Inactivating mutations in the BCKDK gene, which codes for the kinase responsible for the negative regulation of the branched‐chain α‐keto acid dehydrogenase complex (BCKD), have recently been associated with a form of autism in three families. In this work, two novel exonic BCKDK mutations, c.520C>G/p.R174G and c.1166T>C/p.L389P, were identified at the homozygous state in two unrelated children with persistently reduced body fluid levels of branched‐chain amino acids (BCAAs), developmental delay, microcephaly, and neurobehavioral abnormalities. Functional analysis of the mutations confirmed the missense character of the c.1166T>C change and showed a splicing defect r.[520c>g;521_543del]/p.R174Gfs1*, for c.520C>G due to the presence of a new donor splice site. Mutation p.L389P showed total loss of kinase activity. Moreover, patient‐derived fibroblasts showed undetectable (p.R174Gfs1*) or barely detectable (p.L389P) levels of BCKDK protein and its phosphorylated substrate (phospho‐E1α), resulting in increased BCKD activity and the very rapid BCAA catabolism manifested by the patients’ clinical phenotype. Based on these results, a protein‐rich diet plus oral BCAA supplementation was implemented in the patient homozygous for p.R174Gfs1*. This treatment normalized plasma BCAA levels and improved growth, developmental and behavioral variables. Our results demonstrate that BCKDK mutations can result in neurobehavioral deficits in humans and support the rationale for dietary intervention.

This work examined nine patients with creatine deficiency syndrome (CDS): six with a creatine transport (CRTR) defect and three with a GAMT defect. Eleven nucleotide variations were detected: six in SLC6A8 and five in GAMT. These changes were analyzed at the mRNA level and specific alleles (most of which bore premature stop codons) were selected as nulls because they provoked nonsense‐mediated decay activation. The impact of these CDS mutations on metabolic stress (ROS production, p38MAPK activation, aberrant proliferation and apoptosis) was analyzed in patient fibroblast cultures. Oxidative stress contributed toward the severe form of CDS, with increases seen in the intracellular ROS content and the percentage of apoptotic cells. An altered cell cycle was also seen in a number of CRTR and GAMT fibroblast cell lines (mostly those carrying null alleles). p38MAPK activation only correlated with oxidative stress in the CRTR cells. Based on intracellular creatine levels, the contribution of energy depletion toward metabolic stress was demonstrable only in selected CRTR cells. Together, these findings suggest that the apoptotic response to genotoxic damage in the present CDS cells may have been triggered by different cell signaling pathways. They also suggest that reducing oxidative stress could be helpful in treating CDS. Hum Mutat 32:1–10, 2011.

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