Identification of exonic deletions in the PAH gene causing phenylketonuria by MLPA analysis
Lourdes R Desviat1, Belén Pérez, Magdalena Ugarte
1Centro de Biología Molecular Severo Ochoa CSIC-UAM, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
Background:
Multiplex ligation probe amplification (MLPA) is a sensitive and efficient technique for molecular diagnosis of diseases involving deletions or duplications of large genomic regions. In phenylketonuria (PKU), most of the mutant alleles correspond to missense mutations and large deletions have been scarcely identified. In this study, we report for the first time the use of MLPA analysis on PKU patients to detect exonic deletions.
Method:
DNA from 22 unrelated PKU patients with an incomplete genetic diagnosis after standard mutation detection analysis were subjected to MLPA analysis. Deletions were confirmed by long-range PCR and sequence analysis.
Results:
The technique identified two large genomic deletions in the phenylalanine hydroxylase (PAH) gene, of 6.6 kb and 1.8 kb, including exons 3 and 5, respectively. The chromosomal breakpoints were established by long-range PCR and chromosomal walking, confirming the involvement of repetitive sequences in the deletions.
Conclusion:
MLPA may complement routine mutation screening in PKU patients, although, in the sample studied, exonic deletions in the PAH gene do not appear to be a frequent cause of PKU.
Insights
Multiplex ligation probe amplification (MLPA) identified large genomic deletions in the phenylalanine hydroxylase (PAH) gene in phenylketonuria (PKU) patients. While MLPA is effective, these exonic deletions are not a common cause of PKU.
Area of Science:
- Molecular genetics
- Human genetics
Background:
- Multiplex ligation probe amplification (MLPA) is a sensitive technique for detecting large genomic deletions or duplications.
- Phenylketonuria (PKU) is typically caused by missense mutations, with large deletions being rare.
- This study investigates MLPA for identifying exonic deletions in PKU patients.
Purpose of the Study:
- To evaluate the utility of MLPA for detecting exonic deletions in PKU patients.
- To identify large genomic deletions in the phenylalanine hydroxylase (PAH) gene in PKU cases with incomplete genetic diagnoses.
Main Methods:
- MLPA analysis was performed on DNA from 22 unrelated PKU patients.
- Deletions were confirmed using long-range PCR and sequence analysis.
- Chromosomal breakpoints were determined by long-range PCR and chromosomal walking.
Main Results:
- Two large genomic deletions (6.6 kb and 1.8 kb) in the PAH gene were identified, affecting exons 3 and 5.
- Repetitive sequences were implicated in the observed deletions.
- MLPA proved effective in detecting these deletions.
Conclusions:
- MLPA can supplement standard mutation screening for PKU.
- Exonic deletions in the PAH gene are infrequent causes of PKU in the studied cohort.
