The PAH gene and its Allele Arginine-408-Tryptophan

–Use ClinVar to document number of pathogenic variants associated with your gene and how many are expert reviewed.

ClinVar has documented total 139 variants of clinical significance reported regarding the PAH gene. Total 612 results showed up on the screen. There are 85 pathogenic variants, while 40 variants are likely pathogenic, five variants were of uncertain significance, 5 are benign and 4 are likely to be benign. The review status was submitted for all the 85 pathogenic variants of clinical significance. 70 submissions were done single handedly while multiple reviewers or researchers worked together and made 15 submissions.

 

Out of  15 submissions 14 had literature review and clinical testing , while only one had literature review only. 70 clinical variants of pathogenic origin are handled single handedly by researchers. While 20 researchers did literature review and the clinical testing 49 researchers presented the literature review only.

 

40 of the submission in the ClinVar are likely pathogenic and were submitted by a single researcher only. The mutations varied in the submissions and consists of a variety of deletion, duplication, indel, insertion and single nucleotide polymorphism. The variety of the PAH sequence lead to the frameshift, missense, nonsense and splice-site changes in the PAH gene sequence.

http://www.ncbi.nlm.nih.gov/clinvar?term=612349%5BMIM%5D

 

–Document how many labs provide tests related to your gene.

The pathogenic variant of PAH gene can present in the form of phenylalanine hydroxylase deficiency which if severe will lead to inability of the person to utilize essential amino acid phenylalanine in body and its break down into tyrosine leading to phenylketonuria (PKU) which if not diagnosed early will lead to brain damage. Other pathologic variant can also lead to non-phenylketonuria hyperphenylalaninemia (Non-PKU HPA) and variant PKU.

The blood is taken from the heel of the baby called the heel stick test and the phenylketonuria-screening test is done within 24 hrs. of birth of baby to find out the absence of Phenylalanine hydroxylase in order to prevent the excessive accumulation of phenylalanine that in excess can cause brain damage.

 The Genetic Testing Registry (GTR) offer 87 clinical tests for PKU. 44 clinical tests are used for diagnosis, 23 for mutation confirmation, 11 pre-symptomatic, 4 for monitoring and 1 for therapeutic management and 1 for drug response. The sequencing analysis of entire coding region is done along with the deletion/insertion analysis and the variant analysis, full panel quantitative random urine analysis for organic acids, amino-acid analysis of plasma is done is done for the diagnosis of PKU and for mutation confirmation.  For the diagnosis and monitoring of PKU the quantitative analysis of phenylalanine and tyrosine is done.

http://www.ncbi.nlm.nih.gov/gtr/conditions/C0031485/

–Use DNAStar to simulate RFLP.

The first clinically significant variant shown by the ClinVar shows the single point mutation at the position 781. The C > G and the amino acid Arginine is replaced by Glycine. This mutation does not bring about the change in the neighboring restriction enzymes. http://www.ncbi.nlm.nih.gov/clinvar?term=612349%5BMIM%5D

PAH- 781 (C)Arginine

PAH- 781 (G)Glycine

PAH- 781 (C)Arginine The restriciton enzyme BbsI & BbsgI

 

 



PAH- 781 (G) Glycine. The restriciton enzyme BbsI & BbsgI

Many disease associated variants create or destroy restriction enzyme recognition sites. My gene PAH has the allelic variant 0.0002 in OMIM. It is SNP and a point mutation the nucleotide at the position 222 CGG-TGG in exon 12. This mutation will result in the replacement of amino acid Arginine by tryptophan at the position 408. This is a clinically significant variant.

 

PAH-gene-1224-CGG-Arginine & enzyme NmeAIII

 

 

 



 

  

PAH-gene-1224-CGG-Arginine

 

 

PAH-gene-1224-TGG-Tryptophan & enzyme NmeAIII is not seen



The restriction enzyme NmeAIII in PAH gene







 restriction enzyme NmeAIII absent in PAH R-408-W variant

 

 GeneQuest simulation of the agarose gel run with the DNA sequence by the enzyme NmeAIII.

The PAH-gene shows the one cut by NmeAIII enzyme

The PAH-allele (R-408-W) shows no cut by NmeAIII enzyme

The PAH-gene shows the one cut by NmeAIII enzyme



The PAH-allele  shows no cut by NmeAIII enzyme

 

Conclusion:

Allelic variant of my gene PAH  from ClinVar NM_000277.1(PAH):c.781C>G(p.Arg261Gly)

did not show the loss of sites of Restriction Enzyme BbsI and BbsgI.

Allelic variant of gen PAH 0.0002 in OMIM is recognized in ClinVar NM_000277.1(PAH):c.1222C>T (p.Arg408Trp) resulted in the loss of the site for restriciton enzyme NmeAIII.