Characterization of the most frequent ATP7B mutation causing Wilson disease in hepatocytes from patient induced pluripotent stem cells

H1069Q substitution represents the most frequent mutation of the copper transporter ATP7B causing Wilson disease in Caucasian population. ATP7B localizes to the Golgi complex in hepatocytes but moves in response to copper overload to the endo-lysosomal compartment to support copper excretion via bile canaliculi. In heterologous or hepatoma-derived cell lines, overexpressed ATP7B-H1069Q is strongly retained in the ER and fails to move to the post-Golgi sites, resulting in toxic copper accumulation. However, this pathogenic mechanism has never been tested in patients’ hepatocytes, while animal models recapitulating this form of WD are still lacking. To reach this goal, we have reprogrammed skin fibroblasts of homozygous ATP7B-H1069Q patients into induced pluripotent stem cells and differentiated them into hepatocyte-like cells. Surprisingly, in HLCs we found one third of ATP7B-H1069Q localized in the Golgi complex and able to move to the endo-lysosomal compartment upon copper stimulation. However, despite normal mRNA levels, the expression of the mutant protein was only 20% compared to the control because of endoplasmic reticulum-associated degradation. These results pinpoint rapid degradation as the major cause for loss of ATP7B function in H1069Q patients, and thus as the primary target for designing therapeutic strategies to rescue ATP7B-H1069Q function.

substances. The patient did not complain of itching and had no clinical signs of genetic disorders. Growth and neurologic development were normal. Infections, autoimmune hepatitis, celiac disease, thyroid disorders, biliary system disease, cystic fibrosis and myopathy were ruled out.
Wilson disease diagnosis was based on low serum levels of ceruloplasmin, increased basal and after penicillamine urinary copper excretion and genetic analysis (Table 1).
Diagnosis was confirmed by DNA sequencing of the entire family, that indicated the patient as homozygous for the H1069Q mutation of ATP7B, and the mother heterozygous wt/H1069Q (22). The patient was treated with zinc therapy as first line.
A progressive improvement in copper metabolism parameters (urinary copper excretion < 75 mcg/24 h; urinary zinc excretion > 2 g/24 h) was observed in the following 12 months, with a normalization of aminotransferase by 18 months of treatment. The patient remained asymptomatic throughout the entire period of observation and ALT levels persisted normal. Currently he is 18 years old and has normal serum level of aminotransferases.

Mesodermal and ectodermal differentiation of hiPSCs
For mesodermal differentiation to obtain cardiomyocytes we adapted the protocol by Zhang et al. (24). Briefly, control and patient iPSC colonies were detached from the plate using dispase and after sedimentation were resuspended in the following medium: DMEM/F12 (Invitrogen), 20% FBS (Hyclone), 1X Glutamax, 1X μM nonessential amino acids, 50 U/ml (penicillin and 50 mg/ml streptomicin), 50 μM 2mercaptoethanol (all from Invitrogen). After 4 days, the aggregates were plated on Matrigel coated plates and 50 µg/ml of ascorbic acid (Sigma) was added to the medium. The medium was changed on alternative day for further 21 days.
For ectodermal differentiation to obtain neurons we adapted a previously used method (25). Control and patient iPSCs plated on Matrigel were induced to differentiate in RPMI supplemented with 1XN2, 50 U/ml penicillin and 50 mg/ml streptomycin (all from Invitrogen). After 7 days of differentiation 1X B27 (Invitrogen) and 1µm retinoic acid (Sigma) were added and kept for the following 7-18 days. The medium was changed every 2 days.

RNA isolation and quantitative PCR
For quantitative PCR (qPCR), total RNA was extracted by TriSure (Bioline), and first-strand cDNA was synthesized using Mu-MLV RT (New England BioLabs) according to the manufacturer's instructions. qPCR was carried out with the QuantStudio 7 Flex (Thermo Fisher Scientific) using Fast SYBR Green PCR Master Mix (Thermo Fisher Scientific). The housekeeping GAPDH mRNA was used as an internal standard for normalization. Gene-specific primers used for amplification are listed in Supplemental Table S3. qPCR data are presented as fold changes relative to the indicated reference sample using 2DeltaCt comparative analysis.

Preparation of cell extracts, SDS-PAGE and Western blot analysis
After 20

Immuno-electron microscopy.
For pre-embedding immuno-electron microscopy HLCs were fixed, permeabilized and labeled as described previously (16