Objective To explore the clinical and genetic characteristics of patients with hypomyelinating leukodystrophy caused by TMEM163 mutation, track the natural history of the disease, and to construct patient-derived induced pluripotent stem cells, thus laying the foundation for mechanism research.Methods Clinical and genetic data of two patients (Pt1, Pt2) with TMEM163 mutation were collected from 2009 to 2022 in Department of Pediatrics, Peking University First Hospital. The clinical manifestations, genetic data, and protein structure data were analyzed. Peripheral blood of Pt2 was collected to construct induced pluripotent stem cell (iPSC).Results Clinical features: both patients showed early motor and language development retardation and hypomyelination abnormalities from brain magnetic resonance imaging (MRI), however, the symptoms gradually alleviated; nystagmus was the first symptom, abnormal gait, low muscle tone and mild to moderate developmental retardation were observed in both of them; Pt1 has the same growth and development level as children of the same age at 7 years old. Genetic features: both cases were newly detected missense variants at the same locus of TMEM163 c.227T>G p.(L76R), c.227T>C p.(L76P). The differentiation of iPSC induced by peripheral blood monocytes from Pt2 was consistent with the characteristics of iPSC.Conclusion The follow-up study of 2 children with HLD contributes to the understanding of the natural history of HLD caused TMEM163 mutation, expands the understanding of the clinical phenotype of HLD, and constructs TMEM163 c.227T>C p.(L76P) iPSC, which lays a foundation for the mechanism study.