Mutational landscape of phenylketonuria in Iran

To date more than 1000 different variants in the PAH gene have been identified in patients with phenylketonuria (PKU). In Iran, several studies have been performed to investigate the genetics bases of the PKU in different parts of the country. In this study, we have analysed and present an update of the mutational landscape of the PAH gene as well as the population genetics and frequencies of detected variants for each cohort. Published articles on PKU mutations in Iran were identified through a comprehensive PubMed, Google Scholar, Web of Science (ISI), SCOPUS, Elsevier, Wiley Online Library and SID literature search using the terms: “phenylketonuria”, “hyperphenylalaninemia”, and “PKU” in combination with “Iran”, “Iranian population”, “mutation analysis”, and “Molecular genetics”. Among the literature-related to genetics of PKU, 18 studies were on the PKU mutations. According to these studies, in different populations of Iran 1497 patients were included for mutation detection that resulted in detection of 129 different mutations. Results of genetic analysis of the different cohorts of Iranian PKU patients show that the most prevalent mutation in Iran is the pathogenic splice variant c.1066-11G > A, occurring in 19.54% of alleles in the cohort. Four other common mutations were p.Arg261Gln, p.Pro281Leu, c.168 + 5G > C and p.Arg243Ter (8.18%, 6.45%, 5.88% and 3.7%, respectively). One notable feature of the studied populations is its high rate of consanguineous marriages. Considering this feature, determining the prevalent PKU mutations could be advantageous for designing screening and diagnostic panels in Iran.     

Mutational spectrum in the cardioauditory syndrome of Jervell and Lange-Nielsen

Jervell and Lange-Nielsen syndrome (JLNS) is an autosomal recessive syndrome characterised by profound congenital sensorineural deafness and prolongation of the QT interval on the electrocardiogram, representing abnormal ventricular repolarisation. In a study of ten British and Norwegian families with JLNS, we have identified all of the mutations in the KCNQ1 gene, including two that are novel. Of the nine mutations identified in this group of 10 families, five are nonsense or frameshift mutations. Truncation of the protein proximal to the recently identified C-terminal assembly domain is expected to preclude assembly of KCNQ1 monomers into tetramers and explains the recessive inheritance of JLNS. However, study of a frameshift mutation, with a dominant effect phenotypically, suggests the presence of another assembly domain nearer to the N-terminus.     

Mutational spectrum of BRAF gene in colorectal cancer patients in Saudi Arabia

Colorectal cancer (CRC) is one of the topmost causes of death in males in Saudi Arabia. In females, it was also within the top five cancer types. CRC is heterogeneous in terms of pathogenicity and molecular genetic pathways. It is very important to determine the genetic causes of CRC in the Saudi population. BRAF is one of the major genes involved in cancers, it participates in transmitting chemical signals from outside the cells into the nucleus of the cells and it is also shown to participate in cell growth. In this study, we mapped the spectrum of BRAF mutations in 100 Saudi patients with CRC. We collected tissue samples from colorectal cancer patients, sequenced the BRAF gene to identify gene alterations, and analyzed the data using different bioinformatics tools. We designed a three-dimensional (3D) homology model of the BRAF protein using the Swiss Model automated homology modeling platform to study the structural impact of these mutations using the Missense3D algorithm. We found six mutations in 14 patients with CRC. Four of these mutations are being reported for the first time. The novel frameshift mutations observed in CRC patients, such as c.1758delA (E586E), c.1826insT (Q609L), c.1860insA and c.1860insA/C (M620I), led to truncated proteins of 589, 610, and 629 amino acids, respectively, and potentially affected the structure and the normal functions of BRAF. These findings provide insights into the molecular etiology of CRC in general and to the Saudi population. BRAF genetic testing may also guide treatment modalities, and the treatment may be optimized based on personalized gene variations.     

Mutational spectrum of D-bifunctional protein deficiency and structure-based genotype-phenotype analysis

D-bifunctional protein (DBP) deficiency is an autosomal recessive inborn error of peroxisomal fatty acid oxidation. The clinical presentation of DBP deficiency is usually very severe, but a few patients with a relatively mild presentation have been identified. In this article, we report the mutational spectrum of DBP deficiency on the basis of molecular analysis in 110 patients. We identified 61 different mutations by DBP cDNA analysis, 48 of which have not been reported previously. The predicted effects of the different disease-causing amino acid changes on protein structure were determined using the crystal structures of the (3R)-hydroxyacyl-coenzyme A (CoA) dehydrogenase unit of rat DBP and the 2-enoyl-CoA hydratase 2 unit and liganded sterol carrier protein 2-like unit of human DBP. The effects ranged from the replacement of catalytic amino acid residues or residues in direct contact with the substrate or cofactor to disturbances of protein folding or dimerization of the subunits. To study whether there is a genotype-phenotype correlation for DBP deficiency, these structure-based analyses were combined with extensive biochemical analyses of patient material (cultured skin fibroblasts and plasma) and available clinical information on the patients. We found that the effect of the mutations identified in patients with a relatively mild clinical and biochemical presentation was less detrimental to the protein structure than the effect of mutations identified in those with a very severe presentation. These results suggest that the amount of residual DBP activity correlates with the severity of the phenotype. From our data, we conclude that, on the basis of the predicted effect of the mutations on protein structure, a genotype-phenotype correlation exists for DBP deficiency.     

Mutational spectrum analysis of umuC-independent and umuC-dependent gamma-radiation mutagenesis in Escherichia coli

gamma-Radiation mutagenesis (oxic versus anoxic) was examined in wild-type, umuC and recA strains of Escherichia coli K-12. Mutagenesis [argE3(Oc)----Arg+] was blocked in a delta (recA-srlR)306 strain at the same doses that induced mutations in umuC122::Tn5 and wild-type strains, indicating that both umuC-independent and umuC-dependent mechanisms function within recA-dependent misrepair. Analyses of various suppressor and back mutations that result in argE3 and hisG4 ochre reversion and an analysis of trpE9777 (+1 frameshift) reversion were performed on umuC and wild-type cells irradiated in the presence and absence of oxygen. While the umuC strain showed the gamma-radiation induction of base substitution and frameshifts when irradiated in the absence of oxygen, the umuC mutation blocked all oxygen-dependent base-substitution mutagenesis, but not all oxygen-dependent frameshift mutagenesis. For anoxically irradiated cells, the yields of GC----AT [i.e., at the supB and supE (Oc) loci] and AT----GC transitions (i.e., at the argE3 and hisG4 loci) were essentially umuC independent, while the yields of (AT or GC)----TA transversions (i.e., at the supC, supL, supM, supN and supX loci) were heavily umuC dependent. These data suggest new concepts about the nature of the DNA lesions and the mutagenic mechanisms that lead to gamma-radiation mutagenesis.     

Mutational spectrum of Xeroderma pigmentosum group A in Egyptian patients

Xeroderma pigmentosum (XP) is a rare autosomal recessive hereditary disease characterized by hyperphotosensitivity, DNA repair defects and a predisposition to skin cancers. The most frequently occurring type worldwide is the XP group A (XPA). There is a close relationship between the clinical features that ranged from severe to mild form and the mutational site in XPA gene. The aim of this study is to carry out the mutational analysis in Egyptian patients with XP-A. This study was carried out on four unrelated Egyptian XP-A families. Clinical features were examined and direct sequencing of the coding region of XPA gene was performed in patients and their parents. Direct sequencing of the whole coding region of the XPA gene revealed the identification of two homozygous nonsense mutations: (c.553C>T; p.(Gln185*)) and (c.331G>T; p.(Glu111*)), which create premature, stop codon and a homodeletion (c.374delC: p.Thr125Ilefs*15) that leads to frameshift and premature translation termination. We report the identification of one novel XPA gene mutation and two known mutations in four unrelated Egyptian families with Xermoderma pigmentosum. All explored patients presented severe neurological abnormalities and have mutations located in the DNA binding domain. This report gives insight on the mutation spectrum of XP-A in Egypt. This would provide a valuable tool for early diagnosis of this severe disease.     

Mutational spectrum in the recent human genome inferred by single nucleotide polymorphisms

So far, there is no genome-wide estimation of the mutational spectrum in humans. In this study, we systematically examined the directionality of the point mutations and maintenance of GC content in the human genome using approximately 1.8 million high-quality human single nucleotide polymorphisms and their ancestral sequences in chimpanzees. The frequency of C-->T (G-->A) changes was the highest among all mutation types and the frequency of each type of transition was approximately fourfold that of each type of transversion. In intergenic regions, when the GC content increased, the frequency of changes from G or C increased. In exons, the frequency of G:C-->A:T was the highest among the genomic categories and contributed mainly by the frequent mutations at the CpG sites. In contrast, mutations at the CpG sites, or CpG-->TpG/CpA mutations, occurred less frequently in the CpG islands relative to intergenic regions with similar GC content. Our results suggest that the GC content is overall not in equilibrium in the human genome, with a trend toward shifting the human genome to be AT rich and shifting the GC content of a region to approach the genome average. Our results, which differ from previous estimates based on limited loci or on the rodent lineage, provide the first representative and reliable mutational spectrum in the recent human genome and categorized genomic regions.     

Mutational spectrum induced in Saccharomyces cerevisiae by the carcinogen N-2-acetylaminofluorene

The spectrum of mutations induced by the carcinogen N-2-acetylaminofluorene (AAF) was analysed in Saccharomyces cerevisiae using a forward mutation assay, namely the inactivation of the URA3 gene. The URA3 gene, carried on a yeast/bacterial shuttle vector, was randomly modified in vitro using N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) as a model reactive metabolite of the carcinogen AAF. The binding spectrum of AAF to the URA3 gene was determined and found to be essentially random, as all guanine residues reacted about equally well with N-AcO-AAF. Independent Ura^? mutants were selected in vivo after transformation of the modified plasmid into a ura3Δ yeast strain. Plasmid survival decreased as a function of AAF modification, leading to one lethal hit (37% relative survival) for an average of ≈ 50 AAF adducts per plasmid molecule. At this level of modification the mutation frequency was equal to ≈ 70 × 10^?4, i.e. ≈ 50-fold above the background mutation frequency. UV irradiation of the yeast cells did not further stimulate the mutagenic response, indicating the lack of an SOS-like mutagenic response in yeast. Sequence analysis of the URA3 mutants revealed ≈ 48% frameshifts, 44% base substitutions and ≈ 8 % complex events. While most base substitutions (74%) were found to be targeted at G residues where AAF is known to form covalent C8 adducts, frameshift mutations were observed at GC base pairs in only≈ 24% of cases. Indeed, more than 60% of frameshift events occurred at sequences such as 5′-(A/T)_nG-3′ where a short (n = 2 or 3) monotonous run of As or Ts is located on the 5' side of a guanine residue. We refer to these mutations as semi-targeted events and present a potential mechanism that explains their occurrence.     

Mutational spectrum of dihydropyrimidine dehydrogenase gene (DPYD) in the Tunisian population

Dihydropyrimidine dehydrogenase enzyme (DPD) deficiency is a pharmacogenetic syndrome leading to severe side-effects in patients receiving therapies containing the anticancer drug 5-fluorouracil (5-FU). The aim of this population study is to evaluate gene variations in the coding region of the dihydropyrimidine dehydrogenase gene (DPYD) in the Tunisian population. One hundred and six unrelated healthy Tunisian volunteers were genotyped by denaturing HPLC (DHPLC). Twelve variants in the coding region of the DPYD were detected. Allele frequencies of DPYD*5 (A1627G), DPYD*6 (G2194A), DPYD*9A (T85C), A496G, and G1218A were 12.7%, 7.1%, 13.7%, 5.7%, and 0.5%, respectively. The DPYD alleles DPYD*2A (IVS 14+1g>1), DPYD*3 (1897 del C) and DPYD*4 (G1601A) associated with DPD deficiency were absent from the examined subjects. We describe for the first time a new intronic polymorphism IVS 6-29 g>t, found in an allelic frequency of 4.7% in the Tunisian population. Comparing our data with that obtained in Caucasian, Egyptian, Japanese and African-American populations, we found that the Tunisian population resembles Egyptian and Caucasian populations with regard to their allelic frequencies of DPYD polymorphisms. This study describes for the first time the spectrum of DPYD sequence variations in the Tunisian population.     

Mutational spectrum in the neurofibromatosis type 2 gene in sporadic and familial schwannomas

Using a heteroduplex approach and direct sequencing, we have completed the screening of approximately 88% of the neurofibromatosis type 2 (NF2)-coding sequence of DNA extracted from 33 schwannomas from NF2 patients and from 29 patients with sporadic schwannomas. The extensive screening has resulted in the identification of 33 unique mutations. Similarly to other human genes, we have shown that the CpG sites are more highly mutable in the NF2 gene. The frequency, distribution, and types of mutations were shown to differ between the sporadic and familial tumors. The majority of the mutations resulted in protein truncation and were consistent with more severe phenotype, however three missense mutations were identified during this study and were all associated with milder manifestations of the disease. Received: 25 September 1995 / Revised: 19 December 1995     

Mutational spectrum of bleomycin in lacZ mouse kidney: a possible model for mutational spectrum of reactive oxygen species

The mutational spectrum of bleomycin was compared with the spontaneous mutational spectrum in lacZ mouse kidney. Mice were treated with four 20 mg/kg of doses of bleomycin over a two-week period, leading to a mutant fraction several times greater than that of controls. The major class of bleomycin-induced mutations consisted of small deletions, in particular -1 deletions at AT base pairs and hot spots for deletions at 5'-GTC-3' sequences. Smaller, but significant fractions of GC > AT followed by GC > TA substitutions were also observed. In untreated mice, the major class of mutations consisted of GC > AT substitutions followed by GC > TA mutations, and a much smaller fraction of deletions. Other than the specificity of bleomycin for AT base pairs and the 5'-GTC-3' hotspots, the mutational spectrum of bleomycin in mice is similar to that reported for ionizing radiation. However, bleomycin initially mediates the formation of oxidized DNA via reduction of molecular oxygen, as opposed to the radiolysis of water. In this respect mutagenesis induced by bleomycin may be more similar to that induced by endogenous reactive oxygen species (ROS) than mutagenesis induced by ionizing radiation. If bleomycin-induced mutagenesis is an appropriate model for mutagenesis induced by ROS, then, based on the difference between the mutational spectrum of bleomycin and spontaneous mutagenesis, the latter appears not to result predominantly from ROS, at least in mouse kidney.     

Mutational spectrum and recombinogenic effects induced by aminofluorene adducts in bacteriophage M13

Double-stranded replicative form (RFI) DNA of bacteriophage M13 strain M13mp10 which carries partial lacZ gene has been modified in vitro to various extents with N-hydroxy-2-amino-fluorene (N-OH-AF) and then transfected into E. coli cells. High-performance liquid chromatography (HPLC) analysis results demonstrate that the sole adduct (95%) formed in modified DNA is N-(deoxyguanosine-8-yl)-2-aminofluorene (dG-C8-AF). Approximately 20 adducts per RFI molecule constitute 1 lethal event when plaque-forming ability is assayed on E. coli cells which have received no prior SOS induction. The mutagenicity of dG-C8-AF adducts was assayed by measuring loss of beta-galactosidase activity as a function of adducts per molecule. A dose-dependent increase in Lac- mutants was observed, with a 4-fold increase in mutants per survivor at 30 adducts/molecule. The mutations produced, characterized by DNA sequencing, occur predominantly at either G or C positions different from those observed in the spontaneous mutant spectrum. Restriction-mapping results show that in our assay system, dG-C8-AF adducts induce a previously unreported recombinogenic activity.     

Mutational spectrum of phenylalanine hydroxylase deficiency in the population resident in Catalonia: genotype-phenotype correlation

Hyperphenylalaninemia (HPA) is a group of diseases characterized by the persistent elevation of phenylalanine levels in tissues and biological fluids. It is an autosomal recessive disorder affecting 1 in 10,000 individuals in Caucasian populations and about 1 in 6,600 in Catalonia. We report the mutational spectrum of phenylalanine hydroxylase deficiency in the population living in Catalonia and the genotype-phenotype correlation. The molecular study was performed in 383 samples corresponding to 115 patients from 99 unrelated families and 268 relatives. We have characterized 90% of the mutant alleles; there were 57 different mutations, 49 of which have previously been described, 8 being novel mutations and two being large deletions. The 57 mutations detected corresponded to: five nonsense, seven frameshift, and eight splice defects, the remainder being missense mutations. These mutations cause 72 different genotypes in the 83 families characterized, confirming the mutational heterogeneity of phenylketonuria (PKU) in the Mediterranean population. According to our biochemical classification, our HPA population is composed of 40 PKU (35%), 36 variant PKU (31%), and 39 non-PKU HPA (34%). Mutations such as IVS 10, A403 V, and E390G correlated as expected with the phenotype and the predicted residual activity in vitro. However, in four cases (165 T, V388 M, R261Q, and Y414 C), the observed metabolic phenotype was not consistent with the predicted genotypic effect. The identification of the mutations in the PAH gene and the genotype-phenotype correlation should facilitate the evaluation of metabolic phenotypes, diagnosis, implementation of optimal dietary therapy, and determination of prognosis in the patients and genetic counselling for the patient's relatives.     

Mutational spectrum of N-hydroxy-N-acetyl-4-aminobiphenyl at exon 3 of the HPRT gene

4-Aminobiphenyl is a human bladder carcinogen present in many environmental sources, including cigarette smoke. It can be metabolized in two steps to the mutagen N-hydroxy-N-acetyl-4-aminobiphenyl (N-OH-AABP). In this study the mutational spectrum of N-OH-AABP-exposed human lymphoblastoid cells (TK6) was determined using HPRT     

Mutational spectrum of N-hydroxy-N-acetyl-4-aminobiphenyl at exon 3 of the HPRT gene

4-Aminobiphenyl is a human bladder carcinogen present in many environmental sources, including cigarette smoke. It can be metabolized in two steps to the mutagen N-hydroxy-N-acetyl-4-aminobiphenyl (N-OH-AABP). In this study the mutational spectrum of N-OH-AABP-exposed human lymphoblastoid cells (TK6) was determined using HPRT     

Dermatoglyphics in phenylketonuria

Dermatoglyphics of 100 phenylketonurics and 200 controls matched for ethnic origin and sex were compared. The present study of dermatoglyphics in phenylketonurics is consistent with results showing that single gene disorders have fewer and less striking anomalies of ridge development than diseases with gross chromosomal defects.Analysis revealed a decreased frequency of whorl patterns on the finger tips and a wider mean summed atd angle among affected females as compared to normal females. The males showed no significant differences. Limited as they were to one sex, the differences seem unlikely to be attributable to the effect of the phenylketonuric gene.This research was supported by the Minnesota Association for Retarded Children, The Graduate School of the University of Minnesota and United States Public Health Service Grant Number HD 01507-01.     

Mutational spectrum of the lacI gene in Escherichia coli K12 induced by low-energy ion beam

The mutational spectrum of the genomic lacI gene induced by low-energy nitrogen ion irradiation in wild type Escherichia coli strain W3110 were compared with the spontaneous and the vacuum controls. The mutant frequency of irradiated group was dose-dependent and reached 26.3 × 10⁻⁶ at dose of 31.2 × 10¹⁴ ions/cm², which was about 18-fold over the background (1.5 × 10⁻⁶) and 10-fold over the vacuum controls (2.6 × 10⁻⁶). This result indicated that the low-energy ion irradiation was one of many effective mutagens, though the vacuum condition of low-energy ions contributed some low-level gene mutations. It was found that the difference between the spontaneous and the vacuum control was the increases of base-pair substitutions in the vacuum control group. The spectra of irradiated group were quite similar to that of oxygen free-radical induced in the same strain, suggesting free-radicals and other adducts generated by low-energy ions might play an important role in the mutagenesis in vivo. When the spontaneous and the vacuum control group were compared, base-pair substitutions, deletions and additions of the irradiated group were significantly increased, and the +TGGC or −TGGC at hot spot was decreased from 82 to 48%. But the remarkable increase in absolute MF of the +TGGC or −TGGC at hot spot in the irradiated group suggested that low-energy ions did induce the mutations of this type. The spectra of our irradiated group had relative low-level base-pair substitutions, high-level ±TGGC and high proportion additions than those of γ-radiation induced, implying there were some different effects or processes between them.