Martina Zobel’s research while affiliated with National Institute of Molecular Genetics (INGM) and other places

The accurate characterization of triplet repeats, especially the overrepresented CAG repeats, is increasingly relevant for several reasons. First, germline expansion of CAG repeats above a gene-specific threshold causes multiple neurodegenerative disorders; for instance, Huntington’s disease (HD) is triggered by >36 CAG repeats in the huntingtin (HTT) gene. Second, extreme expansions up to 800 CAG repeats have been found in specific cell types affected by the disease. Third, synonymous single nucleotide variants within the CAG repeat stretch influence the age of disease onset. Thus, new sequencing-based protocols that profile both the length and the exact nucleotide sequence of triplet repeats are crucial. Various strategies to enrich the target gene over the background, along with sequencing platforms and bioinformatic pipelines, are under development. This review discusses the concepts, challenges, and methodological opportunities for analyzing triplet repeats, using HD as a case study. Starting with traditional approaches, we will explore how sequencing-based methods have evolved to meet increasing scientific demands. We will also highlight experimental and bioinformatic challenges, aiming to provide a guide for accurate triplet repeat characterization for diagnostic and therapeutic purposes.

Trinucleotide repeats in DNA exhibit a dual nature due to their inherent instability. While their rapid expansion can diversify gene expression during evolution, exceeding a certain threshold can lead to diseases such as Huntington’s disease (HD), a neurodegenerative condition, triggered by >36 C–A–G repeats in exon 1 of the Huntingtin gene. Notably, the discovery of somatic instability (SI) of the tract allows these mutations, inherited from an affected parent, to further expand throughout the patient’s lifetime, resulting in a mosaic brain with specific neurons exhibiting variable and often extreme CAG lengths, ultimately leading to their death. Genome-wide association studies have identified genetic variants—both cis and trans, including mismatch repair modifiers—that modulate SI, as shown in blood cells, and influence HD’s age of onset. This review will explore the evidence for SI in HD and its role in disease pathogenesis, as well as the therapeutic implications of these findings. We conclude by emphasizing the urgent need for reliable methods to quantify SI for diagnostic and prognostic purposes.

Background It is well known that HD patients with similar CAG length show a wide range of variability in motor onset that can account for up to two decades. One possible explanation resides in the fact that the inherited CAG repeats may expand in somatic tissues, especially in post-mitotic neurons, giving rise to a HTT mosaicism that results in longer than inherited CAG tracts in affected tissues, such as the striatum and cortex. This expansion may continue during the lifetime of the individual and contribute to exacerbate neuronal toxicity and selective neuronal degeneration. More recently, trans- and cis- modifiers of age of onset (AOO) have been identified. However, if and how they cause the progressive accumulation of CAG instability is still unclear. Aim To identify new cis and trans modifiers of CAG instability, we aimed to establish an isogenic human stem cell platform that, combined with third generation long-read sequencing, allows to monitor HTT CAG size over time, both during mitotic cell replication and in post-mitotic neurons. Methods Starting from H9 human embryonic stem cell (hES) line, we inserted a monoallelic Recombinant Mediated Exchange Cassette within HTT exon 1, which can be subsequently exchanged with any exon1 variant in an efficient way. We generated a wide variety of exon 1 modified cell lines, which we refer to as the CAGinSTEM platform. Results Our data show that the CAGinSTEM platform is technically robust as for each genotype we have multiple cell lines which have been quality checked. By exploiting the properties of the CAGinSTEM platform, we are testing how CAG length and composition impact on CAG instability in terminally differentiated medium spiny neurons and in active proliferating hES cells. Conclusions The CAGinSTEM platform offers a distinctive biological model system designed to explore genotype-phenotype correlations and investigate the mechanisms underlying CAG instability accumulation in postmitotic human neurons and other cell types.Funded by an ERC Advanced Grant from the European Commission.

Background Huntington’s disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the gene encoding for the huntingtin protein. Recently, Genome Wide Association Studies (GWAS) have identified genomic variations, occurring both at the HTT locus and in genes mostly involved in mismatch repair pathways, to be associated with disease onset and progression. Such mutations correlate with modulations of somatic instability, which is now considered as the main driver of pathogenesis. Accordingly, the set-up of reliable sequencing-based methods for assessing somatic instability is of paramount importance. Aims In this work, we aimed to compare workflows for assessing somatic instability, by identifying strengths and drawbacks of each enrichment, sequencing and analysis method. Methods Based on research studies focusing on Short Tandem Repeats (STR) characterization available in the literature, we compared multiple enrichment methods coupled to various sequencing platforms and data analysis tools. We then performed a preliminary comparison on internal data, and discussed their accuracy in CAG sizing and sensitivity for rare alleles detection. Results We reported much higher enrichment for PCR-based methods, compared to CRISPR/Cas9 enrichment and adaptive sampling. Moreover, unlike long-read sequencing platforms, we described a decrease in sequencing quality at cycles above 300 for Illumina MiSeq. As a last point, we showed a preliminary comparison among sequencing-based methods on internal data, showing the impact of each step towards the most accurate STR characterization. Conclusions We anticipate that long-read sequencing of PCR amplicons incorporating UMIs may represent a valid alternative to PCR-free enrichment methods, providing highly accurate performances in terms of repeat length and rare alleles detection. Nonetheless, we recommend accurate optimization of PCR primers and conditions, as suboptimal number of PCR cycles may result in off-target products and inadequate number of PCR duplicates.