Uncovering pathogenic variants with the founding president of the Thai Society of Human Genetics

What are the aims of Genomics Thailand?

Genomics Thailand represents a significant national endeavour aiming to conduct whole-genome sequencing of 50,000 Thai individuals between 2020 and 2024. The project's approach, focusing on sequencing patients rather than healthy individuals, was likened by Vorasuk to achieving multiple objectives with a single initiative. This strategy not only facilitates the immediate molecular diagnoses for these patients, enhancing their care and treatment options, but also contributes valuable genetic data that can inform broader research and understanding of genetic diseases within the Thai population.

What are the inclusion criteria for these patients with rare diseases?

This question is frequently posed to him. The inclusion criteria are not based on the disease's frequency, but rather on the analytic method used. If the treating physician suspects that a patient's disease could be attributable to a single germline variant, that patient is included in the study. This process is coordinated at the national level by the Thailand Rare & Undiagnosed Disease Network (T-RUN), which is a collaboration among all the major medical centres across the country, united in their mission to leave no patient behind.

Genomics Thailand began this initiative 5 years ago, what technologies are used, and how has this changed during the time period?

Short-read sequencing, employed in Vorasuk's lab, achieves a diagnostic yield of approximately 25% for rare diseases. This prompts the question: what about the remaining cases? In their experience with germline monogenic disorders, two primary challenges contribute to unresolved cases. First, there are instances where candidate variants are detected but lacks sufficient evidence to be conclusively deemed the cause, leading to their classification as Variants of Uncertain Significance (VUS). Second, certain variants may elude detection altogether via short-read sequencing. It is in these scenarios that long sequencing reads emerge as a pivotal tool, offering the potential to clarify and resolve such ambiguous or elusive cases, and has driven Vorasuk’s team to adopt Oxford Nanopore Technologies.

What are some examples of where Oxford Nanopore Technologies has helped in cases of rare disease?

The decision to transition from exome sequencing to nanopore sequencing was driven by considerations of management and efficiency: short-read exome sequencing necessitated combining 40 samples for a single run, whereas the PromethION device allows for individual sample processing, offering greater flexibility. The switch to nanopore sequencing, as Vorasuk notes, has extended benefits beyond mere logistics.

In a comparative study involving acutely and severely ill patients, both exome and nanopore sequencing were employed. One notable case involved a patient with severe myoclonic epilepsy. Long nanopore reads facilitated the identification of pathogenic variants and their cis or trans configuration without sequencing the parents, a level of detail unattainable with short-read sequencing due to the 20 kb distance between variants. This enhanced resolution bolstered the confidence in diagnosing the patient's recessive disease, impacting the ALDH7A1 gene.

Another case highlighted by Vorasuk involved a patient suffering from hypoglycaemia and metabolic acidosis. While exome sequencing identified a single variant, nanopore sequencing revealed a second, 19 kb deletion in the ETFA gene. This additional insight, coupled with phasing information, confirmed the diagnosis of glutaric acidaemia type IIA, an autosomal recessive disorder. These findings have led to a growing confidence in relying solely on nanopore sequencing for such cases in the future and potentially eliminating the need for parallel exome sequencing.

In a further example, a patient experiencing breathing difficulties was assessed using nanopore sequencing, which uncovered a 2.4 kb deletion that might have been overlooked by exome sequencing. Additionally, a novel SNP was detected on the alternate allele. The identification of the deletion by nanopore sequencing was crucial; without it, the novel variant might have been classified as a variant of uncertain significance (VUS) rather than likely pathogenic. This case ultimately led to a confirmed diagnosis of surfactant deficiency, implicating the ABCA3 gene. So far in this proof of concept, Vorasuk and colleagues are observing a diagnostic rate of 75% using the “Singleton Rapid LR-WGS” for acutely and severely ill patients.

Another clear application of long nanopore reads is for repeat expansion and contraction disorders, which Vorasuk showed in the context of Facioscapulohumeral muscular dystrophy (FSHD).

Vorasuk discussed the complex genetics of FSHD, emphasising its intricate diagnosis that goes beyond clinical, biochemical, or standard diagnostic tests, necessitating a molecular approach. FSHD, the third most common inherited muscle disorder, is primarily caused by the contraction of D4Z4 repeat units (each 3.4 kilobases long), from more than 100 units to no more than 10 units. There are many possible molecular defects which makes FSHD genetics complex and challenging. Defects include: a D4Z4 repeat contraction which affects methylation status, single nucleotide variants in other genes, and the necessity for the polyadenylation signal to be from haplotype A, for RNA stabilisation.

The challenge lies in developing a comprehensive genetic test that can assess the repeat size, identify polyadenylation type, detect mutations in other genes, and determine methylation status in one go. Vorasuk highlighted the breakthrough achieved with nanopore sequencing, which allows for such comprehensive analysis in one assay. Using PromethION, they conducted a study including seven individuals with FSHD, revealing that all individuals had contracted D4Z4 repeat units smaller than 10, with haplotype A, contrasting with controls who either had longer repeat units or haplotype B. This study marked a significant advancement in the molecular characterisation of FSHD using whole-genome analysis.

What should the genomics world be looking forward to?

When asked about the future developments he anticipates in the field of genomics, the professor expressed enthusiasm for the transformative potential outlined in the American Journal of Human Genetics' bold predictions for human genomics by 2030. He highlighted the prospect of genomic testing becoming as commonplace as a total blood cell count, a development he finds particularly thrilling from a physician’s standpoint. However, his excitement extends beyond the normalisation of genomic testing. He is particularly keen on the advancements in long-read capable sequencing technologies that are expected to enable access to phased, telomere-to-telomere (T2T) genomic testing. This technological leap promises to enhance the precision and scope of genomic analysis, potentially revolutionising the diagnosis and treatment of genetic disorders.

Yeetong, P. et al. Long-read nanopore sequencing identified D4Z4 contractions in patients with facioscapulohumeral muscular dystrophy. Neuromuscular Disorders. 33(7): P551-556 (2023). DOI: https://doi.org/10.1016/j.nmd.2023.05.004