Objective: This study investigated a NIID patient with typical clinical and imaging features but no detectable NOTCH2NLC GGC expansion using standard testing. Whole-genome long-read sequencing (LRS) was employed to identify pathogenic variants, establish a molecular diagnosis, and analyze mutation characteristics near the NOTCH2NLC GGC repeat region.

Background: Neuronal intranuclear inclusion disease (NIID) is a rare neurodegenerative disorder with genetic heterogeneity, presenting cognitive decline, parkinsonism, ataxia, autonomic dysfunction, and neuropathy. Diagnosis relies on detecting NOTCH2NLC GGC repeat expansions or intranuclear inclusions via skin biopsy. However, some NIID patients lack detectable GGC expansions, suggesting undiscovered genetic factors.

Method: The patient’s clinical features, initial genetic test results, and pathological diagnosis were reviewed. Whole-genome LRS was performed, and new RP-PCR and fragment PCR primers were designed and tested on the patient and 501 essential tremor patients. Additionally, 361 LRS datasets were analyzed to study sequence characteristics near the NOTCH2NLC GGC repeat region.

Results: The patient exhibited cognitive decline, motor dysfunction, autonomic dysfunction, and multi-organ involvement, with typical imaging features. Pathological biopsy confirmed NIID, but conventional genetic testing was negative. Whole-genome LRS identified a GGC repeat expansion and three upstream mutations in NOTCH2NLC, which interfered with initial RP-PCR primer binding. New primers confirmed the expansion (137 repeats). When the new primers were used to test 501 patients with essential tremor, no repeat expansion of the NOTCH2NLC gene was found. In the 361 whole-genome LRS datasets, four datasets were found to have a 27-bp repeat at the binding site of the traditional primers, which may lead to false-positive results, while no rare or polymorphic sites were found at the new primer sites.

Conclusion: Whole-genome LRS identified rare NOTCH2NLC variants causing false-negative results, highlighting its value in detecting variations missed by conventional methods. This study provides insights for diagnosing similar NIID cases and underscores the importance of advanced sequencing technologies in preventing misdiagnosis.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/detection-and-characterization-of-rare-variants-in-notch2nlc-causing-false-negative-molecular-diagnosis-through-long-read-sequencing/