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Summary: Genetic variation of the 16p11.2 deletion locus containing the KCTD13 gene and of CUL3 is linked with autism. This genetic connection suggested that substrates of a CUL3-KCTD13 ubiquitin ligase may be involved in disease pathogenesis. Comparison of Kctd13 mutant (Kctd13−/−) and wild-type neuronal ubiquitylomes identified adenylosuccinate synthetase (ADSS), an enzyme that catalyzes the first step in adenosine monophosphate (AMP) synthesis, as a KCTD13 ligase substrate. In Kctd13−/− neurons, there were increased levels of succinyl-adenosine (S-Ado), a metabolite downstream of ADSS. Notably, S-Ado levels are elevated in adenylosuccinate lyase deficiency, a metabolic disorder with autism and epilepsy phenotypes. The increased S-Ado levels in Kctd13−/− neurons were decreased by treatment with an ADSS inhibitor. Lastly, functional analysis of human KCTD13 variants suggests that KCTD13 variation may alter ubiquitination of ADSS. These data suggest that succinyl-AMP metabolites accumulate in Kctd13−/− neurons, and this observation may have implications for our understanding of 16p11.2 deletion syndrome.

To achieve high-throughput analysis of allele frequencies in human SNPs, we have developed automated methods for designing PCR and DNA sequencing primers. We found we could run the PCR assays at quite stringent, uniform conditions. The design process used freely available databases, including dbSNP, SNPper, and TSC, and publicly available software including RepeatMasker and Primer3. We describe parameters for the software and other considerations that increase experimental success. As anticipated, some assays failed at the design stage due primarily to the genomic locations of repetitive sequences, extreme GC content regions, or lack of sufficient sequence. However, over 23 000 assays, including 96% of those recently analyzed, have been experimentally successful. Similar design methods could be used for PCR assays in any organism with substantial available sequence.