Coding and nonnoncoding variants in the SHOX 2

Coding and non-noncoding variants in the SHOX 2 gene in patients with early-onset of atrial fibrillation Presented by Paa Kwesi Ankrah December 1, 2020 BIOL 6564 – Advanced Genetics, Fall 2020 1

KEY TERMS • ATRIAL FIBRILLAT ION Advanced Genetics, Fall 2020 • SHOX 2 • CODING & NONCODING VARIANTS • MIRNA 2

OUTLINE • Background • Results & Methodology • Summary Advanced Genetics, Fall 2020 3

BACKGROUND ATRIAL FIBRILLATION Irregular, usually fast heart rate. Characterized by disorganized electrical activity of the atria. Causes may include genetic mutations, damage to heart structure, high blood pressure, etc. Early onset – 14 to 60 years. Advanced Genetics, Fall 2020 4

BACKGROUND CONT’D • SHOX 2 (Short-stature Homeobox 2) • Encode proteins with a 60 -amino acid residue that represents a DNAbinding domain • Are transcriptional regulators • Shox 2 activates promoters of the BMP 4 and ISL-1 genes in heart development. • Autosomal: Located on chromosome 3 • Human SHOX 2 has homologs in other mammals: Shox 2 (mouse and zebra fish) Advanced Genetics, Fall 2020 5

CODING AND NON-CODING VARIANTS HOW? ▪ SEQUENCING ▪ GENOTYPING Determine the exact sequence of shox 2 gene in AF patients Compare the Shox 2 sequence of AF patients to healthy individuals ✓ ✓ TAQMAN SNP GENOTYPING ASSAY GATC BIOTECH Advanced Genetics, Fall 2020 6

SHOX 2 GENE IN AF PATIENTS Advanced Genetics, Fall 2020 7

Coding variants: ▪ Two missense mutations • Transition (G >A) in exon 1 • Transversion (C>A) in exon 6 • Found in each patient Non-coding variant: ▪ Transition (T>C) in 3’ UTR Advanced Genetics, Fall 2020 • Found in subgroup of A. F and in healthy. 8

WILDTYPE AND AF SHOX 2 Advanced Genetics, Fall 2020 9

FUNCTIONAL RELEVANCE NON-CODING VARIANT ? Advanced Genetics, Fall 2020 CODING VARIANTS ? 10

NON- CODING VARIANT • Creates a novel binding site in the 3’ UTR for the mi. RNA, hsa-mi. R-92 b-5 p. • Reduced hsa-mi. R-92 b-5 p in circulation. • Individuals with this 3’ UTR variant had prolonged PR intervals, (over 200 ms) and increased susceptibility to A. F • Luciferase Reporter Assay used. Advanced Genetics, Fall 2020 11

PR Interval in A. F. patients Advanced Genetics, Fall 2020 Luciferase Reporter Mechanism 12

CODING VARIANTS ▪ Impact of mutations on SHOX 2: • Transition mutation (G>A) has transactivation activity like WT • Transversion mutation (C>A) lacks transactivation activity ▪ Luciferase reporter assay used Advanced Genetics, Fall 2020 13

CODING VARIANT cont’d Targeted knockdown of zebra fish shox 2 using morpholino-modified antisense oligonucleotides. Shox 2 morphant (MO) causes pericardial edema. Wildtype Shox 2 could rescue Shox 2 morphant but the transversion mutant could not. Advanced Genetics, Fall 2020 14

SHOX 2 EXPRESSION Expression analysis revealed reduced shox 2 transcripts in A. F patients Tissue used was right atrial appendages. Advanced Genetics, Fall 2020 15

SUMMARY ✓Two missense mutations identified in exons 1 and 6 of the coding region • The transversion mutation (C>A) could not transactivate target genes. ✓The 3’ UTR variant had longer PR intervals and increased susceptibility to A. F • This variant creates a functional binding site for hsa-mi. R-92 b-5 p ✓Reduced SHOX 2 expression in right atrial cartilage of AF patients compared to healthy individuals. These results suggest a genetic contribution of SHOX 2 in Atrial 16 Advanced Genetics, Fall 2020 Fibrillation.

FUTURE DIRECTIONS ▪ How does the mi. RNA bind to the 3’ UTR ? Total homology (degradation) or Partial homology (inhibition) ▪ How other transcription factor genes contribute to A. F. development ? ▪ SHOX 2 variants in other diseased organs that are also SHOX 2 Advanced Genetics, Fall 2020 dependent. 17

REFERENCES • Liu H. , Chen C. H. , Espinoza-Lewis R. A. , Jiao Z. , Sheu L. , Hu X. , Lin M. , Zhang Y. , Chen Y. (2011) Functional Redundancy between Human SHOX and Mouse shox 2 Genes in the Regulation of Sinoatrial Node Formation and Pacemaking Function. J biol chem 286: 17029– 17038. Doi: 10. 1074/jbc. M 111. 234252 • Hoffmann S. , Berger I. M. , Glaser A. , Bacon C. , Li L. , Gretz N. , Steinbeisser H. , Rottbauer W. , Just S. , Rappold G. (2013) Islet 1 is a Direct Transcriptional Target of the Homeodomain Transcription Factor shox 2 and rescues the shox 2 -mediated bradycardia. Basic Res cardiol 108: 339. Doi: 10. 1007/s 00395 -013 -0339 z • Cobb, J. , Dierich, A. , Huss-garcia, Y. , & Duboule, D. (2006). A mouse model for human shortstature syndromes identifies shox 2 as an upstream regulator of runx 2 during long-bone development. Proceedings of the national academy of sciences, 103(12), 4511 -4515. • Hoffmann, S. , Clauss, S. , Berger, I. M. , Weiß, B. , Montalbano, A. , Röth, R. , . . . & Schulze-bahr, e. (2016). Coding and non-coding variants in the SHOX 2 gene in patients with early-onset atrial Advanced Genetics, Fall 2020 18 fibrillation. Basic research in cardiology, 111(3), 36.