Exon deletions of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemics

Abstract

A consistent finding of many studies describing the spectrum of mutant phenylalanine hydroxylase (PAH) alleles underlying hyperphenylalaninemia is the impossibility of achieving a 100% mutation ascertainment rate using conventional gene-scanning methods. These methods include denaturing gradient gel electrophoresis (DGGE), denaturing high performance liquid chromatography (DHPLC), and direct sequencing. In recent years, it has been shown that a significant proportion of undetermined alleles consist of large deletions overlapping one or more exons. These deletions have been difficult to detect in compound heterozygotes using gene-scanning methods due to a masking effect of the non-deleted allele. To date, no systematic search has been carried out for such exon deletions in Italian patients with phenylketonuria or mild hyperphenylalaninemia. We used multiplex ligation-dependent probe amplification (MLPA), comparative multiplex dosage analysis (CMDA), and real-time PCR to search for both large deletions and duplications of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemia patients. Four deletions removing different phenylalanine hydroxylase (PAH) gene exons were identified in 12 patients. Two of these deletions involving exons 4-5-6-7-8 (systematic name c.353-?_912 + ?del) and exon 6 (systematic name c.510-?_706 + ?del) have not been reported previously. In this study, we show that exon deletion of the PAH gene accounts for 1.7% of all mutant PAH alleles in Italian hyperphenylalaninemics.

Novel deletion of the E3A ubiquitin protein ligase gene detected by multiplex ligation-dependent probe amplification in a patient with Angelman syndrome

“…However, at the same time we need to claim about some limitations in the performance of MLPA for quantification analysis possibly leading to false positives as documented in our previous study (Calì et al, 2010). These observations thus make indispensable the use of other gene dosage analysis methods to confirm the preliminary MLPA findings.…”

“…MLPA analysis was carried out essentially as described by Schouten et al (2002) and Calì et al (2010). PCR products were identified and quantified by capillary electrophoresis on an ABI 3130 genetic analyzer, using the Gene Mapper software from Applied Biosystems, Foster City, CA.…”

Multiplex ligation-dependent probe amplification detection of an unknown large deletion of the CREB-binding protein gene in a patient with Rubinstein-Taybi Syndrome

“…In these cases, MLPA is a suitable alternative. Moreover, MLPA can detect gene deletions in heterozygotes whereas DNA sequencing would fail due to the masking effect of the non-deleted allele (Calì et al, 2010). Based on the above considerations, we genotyped patient RSTS01A by MLPA instead of DNA sequencing, using the P313-A1 CREBBP kit (MRC-Holland), containing probes for all 31 exons of the CREBBP gene.…”

“…The latter ratio was then used to define the following categories: i) ~ 1 for the non-deleted/ non-duplicated gene region, ii) ~ 0.5 if deleted, and iii) ~ 1.5 if duplicated. Other details regarding the normalizing method and quality test have been described previously by Calì et al (2010).…”

“…CMDA was performed as previously described by Gable et al (2003) and Calì et al (2010) to confirm the deletion of the exons 17-21 of the CREBBP gene identified by MLPA. Briefly, only exons 16 and 22 (expected normal dosage) and 17 and 21 (expected heterozygous deletion) of the CREBBP gene were co-amplified in a fluorescent multiplex PCR with myelin protein zero (PROZ), mutL homolog 1 (MLH1), and neuroligin 3 (NLGN3) as control genes.…”