To validate the analytical validity of next-generation sequencing methods, it is necessary to examine whether the variants can be correctly detected using genomic DNA with known variant information. The benchmark human genome variants in the human reference genome published by the Genome In A Bottle (GIAB) consortium at the US National Institute of Standards and Technology (NIST) are ideal for verifying analytical validity. Here, we present the methods and results of analytical validation of next-generation sequencing method that we conducted using GIAB data and standard human genome DNA, and demonstrate that analytical validation is possible at facilities in Japan. In addition, to verify the analytical validity of the data as an institution, i.e., whether pathological variants can be correctly detected based on the patient phenotype, we present an example of our use of an external quality assessment (EQA), GenQA in the UK. This study discusses the method for verifying the analytical validity of next-generation sequencing methods.

Background and Purpose: The prevalence of migraine in Japan is 8%. However, the diagnosis of migraine by genetic testing has not been actively conducted nor covered by insurance. In this study, patients diagnosed with hemiplegic migraine （HM） were subjected to genetic testing of genes for migraine-associated diseases and channelopathies. We report on the genes and variant evaluation detected in that analysis.

Methods: Comprehensive genetic testing was performed on about 320 genes, including genes involved in channelopathies, epilepsy and neurological diseases, including causative genes （CACNA1A, ATP1A2, SCN1A） among familial hemiplegic migraine （FHM） in 45 patients diagnosed with HM. Genetic testing was performed using DNA derived from peripheral blood and analyzed with a next-generation sequencer.

Results: Analysis revealed that variants of ATP1A2 and SCN1A, which are considered to be causative genes of FHM, were detected in five cases and four cases, respectively. Variants of other genes involved in channelopathies were also detected. The variants detected were evaluated as Variant of uncertain significance （VUS）.

Discussion: In this study, we performed genetic testing for FHM and other epilepsy and neurological disorders, and detected multiple variants, which were variants of VUS evaluation. From these, we believe that accumulating variants and clarifying the correlation between genotype and phenotype will lead to optimal treatment selection and prophylactic testing for clinical symptoms other than headache.