Molecular Analysis of PKU-Associated PAH Mutations: A Fast and Simple Genotyping Test

Abstract

Neonatal screening for phenylketonuria (PKU, OMIM: 261600) was introduced at the end of the 1960s. We developed a rapid and simple molecular test for the most frequent phenylalanine hydroxylase (PAH, Gene ID: 5053) mutations. Using this method to detect the 18 most frequent mutations, it is possible to achieve a 75% detection rate in Italian population. The variants selected also reach a high detection rate in other populations, for example, 70% in southern Germany, 68% in western Germany, 76% in Denmark, 68% in Sweden, 63% in Poland, and 60% in Bulgaria. We successfully applied this confirmation test in neonatal screening for hyperphenylalaninemias using dried blood spots and obtained the genotype in approximately 48 h. The method was found to be suitable as second tier test in neonatal screening for hyperphenylalaninemias in neonates with a positive screening test. This test can also be useful for carrier screening because it can bypass the entire coding sequence and intron–exon boundaries sequencing, thereby overcoming the questions that this approach implies, such as new variant interpretations.

Phenylketonuria: Genes in phenylketonuria, diagnosis, and treatments

“…The Life Technologies can help for the sample preparation and SS. The BigDye Terminator v1.1 Cycle Sequencing kit (Gu et al, 2014) and by an ABI PRISM 3130XL (Thermo Fisher Scientific) (Tolve et al, 2018) can perform SS. An ABI 3730 can run sequencing after ethanol purification.…”

“…The NGS procedures can also use Nextera Rapid Capture Enrichment Reference Guide. Runs can use Miseq DX platform and data analysis can use BaseSpace Variant Interpreter (Illumina, San Diego, CA, USA) (Tolve et al, 2018). NGS is useful for the clinical genetic analysis of IEMs diagnosis (Chaiyasap et al, 2017).…”

“…After metabolic screening tests, about 2 mL urine specimens were stored at -70º C for second-tier tests such as metabolic profiling using gas chromatography-mass spectrometry (Sumaily and Mujamammi, 2017). Tolve et al (2018) suggested a procedure for diagnosing PKU: minisequencing. The authors have optimized the detection of the 24 probes in four multiplex PCR followed by four single-nucleotide extension reactions and four electropherograms.…”

“…Tolve et al (2018) successfully tested the method on dried blood spots. This genotyping assay needs 48 hours to do the test and is able to characterize both alleles in more than 50% of the PKU subjects (Tolve et al, 2018). This method is a useful tool for detecting PKU disorder.…”

“…This disorder affects about 1 in 2,500 (Wang et al, 2019) to 100,000 births (Muntau et al, 2019). The carrier frequency is 1 in 50 (Tolve et al, 2018). PKU has a high prevalence in Turkey, Ireland, and the United Kingdom.…”