Genetic Disorders Diagnosed More Quickly via Long-Read Sequencing Method

Despite rapid advances in genetic testing over recent decades, about half of people with a suspected Mendelian genetic disorder have no accurate diagnosis, while others may have to wait years for their diagnosis.

Researchers headed by a team at the King Abdullah University of Science and Technology (KAUST) have now developed a long-read sequencing (LRS) platform, known as NanoRanger (nanopore-based rapid acquisition of neighboring genomic regions) that they claim offers an accurate, rapid and low cost method for diagnosing Mendelian genetic disorders within just a few hours. NanoRanger works with routine clinical samples (e.g., peripheral blood) and resolves large or complex genomic alterations at base-pair resolution.

“Precise, efficient genomic diagnosis is urgently needed to improve patient outcomes and facilitate carrier screening,” said Yingzi Zhang a PhD candidate at KAUST, who is supervised by research lead Mo Li, PhD. “Using our custom-developed data analysis tool, NanoRanger accurately maps breakpoints at single base-pair resolution, providing a detailed picture that helps diagnose the genetic disorder. Diagnosis can be as fast as 12 minutes after initial sequencing, which is a game-changer.”

Zhang, Li and colleagues reported on the development of NanoRanger, and its application in a small human study, in Med. In their report, titled “NanoRanger enables rapid single-base-pair resolution of genomic disorders,” the team stated, “We developed a simple and effective approach, NanoRanger, which shows paradigm-shifting reliability in resolving disease-causing breakpoints that are missed by conventional clinical testing … Our results demonstrate that NanoRanger is a rapid and cost-effective approach with wide clinical applicability.”

Over 400 million people worldwide have a rare disease, and 80% of rare diseases are estimated to have a genetic origin,” the authors noted. “Despite advances in routine genetic testing in both clinical and research settings, approximately half of the individuals worldwide suspected of having a Mendelian disorder remain without an accurate diagnosis. For those fortunate enough to receive an accurate diagnosis, it takes an average of 6 years.” And perhaps 80% of individuals with a rare disease will experience at least one misdiagnosis.

Mendelian disorders—including nervous system and intellectual developmental conditions— are caused by either an alteration in one particular gene or an abnormal rearrangement in one segment of the genome. Each disease has a specific “breakpoint”—the genomic location of a structural variant where DNA is deleted, rearranged or inverted. While these variants may be identified using traditional screening techniques, the sheer complexity of the rearrangements means they are often missed. “The failure of exome sequencing (ES) to secure a molecular diagnosis in at least 50% of patients with suspected Mendelian diseases in large cohorts has been attributed by some to its limited coverage compared to genome sequencing,” the team continued.

Mendelian diseases are heritable, particularly if both parents are carriers of the same faulty segment. Such diseases are more prevalent in regions where it is common for marriage between related individuals (consanguinity). The investigators developed NanoRanger to overcome the limitations of current techniques and enable rapid, accurate detection of various types of structural variants (SVs) in expanded genomic regions at base resolution.

The technique is cost-effective and requires only a tiny amount of DNA from a patient or suspected carrier. NanoRanger takes a sample of genomic DNA and uses molecular scissors called restriction enzymes to fragment the DNA into pieces with the same end sequences. These pieces are then self-joined into circles and amplified, which makes it easier to target and sequence the genomic regions of interest using Oxford Nanopore Tecnologies’ long-read sequencing technology.

Compared with other targeted long-read sequencing (T-LRS) approaches, the scientists noted, “NanoRanger requires significantly less DNA and a fraction of the flow-cell capacity while achieving tens of thousands-fold higher sequence coverage.” Li explained, “NanoRanger uses simple molecular biology strategies to ‘fish out’ genomic regions that are suspected of harboring complex mutations, deletions or rearrangements.”

In trials carried out in collaboration with a group of Saudi clinicians led by Fowzan Alkuraya, MD, at King Faisal Specialist Hospital & Research Center, NanoRanger successfully identified precise breakpoints in 13 familial cases of genomic disorders that were missed by conventional genetic tests. Using these breakpoints, the researchers then screened the carrier status of related family members and 1,000 healthy Saudi individuals. “NanoRanger identified the breakpoints with single-base-pair resolution, enabling accurate determination of the carrier status of unaffected family members as well as the founder nature of these genomic lesions and their frequency in the local population,” they reported.

The testing method prompted one Saudi couple in the trial to opt for in vitro fertilization after they were both found to carry the genomic deletion for an inherited Mendelian disease. “We have filed for a patent, and plan to integrate NanoRanger into standard diagnostic routines to provide a comprehensive toolkit for clinical settings, both here in Saudi Arabia and across the world,” concluded Li. The authors concluded, “We anticipate that multiplex NanoRanger could serve as a complementary diagnostic tool for genetic disorders with well-defined candidate genes and as a cost-effective premarital screening test for common genetic disorders.”