Short‐chain 3‐hydroxyacyl‐CoA dehydrogenase (SCHAD), encoded by the HADH gene, is a ubiquitously expressed mitochondrial enzyme involved in fatty acid oxidation. This protein also plays a role in insulin secretion as recessive HADH mutations cause congenital hyperinsulinism of infancy (CHI) via loss of an inhibitory interaction with glutamate dehydrogenase (GDH). Here, we present a functional evaluation of 16 SCHAD missense variants identified either in CHI patients or by high‐throughput sequencing projects in various populations. To avoid interactions with endogenously produced SCHAD protein, we assessed protein stability, subcellular localization, and GDH interaction in a SCHAD knockout HEK293 cell line constructed by CRISPR‐Cas9 methodology. We also established methods for efficient SCHAD expression and purification in E. coli, and tested enzymatic activity of the variants. Our analyses showed that rare variants of unknown significance identified in populations generally had similar properties as normal SCHAD. However, the CHI‐associated variants p.Gly34Arg, p.Ile184Phe, p.Pro258Leu, and p.Gly303Ser were unstable with low protein levels detectable when expressed in HEK293 cells. Moreover, CHI variants p.Lys136Glu, p.His170Arg, and p.Met188Val presented normal protein levels but displayed clearly impaired enzymatic activity in vitro, and their interaction with GDH appeared reduced. Our results suggest that pathogenic missense variants of SCHAD either make the protein target of a post‐translational quality control system or can impair the function of SCHAD without influencing its steady‐state protein level. We did not find any evidence that rare SCHAD missense variants observed only in the general population and not in CHI patients are functionally affected.

DNA methylation is an epigenetic mechanism that can alter gene expression without modifying the DNA sequence. Reactivation of tumor suppressor genes that have been epigenetically silenced in cancer could be a potential therapeutic approach. In this study, we performed long-term treatment of the breast cancer cell line SKBR3, with Olaparib or Doxorubicin in order to assess the methylation status of tumor suppressor genes, after the cell cultures had become resistant to these drugs. Using bisulphite conversion and massive parallel sequencing we examined CpG islands in promoter regions of 283 tumor suppressor genes at several time points under the two treatment regiments. Methylation analysis was carried out for each sample using our in-house bioinformatic pipeline established in collaboration with Roche and publicly available tools. We assessed differentially methylated CpG’s for each drug, compared to control samples treated with dimethyl sulfoxide (DMSO) using the R package methylKit. Gene ontology and pathway analysis revealed hypermethylation in genes belonging to homologous recombination pathway and hypomethylation in genes responding to cellular stress and protein kinase signaling in Olaparib-treated samples. In Doxorubicin-treated samples, we identified genes involved in inter-strand cross-link repair to be hypomethylated, while other genes associated with more general cellular response to DNA damage were hypermethylated. While in Olaparib-treated samples, differential methylation seemed to be reversible and dependent on the dosage of Olaparib, methylation patterns in Doxorubicin-treated samples appeared to change in an incremental manner, as a function of exposure time to the drug. Citation Format: Zuzana Sichmanova, Asta Ottesen, Per Eystein Lonning, Elisabet Ognedal Berge, Stian Knappskog. Changes in tumor suppressor methylation in SKBR3 cells during treatment with Olaparib or Doxorubicin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 818.

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