Cases 2 and 3
Sequence analysis of coding exons, including flanking intronic regions,
as well as the promoter and enhancer region were carried out as
described before (Williams et al., 2018). Sequencing of the regulatory
element in the NAGS intron 1 was performed using the BigDye
Terminator Cycle Sequencing kit version 1.1 and an ABI 3130 genetic
analyzer (Applied Biosystems by Life Technologies Europe BV, Zug,
Switzerland) (Williams et al., 2018) after amplification with 5’-gca gga
tac gct gcg ggc tc-3’ and 5’-gtg ggc cag acg tgg tgc tc-3’ primers and
following amplification conditions: 15 min initial denaturation at 95°C;
38 cycles of denaturation (20 s at 95°C), annealing (20 s at 58°C) and
extension (1 min at 72°C); and a final extension at 72°C for 7 min. If
not provided, genomic DNA was extracted from peripheral blood
leukocytes.