyopathy; SUD, sudden unexplained death; Fhx, family history; SCD, sudden cardiac death The TNNT2 c.571-1G > A (rs483352835) variant disrupts the canonical splice site of Exon 12 (NM_001001430.3), an alternatively spliced exon with just three amino acids expressed in 7/13 TNNT2 transcripts2 (Figure 1Bi). SpliceAI predicts it abolishes the canonical acceptor site and strengthens a cryptic acceptor downstream (Figure 1Bii). RNA studies confirm activation of this cryptic acceptor, resulting in an in-frame deletion of one amino acid, p.Gln191del (Figure 1Biii). This cryptic site is present in 25% of RNA sequencing samples from SpliceVault. Reduced expression of Exon 12, supported by proportion expressed across transcripts (pext) scores, suggests alternative splicing. The weak conservation of the three encoded amino acids and the absence of pathogenic missense variants in ClinVar indicate that p.Gln191del is a common, tolerated event. The c.571-1G>A variant likely increases the proportion of transcripts with p.Gln191del, with minimal impact on TNNT2 function. The TNNT2 c.571-1G>A (rs483352835) variant appears in gnomAD v4.0.0 at a frequency of 13/780 762 alleles (0.002%), with the highest frequencies in South Asian (0.007%) and East Asian (0.002%) populations (Figure 1C).3 In the Genome Asia 100K Project, the variant is present in 6/148 (4.1%) Oceanian chromosomes, with a higher frequency in Papuans and two Australian participants (50%, one homozygote).4 In All of Us (n = 245 388), the variant was found in fewer than 20 individuals, 59% of whom identified as Native Hawaiian or Other Pacific Islander, none with cardiomyopathy. In the UK Biobank (n = 46 983), one South-East Asian participant had the variant but no cardiomyopathy. We also contacted research groups with ancestry-matched participants and sequencing data unavailable in the public domain. The variant was absent in the Taiwanese Biobank (0/3032; 0%), which primarily consists of Han Chinese individuals and excludes indigenous Taiwanese (n = 1517).5 Within low-coverage genomes and single nucleotide polymorphism arrays with imputation from the Pacific Islands Rheumatic Heart Disease Genetics Network,6 the variant was present at a frequency of 0.088 (8.8%) within the Polynesian sub-group and 0.035 (3.5%) across the wider group, including Melanesians and South Asians. In a genome sequencing data set (low-pass sequencing followed by imputation),7 of participants of Aotearoa New Zealand Māori and Pacific Islander ancestry recruited to a Health and Disability study (MEC/05/10/130), the variant was present at an allele frequency of 4.0% in East Polynesian individuals (n = 139), and 3.6% among West Polynesian individuals (n = 55; Figure 1C). Indigenous Papuan and Australian people derive >3% of their DNA from Neanderthal ancestry, a higher percentage than for Eurasian populations.8 TNNT2:c.571-1G>A (rs483352835) was shown to be present in two archaic genomes (Vindija and Altai Neanderthal), but not the Altai Denisovan,8–10 suggesting it might have arisen ≥130–145 thousand years ago after the Neanderthal populations diverged from modern humans. This likely explains the increased frequency of TNNT2:c.571-1G>A (rs483352835) in Oceanian populations. According to the ACMG/AMP guidelines, a minor allele frequency >0.05 for an autosomal dominant gene can be considered stand-alone evidence of benign impact (BA1 criterion). Considering the allele frequencies among individuals with Oceanian ancestry, we applied the BA1 criterion and classified TNNT2:c.571-1G>A (rs483352835) as a benign variant, which is predicted to lead to a tolerated in-frame deletion of a single amino acid. We highlight the challenges of interpreting ‘rare’ monogenic variants among poorly represented ancestry groups. Our analysis of TNNT2:c.571-1G>A (rs483352835), a common single nucleotide variant within Oceanian populations, underscores the critical need for large, diverse, and openly accessible genomic refer

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