Screening for Tay-Sachs disease carriers by full-exon sequencing with novel variant interpretation outperforms enzyme testing in a pan-ethnic cohort

Abstract

Background: Pathogenic variants in HEXA that impair β-hexosaminidase A (Hex A) enzyme activity cause Tay-Sachs Disease (TSD), a severe autosomal-recessive neurodegenerative disorder. Hex A enzyme analysis demonstrates near-zero activity in patients affected with TSD and can also identify carriers, whose single functional copy of HEXA results in reduced enzyme activity relative to noncarriers. Although enzyme testing has been optimized and widely used for carrier screening in Ashkenazi Jewish (AJ) individuals, it has unproven sensitivity and specificity in a pan-ethnic population. The ability to detect HEXA variants via DNA analysis has evolved from limited targeting of a few ethnicity-specific variants to next-generation sequencing (NGS) of the entire coding region coupled with interpretation of any discovered novel variants.

Methods: We combined results of enzyme testing, retrospective computational analysis, and variant reclassification to estimate the respective clinical performance of TSD screening via enzyme analysis and NGS. We maximized NGS accuracy by reclassifying variants of uncertain significance and compared to the maximum performance of enzyme analysis estimated by calculating ethnicity-specific frequencies of variants known to yield false-positive or false-negative enzyme results (e.g., pseudodeficiency and B1 alleles).

Results: In both AJ and non-AJ populations, the estimated clinical sensitivity, specificity, and positive predictive value were higher by NGS than by enzyme testing. The differences were significant for all comparisons except for AJ clinical sensitivity, where NGS exceeded enzyme testing, but not significantly.

Conclusions: Our results suggest that performance of an NGS-based TSD carrier screen that interrogates the entire coding region and employs novel variant interpretation exceeds that of Hex A enzyme testing, warranting a reconsideration of existing guidelines.

Keywords: HEXA enzyme testing; Tay-Sachs disease; VUS reclassification; carrier screening; variant interpretation.

Figure 1

Sources of false positives and false negatives in Hex A enzyme‐based and HEXA NGS‐based carrier screening. Enzyme screening can yield false results due to statistical outliers (e.g., an impaired enzyme that randomly happens to yield activity above the assay threshold when tested) or well‐established variants (e.g., pseudodeficiency and B1 alleles) that are incompatible with the assay. NGS may produce false results due to incorrect variant classifications or analytical errors. Not depicted are additional potential sources of false screening results such as inconsistencies in enzyme level reference ranges across non‐AJ ethnicities and unidentified biologic factors. Enzyme activity levels near the cutoff are sometimes reported as “inconclusive” due to the statistical ambiguity. Abbreviations: FP, false positive; FN, false negative; NGS, next‐generation sequencing; VUS, variant of uncertain significance

Figure 2

Two approaches to HEXA VUS reclassification. Study workflow demonstrating two approaches to variant reclassification. (a) Hex A enzyme analysis was performed in 29 individuals, each carrying one of six selected HEXA variants classified as a VUS at the time of testing. Enzyme results were used as functional evidence to satisfy the BS3 criterion in ACMG‐AMP guidelines during reclassification (Richards et al., 2015). (b) All HEXA variants in the Myriad Women's Health database were reevaluated. A standardized set of classification rules was applied to all variants, prompting some downgrades from VUS to likely benign. The remainder of VUSs were tabulated, and 40 VUSs were manually reevaluated in ethnicities with the highest VUS rates. Abbreviations: ACMG‐AMP, American College of Medical Genetics and Genomics‐Association for Molecular Pathology; AA, African American; EA, East Asian; SA, South Asian; SEA, Southeast Asian; VUS, variant of uncertain significance. †Variants shown in Table 2. ‡Classification framework described previously (Beauchamp et al., 2017)

Figure 3

Clinical test performance of sequencing‐based HEXA carrier screening matches or exceeds that of enzyme‐based Hex A screening. (a) Population‐specific HEXA carrier rate estimated from NGS data. (b) The clinical sensitivity, specificity and positive predictive values of sequencing‐ and enzyme‐based HEXA carrier screening. The sensitivity and specificity of sequencing‐based carrier screening is reduced by potentially incorrect variant classifications, while enzyme‐based carrier screening has false negatives due to the B1 allele and false positives due to pseudodeficiency alleles (see Methods). Abbreviations: NS, not significant; PPV, positive predictive value. Error bars indicate 95% CI. Asterisk (*) indicates statistical significance (p < 0.05, see Methods)