الفهرس 
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Abstract
Phenyl ketonuria(PKU) is an autosomal recessive inherited inborn error of metabolism that results from the impairment of phenylalanine hydroxylases(PAH)action due to either a mutation in PAH- gene which lead to nonfunctional enzyme which lead to dramatically increase of phenylalanine (Phe) level an decrease of tyrosine(Tyr) level which give the dramatically neurological symptoms of PKU or a defect in the Enzyme co-factor tetrahydrobiopterin biosynthesis (BH4) (Atypical PKU) (Malignant PKU), the main cause of BH4 biosynthesis defect is the 6-pyrovoil tetrahydrobiopterin syntheses ( PTPS-gene) mutations .
The present study aims to investigate the mutations of PTPS – gene coding sequence in atypical PKU Egyptian patients and molecular analysis of novel mutations to examine their spectrum and explore the possibility of a molecular diagnosis and investigate the levels of Phe and Tyr by Tandem mass spectroscopy (MS/MS) .
Samples were collected from 13 Egyptian patients of unrelated families, were previously diagnosed with Atypical PKU and followed up previously .
Total RNA was extracted from the peripheral blood of the patient by using GeneJETᵀᴹ Whole Blood RNA Purification Mini Kit) and was converted to cDNA by using the QuantiTect Reverse Transcription Kit. cDNA was amplified by PCR using Go Taq® green master mix, 2X. one set of primers (forward and reverse) was designed for amplification of specific sits covering entire length of cDNA of PTPs-gene. These primers were designed by using web based primer-blast tool, NCBI (National center for biotechnology information). Agarose gel electrophoresis was used for detection of DNA PCR product. Sequencing of the PCR product in both directions was carried out then the results were used for nucleotide blast by the online program on NCBI web site to make alignment with normal PTPS- gene for detection any mutations. All detected mutations were submitted to online web server, Mutalyzer Name Checker for detection of the effect of the mutation on the amino acids sequence of protein. The amino acids sequence are used then to detect the expected 3- dimension structure of the PTPs protein by using the online web server, RaptorX and I-TASSER software Structure Prediction. In the end we submitted the mutated sequence to Polyphene-2 online web server to predict the degree of severity of the mutation on the protein to know if it is a benign mutation or pathogenic.
The 4 new mutations detected in this study were submitted to Gene Bank with accession numbers : MH373432-MH373433 - MH373434 - MH373435.
This study detected six mutations, 4 of them are novel mutations (c.164_186del(p.Val55Aspfs*2), c.273G>A (p.91Lys= ) c.86A>T(p.Lys29Ile), c.22C>T(p.Arg8Cys) , and two was reported previously ) c.405T>C(p.135Thr=) and c.200C>Tp. Thr67Met).
In Patient no.1 identified Two different mutations. One of them is a homozygous deletion mutation of all of exon 3, c.164-186 del. (p.Val55Aspfs*2). This is identified as a pathogenic mutation which also found in 11 other patients with prevalence of (92.3 %) of total patients and it is considered as the most common mutation detected in our study. The other mutation, c.405T>C (p.Thr135=), was a heterozygous synonymous mutation.
Patient no.4 has one mutation, c.200C>T (p. Thr67Met), which is a pathogenic homozygous missens mutation.
Patients no.2, 3, 5, 6, 7, 8, 9, 10, 11, 12 and 13 have the same deletion mutation as in patient no. 1 which is c.164-186 del. (p.Val55Aspfs*2), In patient no. 13 , the deletion mutation is combined with another mutation, c.273G>A (p.91Lys= ), a heterozygous synonymous mutation. In patient 6, 8 and 10, the deletion mutation was combined with c.200C>T (p. Thr67Met). In patients no. 7 and 9 the deletion mutation is combined with c.86A>T (p. Lys29Ile ), a benign missens mutation, in patient no.11, the deletion mutation was combined with c.22C>T (p.Arg8Cys), which is a homozygous benign missins mutation.
Identification of these mutations in PTPS-gene in atypical PKU Egyptian patients will gives a well view of the cause of PKU in each case and the link between the severity of each case and its mutation. The early determination of Phe/Tyr levels by (MS/MS) prevents the accumulation of phenyl ketons and the neurological delay of patient. Also facilitate differential confirmatory diagnosis, which is important for appropriate treatments. It will also aid carrier detection, genetic counseling, and subsequent prenatal diagnosis among Egyptian families who have history of disease.