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Investigating the Relationship Between LUNATIC FRINGE Variants and Spondylocostal Dysostosis Type-III
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- Author / Creator
- Wengryn, Parker C
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The vertebral column’s bilateral symmetry plays a crucial role in respiration, ambulation, and weight bearing. Scoliosis, a lateral curvature of the spine of >10°, disrupts this anatomy and can lead to pain, poor quality of life, and in severe cases, mortality. The prevalence of scoliosis is estimated to be as high as 1-in-33, and treatment ranges from physiotherapy and structural bracing to corrective surgery. In most cases, early diagnosis and proactive care are key to limiting curve progression and minimizing treatment invasiveness. However, the underlying cause of most scoliosis cases is unknown, and screening may not occur until the patient is symptomatic. The study of monogenic causes of scoliosis can lead to the identification of new diagnostic targets within the affected cellular pathways. The NOTCH signalling pathway is critical for somite (i.e. embryonic vertebral precursor) formation and is implicated in many diseases that cause scoliosis. Spondylocostal Dysostosis Type 3 (SCD3) is caused by pathogenic variants in the NOTCH pathway gene Lunatic Fringe (LFNG). LFNG is a Golgi-resident β-1,3-N-Acetylglycosaminyltransferase that glycosylates NOTCH receptors to regulate the size, shape, and symmetry of somites and thus vertebral anatomy. This monogenic condition is characterized by abnormal anatomy of the vertebrae and ribs which leads to short stature, various extents of scoliosis, and restrictive lung disease. Very few cases have been identified, and little is known about how each causative variant affects LFNG function; however, case-to-case phenotypic variability may hint at a genotype-phenotype relationship.
In hopes of determining whether there is a relationship between SCD3 phenotype and LFNG variant, the first goal of this thesis was to investigate the functional consequences of two novel LFNG variants (c.521G>A [p.R174H]; c.766G>A [p.G256S]) presenting in trans in a proband with SCD phenotype with mild scoliosis, but not clinically defined short stature nor restrictive lung disease. I assessed both variants for impaired glycosyltransferase activity, subcellular mislocalization, and aberrant protein processing. Our results indicate that the p.G256S variant is enzymatically non-functional, while the p.R174H variant is functionally less effective. Both variants were not different from wildtype (WT) in localization and processing. Our findings suggest that the hypomorphic variant (p.R174H) may have partially improved the patient's stature, as evidenced by a lower arm span-to-height ratio, increased height, and more vertebrae. However, this variant did not appear to have any effect on the severity of vertebral malformations, including scoliosis. The lack of molecular characterization studies for LFNG variants of other SCD3 probands hinders the generalizability of this conclusion.
To address this lack of functional testing, nine LFNG missense variants associated with SCD3 published before 2023, c.564C>A, c.583T>C, c.842C>A, c.467T>G, c.856C>T, c.601G>A, c.446C>T, c.521G>A, and c.766G>A, were assessed in vitro for subcellular localization and protein processing. Glycosyltransferase activity was quantified for the first time in the c.583T>C, c.842C>A, and c.446C>T variants. Primarily, this work indicates that all variants that prevent Golgi localization also impair protein processing, suggesting the two methods evaluate LFNG trafficking from different perspectives. It appears that the FRINGE domain is responsible for aberrant trafficking. Secondly, our data suggests that variant proximity to the catalytic residue may influence whether LFNG is improperly trafficked and/or enzymatically dysfunctional. Finally, the phenotype of the axial skeleton, but not elsewhere, may be modulated in a variant-specific fashion, supporting our previous work. More reports are needed to continue testing this hypothesis. We anticipate our data will be used as a basis for the discussion of genotype-phenotype correlations in SCD3.
In sum, this work led to the discovery of the first two disease-implicated LFNG alleles with partial enzyme activity, the first LFNG allele to partially traffic to the Golgi and contributed to determining the effect of nine LFNG variants on glycosyltransferase activity, processing, and localization. With these discoveries, this work has provided support for a loci-dependant pattern of LFNG functional perturbation, a clearer perspective of LFNG trafficking experiments, and associated the larger vertebral number of an SCD3 proband with the only hypomorphic LFNG allele known to date. The vertebral number of SCD3 probands is likely modulated in a variant-specific fashion, and thus there does appear to be a genotype-phenotype relationship in SCD3. -
- Graduation date
- Fall 2024
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- Type of Item
- Thesis
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- Degree
- Master of Science
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- License
- This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.