ABSTRACT
Variants in SCYL1 can cause a syndrome with low γ-glutamyl-transferase cholestasis, acute liver failure, and neurodegeneration (CALFAN). The encoded protein is involved in intracellular trafficking between Golgi and ER, specific mechanisms are still to be elucidated. We reprogrammed fibroblasts of a 2â¯years old male patient with CALFAN Syndrome due to a homozygous nonsense variant in SCYL1 (c.[1882Câ¯>â¯T]; c.[1882Câ¯>â¯T]/p.[Gln628*]; p.[Gln628*]) and generated DHMCi005-A using the Cytotune®-iPS 2.0 Sendai Reprogramming Kit (Invitrogen). Cells showed a normal karyotype. Pluripotency was proven using immunohistochemistry, RT-PCR, and flow cytometry. Differentiation into all germ layers was shown using the STEMdiff™ Trilineage Differentiation Kit (Stemcell Technologies).
Subject(s)
Adaptor Proteins, Vesicular Transport/genetics , Cell Differentiation , Cellular Reprogramming , DNA-Binding Proteins/genetics , Fibroblasts/pathology , Induced Pluripotent Stem Cells/pathology , Liver Failure, Acute/genetics , Mutation , Cells, Cultured , Child, Preschool , Cholestasis/genetics , Cholestasis/pathology , Fibroblasts/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Liver Failure, Acute/pathology , Male , Nerve Degeneration/genetics , Nerve Degeneration/pathology , gamma-Glutamyltransferase/deficiencyABSTRACT
Skin fibroblasts were isolated from a male patient with DNAJC12 deficiency and reprogrammed to iPSCs using the Cytotune®-iPS 2.0 Sendai Reprogramming Kit (Invitrogen). Two clones, DHMCi003-A and DHMCi003-B, were characterized for expression of pluripotency marker genes (Oct4, Nanog, Lin28, SSEA-4, TRA-1-60) and differentiated into all three germ layers using embryoid body (EB) formation. Karyotype of both clones was normal and presence of the homozygous mutation in the DNAJC12 gene was verified by PCR and Sanger sequencing. Both clones represent a useful tool to study the pathomechanisms underlying the deficiency.
Subject(s)
Clone Cells , Induced Pluripotent Stem Cells , Repressor Proteins/genetics , Cell Differentiation , Cellular Reprogramming Techniques , Fibroblasts , Genotype , Homozygote , Humans , Karyotype , Male , SkinABSTRACT
Fibroblasts of a patient with Infantile Liver Failure Syndrome 2 (OMIM #616483) due to a homozygous missense variant in the neuroblastoma amplified sequence gene (NBAS; c.[2708T>G]; c.[2708T>G]/p.[Leu903Arg]; p.[Leu903Arg]) were reprogrammed to iPSCs using the Cytotune®-iPS 2.0 Sendai Reprogramming Kit (Invitrogen) delivering the reprogramming factors Oct3/4, Sox2, c-Myc and Klf4. Cells showed a normal karyotype. Pluripotency of DHMCi004-A was proven using immunohistochemistry, RT-PCR analysis, flow cytometry and differentiation into all three germ layers using the STEMdiff™ Trilineage Differentiation Kit (Stemcell Technologies). DHMCi004-A represents the first iPS-based cell model system to elucidate the pathomechanism underlying this disease.
