Lysoplex: An efficient toolkit to detect DNA sequence variations in the autophagy-lysosomal pathway

Abstract

The autophagy-lysosomal pathway (ALP) regulates cell homeostasis and plays a crucial role in human diseases, such as lysosomal storage disorders (LSDs) and common neurodegenerative diseases. Therefore, the identification of DNA sequence variations in genes involved in this pathway and their association with human diseases would have a significant impact on health. To this aim, we developed Lysoplex, a targeted next-generation sequencing (NGS) approach, which allowed us to obtain a uniform and accurate coding sequence coverage of a comprehensive set of 891 genes involved in lysosomal, endocytic, and autophagic pathways. Lysoplex was successfully validated on 14 different types of LSDs and then used to analyze 48 mutation-unknown patients with a clinical phenotype of neuronal ceroid lipofuscinosis (NCL), a genetically heterogeneous subtype of LSD. Lysoplex allowed us to identify pathogenic mutations in 67% of patients, most of whom had been unsuccessfully analyzed by several sequencing approaches. In addition, in 3 patients, we found potential disease-causing variants in novel NCL candidate genes. We then compared the variant detection power of Lysoplex with data derived from public whole exome sequencing (WES) efforts. On average, a 50% higher number of validated amino acid changes and truncating variations per gene were identified. Overall, we identified 61 truncating sequence variations and 488 missense variations with a high probability to cause loss of function in a total of 316 genes. Interestingly, some loss-of-function variations of genes involved in the ALP pathway were found in homozygosity in the normal population, suggesting that their role is not essential. Thus, Lysoplex provided a comprehensive catalog of sequence variants in ALP genes and allows the assessment of their relevance in cell biology as well as their contribution to human disease.

Keywords: ALP, autophagy-lysosomal pathway; LSDs, lysosomal storage disorders; NCL, neuronal ceroid lipofuscinosis; NGS, next-generation sequencing; WES, whole exome sequencing; WGS, whole genome sequencing.; autophagy; genetic variants; lysosomal storage disorders; neuronal ceroid lipofuscinoses; next-generation sequencing.

Figure 1.

Subsets of genes that are part of the ALP gene list. Schematic diagram showing the composition of the Lysoplex gene list (n = 891 genes). Please note that some of the selected genes belong to more than one category.

Figure 2.

Performance of the Lysoplex toolkit. (A) Average yield of the Lysoplex capture. More than 92% of sequence reads truly map on ALP targets, indicating a very limited off-targeting in comparison with other methods. (B) Comparison between Lysoplex and WES. Comparison of the coverage distribution on all the 891 ALP genes between whole exome sequencing (blue boxes) and Lysoplex (red boxes). (C, D) Comparison of sequence coverage between WES and Lysoplex in 2 disease genes, namely CLN3 (C) and MTMR2 (D). Red boxes, Lysoplex; blue boxes, WES. Bold lines represent the median of all samples tested, the boxes include 50% of the values and the whiskers represent minimum and maximum values, excluding the outliers that are plotted individually.

Figure 3.

Success rate of Lysoplex in NCL patients. Schematic diagram summarizing the results of the Lysoplex procedure in the NCL patients analyzed. An overall detection rate of 67% was observed. In particular, mutations were identified in 8 NCL known genes. In one case, we found pathogenic mutations in the GLB1 gene (see text for details).

Figure 4.

Comparison between the number of sequence variations found by Lysoplex and WES. Plotted data indicate the log of the number of variations/kb found in each gene by Lysoplex (Y-axis) and by public database (X-axis). If the sensitivity was identical, dots should tend to align. Dots above the line indicate a superior sensitivity of Lysoplex. Red dots indicate NCL genes and blue dots other disease genes. (A) synonymous variations; (B) missense variations; (C) loss-of-function variations.