Human gain-of-function variants in HNF1A confer protection from diabetes but independently increase hepatic secretion of atherogenic lipoproteins.

Loss-of-function mutations in hepatocyte nuclear factor 1A (HNF1A) are known to cause rare forms of diabetes and alter hepatic physiology through unclear mechanisms. In the general population, 1:100 individuals carry a rare, protein-coding HNF1A variant, most of unknown functional consequence. To characterize the full allelic series, we performed deep mutational scanning of 11,970 protein-coding HNF1A variants in human hepatocytes and clinical correlation with 553,246 exome-sequenced individuals. Surprisingly, we found that ∼1:5 rare protein-coding HNF1A variants in the general population cause molecular gain of function (GOF), increasing the transcriptional activity of HNF1A by up to 50% and conferring protection from type 2 diabetes (odds ratio [OR] = 0.77, p = 0.007). Increased hepatic expression of HNF1A promoted a pro-atherogenic serum profile mediated in part by enhanced transcription of risk genes including ANGPTL3 and PCSK9. In summary, ∼1:300 individuals carry a GOF variant in HNF1A that protects carriers from diabetes but enhances hepatic secretion of atherogenic lipoproteins.

Combining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation.

Cis-regulatory elements (CREs) can be classified by the shapes of their transcription start site (TSS) profiles, which are indicative of distinct regulatory mechanisms. Massively parallel reporter assays (MPRAs) are increasingly being used to study CRE regulatory mechanisms, yet the degree to which MPRAs replicate individual endogenous TSS profiles has not been determined. Here, we present a new low-input MPRA protocol (TSS-MPRA) that enables measuring TSS profiles of episomal reporters as well as after lentiviral reporter chromatinization. To sensitively compare MPRA and endogenous TSS profiles, we developed a novel dissimilarity scoring algorithm (WIP score) that outperforms the frequently used earth mover's distance on experimental data. Using TSS-MPRA and WIP scoring on 500 unique reporter inserts, we found that short (153 bp) MPRA promoter inserts replicate the endogenous TSS patterns of ∼60% of promoters. Lentiviral reporter chromatinization did not improve fidelity of TSS-MPRA initiation patterns, and increasing insert size frequently led to activation of extraneous TSS in the MPRA that are not active in vivo. We discuss the implications of our findings, which highlight important caveats when using MPRAs to study transcription mechanisms. Finally, we illustrate how TSS-MPRA and WIP scoring can provide novel insights into the impact of transcription factor motif mutations and genetic variants on TSS patterns and transcription levels.

Generation of Neural Progenitor Cells From Canine Induced Pluripotent Stem Cells and Preliminary Safety Test in Dogs With Spontaneous Spinal Cord Injuries.

Advances in stem cell technology, including the use of induced pluripotent stem cells (iPSC) to produce neurons and glial cells, offer new hope for patients with neurological disease and injuries. Pet dogs with spinal cord injuries provide an important spontaneous animal model for evaluating new approaches to stem cell therapy. Therefore, studies were conducted to identify optimal conditions for generating neural progenitor cells (NPC) from canine induced pluripotent stem cells (iPSC) for preliminary evaluation in animals with spinal cord injury. We found that canine NPC could be induced to differentiate into mature neural cells, including glia and neurons. In addition, canine NPC did not form teratomas when injected in NOD/SCID mice. In a pilot study, two dogs with chronic spinal cord injury underwent fluoroscopically guided intrathecal injections of canine NPC. In follow-up MRI evaluations, tumor formation was not observed at the injection sites. However, none of the animals experienced meaningful clinical or electrophysiological improvement following NPC injections. These studies provide evidence that canine iPSC can be used to generate NPC for evaluation in cellular therapy of chronic spinal cord injury in the dog spontaneous injury model. Further refinements in the cell implantation procedure are likely required to enhance stem cell treatment efficacy.