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.

Keywords: PKU mutation analysis; PKU screening; phenylalanine hydroxylase deficiency; phenylketonuria.

Figure 1

Compound heterozygosity electropherogram of p.Arg243* and p.Arg261* of Multiplex SNaPshot Reaction I. (1) and (2) heterozygosity of R1P1probe (p.Arg243*, c.727C>T); (3) and (4) heterozygosity of R1P2 probe (p.Arg261*, c.781C>T); (5) wild type R1P3r (p.Arg252Trp, c.754C>T); (6) wild type R1P4r (c.842+1G>A); (7) wild type R1P5r (p.Arg261Gln, c.782G>A); (8) wild type R1P6 (p.Pro281Leu, c.842C>T and p.Pro281Arg, c.842C>G).

Figure 2

Homozygosity electropherogram of p.Leu48Ser of Multiplex SNaPshot Reaction II. (1) wild type R2P1r probe (p.Ala300Ser, c.898G>T); (2) homozygosity R2P2 probe (p.Leu48Ser, c.143T>C); (3) wild type R2P3r probe (p.Arg158Gln, c.473G>A); (4) wild type R2P4r probe (p.Phe55del, c.163_165delTT); (5) wild type R2P5 probe (c.441+5G>T).

Figure 3

Heterozygosity electropherogram of p.Arg408Trp of Multiplex SNaPshot Reaction III. (1) wild type R3P1 probe (p.Ala403Val, c.899C>T); (2) wild type R3P2r probe (p.Tyr414Cys, c.1241A>G); (3) and (4) heterozygosity of R3P3r probe (p.Arg408Trp; c.1222C>T); (5) wild type R3P4 probe (p.Glu390Gly, c.1169A>G); (6) wild type R3P5 probe (c.1315+1G>A); 7) wild type R3P6r probe (c.1066-11G>A).

Figure 4

Wild type electropherogram of Multiplex SNaPshot Reaction IV. (1) wild type R4P1r probe (p.Val245Ala, c.734T>C and p.Val245Glu, c.734T>A); (2) wild type R4P2 probe (p.Ala300Val, c.899C>T); (3) wild type R4P3 probe (p.Tyr414Tyr; c.1242C>T); (4) wild type R4P4 (p.Val245Ile, c.733G>A and p.Val245Leu, c.733G>C); (5) wild type R4P5 probe (p.Arg408Gln, c.1223G>A); (6) wild type R4P6 probe (c.842+2T>A); (7) wild type R4P7 probe (p.Pro281Ser, c.841C>T andp.Pro281Ala, c.841C>G).

Figure 5

Aberrant Pattern: the picture shows how the use of more than one probe can be helpful to reveal correct genotype in case of an ambiguous pattern. When probe R1P4r shows a T/T on the electropherogram two genotypes are possible: c.842+1G>A and c.841_842delCCG. Using other probes present in Reaction mix I and IV we can distinguish the two genotypes: If c.842+1G>A is present, R4P7r shows C/C genotype (c) and R1P6 C/C genotype (a). If c.841_842delCCG is present R4P7 will be T/T (d), R1P4r T/T and peak corresponding to RIp6 will be absent (b).