Systematic alanine scanning of PAX8 paired domain reveals functional importance of the N-subdomain.

Thyroid-specific transcription factor PAX8 has an indispensable role in the thyroid gland development, which is evidenced by the facts that PAX8/Pax8 mutations cause congenital hypothyroidism in humans and mice. More than 90% of known PAX8 mutations were located in the paired domain, suggesting the central role of the domain in exerting the molecular function. Structure-function relationships of PAX8, as well as other PAX family transcription factors, have never been investigated in a systematic manner. Here, we conducted the first alanine scanning mutagenesis study, in which 132 alanine variants located in the paired domain of PAX8 were created and systematically evaluated in vitro. We found that 76 alanine variants (55%) were loss of function (LOF) variants (defined by <30% activity as compared with wild type PAX8). Importantly, the distribution of LOF variants were skewed, with more frequently observed in the N-subdomain (65% of the alanine variants in the N-subdomain) than in the C-subdomain (45%). Twelve out of 13 alanine variants in residues that have been affected in patients with congenital hypothyroidism were actually LOF, suggesting that the alanine scanning data can be used to evaluate the functional importance of mutated residues. Using our in vitro data, we tested the accuracy of seven computational algorithms for pathogenicity prediction, showing that they are sensitive but not specific to evaluate on the paired domain alanine variants. Collectively, our experiment-based data would help better understand the structure-function relationships of the paired domain, and would provide a unique resource for pathogenicity prediction of future PAX8 variants.

[Maternal Crohn's disease-related vitamin B12 deficient megaloblastic anemia in an infant].

We report an 11-month-old breast-fed boy with feeding difficulties, lethargy, and developmental delay. Blood examination showed pancytopenia and decreased serum levels of vitamin B12. Anisocytosis and poikilocytes were detected in his peripheral blood, and increased megaloblastosis without leukemic cells was detected in his bone marrow. After the diagnosis of megaloblastic anemia due to vitamin B12 deficiency, symptoms were improved by vitamin B12 administration. Further investigation of the mother identified Crohn's disease and suggested that the supply of vitamin B12 from the mother to the infant, via the placenta during pregnancy and via breast milk after birth, was decreased due to impaired absorption of vitamin B12 in the mother's small intestine. Magnetic resonance imaging of the boy's brain on admission showed cerebral cortex atrophy which had improved by the age of 1 year and 10 months after vitamin B12 treatment, though developmental delay was still evident at the age of 3 years. Infantile vitamin B12 deficiency often presents with nonspecific manifestations, such as developmental delay and failure to thrive, in addition to anemia and is thus not easily diagnosed. To prevent severe neurological sequelae, this condition must be rapidly diagnosed, because a prolonged duration increases the risk of permanent disabilities.