Abstract
Biotinidase deficiency is an autosomal recessive syndrome caused by defects in the
biotinidase gene, the product of which affects biotin metabolism. Newborn screening
(NBS) for biotinidase deficiency can identify affected infants prior to onset of symptoms;
biotin supplementation can resolve or prevent the clinical features. In NBS, dry blood
spots (DBS) are usually tested for biotinidase enzyme activity by colorimetric analysis.
By taking advantage of the multiplexing capabilities of the Luminex platform, we have
developed a microsphere-based array genotyping method for the simultaneous detection
of six disease causing mutations in the biotinidase gene, thereby permitting a second
tier of molecular analysis. Genomic DNA was extracted from 3.2 mm DBS. Biotinidase
gene sequences, containing the mutations of interest, were amplified by multiplexed
polymerase chain reaction, followed by multiplexed allele-specific primer extension
using universally tagged genotyping primers. The products were then hybridized to
anti-tag carrying xTAG microspheres and detected on the Luminex platform. Genotypes
were verified by sequencing. Genotyping results of 22 known biotinidase deficient
samples by our xTAG biotinidase assay was in concordance with the results obtained
from DNA sequencing, for all 6 mutations used in our panel. These results indicate
that genotyping by an xTAG microsphere-based array is accurate, flexible, and can
be adapted for high-throughput. Since NBS for biotinidase deficiency is by enzymatic
assay, less than optimal quality of the DBS itself can compromise enzyme activity,
while the DNA from these samples mostly remains unaffected. This assay warrants evaluation
as a viable complement to the biotinidase semi-quantitative colorimetric assay.
Keywords
Newborn screening - biotinidase deficiency - multiplexed genotyping
