Functional Assays Are Essential for Interpretation of Missense Variants Associated with Variable Expressivity

Raraigh, Karen S.; Han, Sejin; Davis, Emily; Evans, Taylor A.; Pellicore, Matthew J.; McCague, Allison F.; Joynt, Anya T.; Lu, Zhongzhou; Atalar, Melis; Sharma, Neeraj; Sheridan, Molly B.; Sosnay, Patrick R.; Cutting, Garry R.

doi:10.1016/j.ajhg.2018.04.003

Cited by 75 publications

(85 citation statements)

References 66 publications

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“…The above notwithstanding, to date, the actual scale of canonical 5′SSs capable of generating wild‐type transcripts in the case of GT>GC substitutions, both in the context of the frequency of such substitutions and the level of wild‐type transcripts generated by such substitutions, remains unknown owing to the intrinsic complexity of splicing (Boehm et al, ; De Conti, Baralle, & Buratti, ; Krainer, ; Wong et al, ; Zhang, Arias, Ke, & Chasin, ) and the lack of suitable model systems for study. This issue has important implications for medical genetics as mutant genotypes retaining even a small fraction of their normal function may differ significantly from null genotypes in terms of their associated clinical phenotypes (e.g., 5% normal CFTR gene expression is sufficient to prevent the lung manifestations of cystic fibrosis [Ramalho et al, ; Raraigh et al, ]; for hemophilia B and other coagulation factor deficiencies, raising plasma levels above 5% normal often results in milder bleeding phenotypes [Den Uijl et al, ; Scalet et al, ]). Herein, we attempted to address this issue by employing two distinct but complementary approaches in concert.…”

Section: Introductionmentioning

confidence: 99%

First estimate of the scale of canonical 5′ splice site GT>GC variants capable of generating wild‐type transcripts

et al. 2019

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It has long been known that canonical 5′ splice site (5′SS) GT>GC variants may be compatible with normal splicing. However, to date, the actual scale of canonical 5′SSs capable of generating wild‐type transcripts in the case of GT>GC substitutions remains unknown. Herein, combining data derived from a meta‐analysis of 45 human disease‐causing 5′SS GT>GC variants and a cell culture‐based full‐length gene splicing assay of 103 5′SS GT>GC substitutions, we estimate that ~15–18% of canonical GT 5′SSs retain their capacity to generate between 1% and 84% normal transcripts when GT is substituted by GC. We further demonstrate that the canonical 5′SSs in which substitution of GT by GC‐generated normal transcripts exhibit stronger complementarity to the 5′ end of U1 snRNA than those sites whose substitutions of GT by GC did not lead to the generation of normal transcripts. We also observed a correlation between the generation of wild‐type transcripts and a milder than expected clinical phenotype but found that none of the available splicing prediction tools were capable of reliably distinguishing 5′SS GT>GC variants that generated wild‐type transcripts from those that did not. Our findings imply that 5′SS GT>GC variants in human disease genes may not invariably be pathogenic.

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Section: Introductionmentioning

confidence: 99%

First estimate of the scale of canonical 5′ splice site GT>GC variants capable of generating wild‐type transcripts

et al. 2019

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“…In silico prediction algorithms have been used to identify likely disease‐contributing CFTR variants, but the utility of predictive algorithms is controversial as they cannot differentiate between variants that caused severe, moderate, or minimal reduction in CFTR function . Our study similarly found inconsistent predictions as the algorithms predicted five variants to be deleterious but were annotated by CFTR2 as “non‐CF‐causing” (Table S1).…”

Section: Discussionmentioning

confidence: 81%

Identification of CFTR variants in Latino patients with cystic fibrosis from the Dominican Republic and Puerto Rico

Zeiger

McGarry

Mak

et al. 2019

Pediatric Pulmonology

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Background In cystic fibrosis (CF), the spectrum and frequency of CFTR variants differ by geography and race/ethnicity. CFTR variants in White patients are well‐described compared with Latino patients. No studies of CFTR variants have been done in patients with CF in the Dominican Republic or Puerto Rico. Methods CFTR was sequenced in 61 Dominican Republican patients and 21 Puerto Rican patients with CF and greater than 60 mmol/L sweat chloride. The spectrum of CFTR variants was identified and the proportion of patients with 0, 1, or 2 CFTR variants identified was determined. The functional effects of identified CFTR variants were investigated using clinical annotation databases and computational prediction tools. Results Our study found 10% of Dominican patients had two CFTR variants identified compared with 81% of Puerto Rican patients. No CFTR variants were identified in 69% of Dominican patients and 10% of Puerto Rican patients. In Dominican patients, there were 19 identified CFTR variants, accounting for 25 out of 122 disease alleles (20%). In Puerto Rican patients, there were 16 identified CFTR variants, accounting for 36 out of 42 disease alleles (86%) in Puerto Rican patients. Thirty CFTR variants were identified overall. The most frequent variants for Dominican patients were p.Phe508del and p.Ala559Thr and for Puerto Rican patients were p.Phe508del, p.Arg1066Cys, p.Arg334Trp, and p.I507del. Conclusions In this first description of the CFTR variants in patients with CF from the Dominican Republic and Puerto Rico, there was a low detection rate of two CFTR variants after full sequencing with the majority of patients from the Dominican Republic without identified variants.

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“…Studies in CF pig models have shown that CFTR function, and not the presence of chronic infection, leads to sinus hypoplasia, whereby older CF pigs spontaneously develop sinusitis regardless of infection 32 . Chloride conductance has been shown to accurately reflect CFTR function 20,21 and was used in this study. CFTR function, dichotomized to >1% or ≤1%, correlated with a history of surgical intervention for sinus disease.…”

Section: Discussionmentioning

confidence: 99%

“…CFTR genotype was determined from medical records or through molecular testing at the Johns Hopkins Diagnostic Laboratory. Functionality of CFTR missense or in‐frame deletion variants expected to produce protein was determined in vitro using heterologous expression systems in Fisher rat thyroid (FRT) or cystic fibrosis bronchial epithelial (CFBE) cell lines bearing individual variants 20,21 . Short‐circuit current was measured to assess chloride conductance from mutant CFTR and each variant was subsequently assigned a percentage of wild‐type CFTR function.…”

Section: Methodsmentioning

confidence: 99%

Cystic fibrosis transmembrane conductance regulator function, not TAS2R38 gene haplotypes, predict sinus surgery in children and young adults with cystic fibrosis

Dalesio

Aksit

Ahn

et al. 2020

Int Forum Allergy Rhinol

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Background Chronic rhinosinusitis symptomatology begins in early childhood individuals with cystic fibrosis (CF). Cystic fibrosis transmembrane conductance regulator (CFTR) function contributes to sinus development and disease. Genetic variants of the bitter taste receptor TAS2R38 have been suggested to contribute to sinus disease severity in individuals without CF. Our objective was to explore whether functional TAS2R38 haplotypes and CFTR function are associated with sinus disease or the need for sinus surgery in individuals with CF. Methods We conducted a retrospective study using prospectively collected data from the CF Twin‐Sibling Study. The function of CFTR was assessed via chloride conductance. Genotyping of the TAS2R38 gene identified patients who were homozygous for the functional haplotype, heterozygous, or homozygous for nonfunctional haplotypes. Clustered multivariate logistic regression was performed, controlling for sex and family relationship. Results A total of 1291 patients were evaluated. Patients with ≤1% CFTR function were 1.56 times more likely to require sinus surgery than those with >1% CFTR function (p = 0.049). CFTR function did not correlate significantly with the presence of sinus disease (p = 0.30). In addition, there were no statistically significant differences in diagnosis of sinus disease or need for sinus surgery between patients with functional and nonfunctional TAS2R38 haplotypes. Conclusion CFTR function correlates with need for sinus surgery, whereas TAS2R38 function does not appear to contribute to sinus disease or the need for sinus surgery in patients with CF.

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Functional Assays Are Essential for Interpretation of Missense Variants Associated with Variable Expressivity

Cited by 75 publications

References 66 publications

First estimate of the scale of canonical 5′ splice site GT>GC variants capable of generating wild‐type transcripts

First estimate of the scale of canonical 5′ splice site GT>GC variants capable of generating wild‐type transcripts

Identification of CFTR variants in Latino patients with cystic fibrosis from the Dominican Republic and Puerto Rico

Cystic fibrosis transmembrane conductance regulator function, not TAS2R38 gene haplotypes, predict sinus surgery in children and young adults with cystic fibrosis

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