Multiplex Snapshot minisequencing for the detection of common PAH gene mutations in Iranian patients with Phenylketonuria

Background: Phenylketonuria is a common inborn defect of amino acid metabolism in the world. This failure is caused by an autosomal recessive insufficiency of the hepatic enzyme hyperphenylalaninemia (PAH), which catalyzes the irreversible hydroxylation of phenylalanine to tyrosine. More than 1,040 different disease-causing mutations have already been identified in the PAH gene. The most prominent complication of Phenylketonuria, if not diagnosed and treated, is severe mental retardation. Hence, early diagnosis and initiation of nutritional therapy are the most significant measures in preventing this mental disorder. Given these data, we developed a simple and rapid molecular test to detect the most frequent PAH mutations. Methods: Multiplex assay was developed based on the SNaPshot minisequencing approach to simultaneously perform genotyping of the 10 mutations at the PAH gene. We optimized detection of these mutations in one multiplex PCR, followed by 10 single-nucleotide extension reactions. DNA sequencing assay was also used to verify genotyping results obtained by SNaPshot minisequencing. Result: All 10 genotypes were determined based on the position and the fluorescent color of the peaks in a single electropherogram. Sequencing results of these frequent mutations showed that by using this method, a 100% detection rate could be achieved in the Iranian population. Conclusion: SNaPshot minisequencing can be useful as a secondary test in neonatal screening for HPA in neonates with a positive screening test, and it is also suitable for carrier screening. The assay can be easily applied for accurate and time- and cost-efficient genotyping of the selected SNPs in various population.

The human RIT2 core promoter short tandem repeat predominant allele is species-specific in length: a selective advantage for human evolution?

Evolutionary analyses of the critical core promoter interval support a selective advantage for expanding the length of certain short tandem repeats (STRs) in humans. We recently reported genome-wide data on human core promoter STRs that are "exceptionally long" (≥6-repeats). Near the top of the list, the neuron-specific gene, RIT2, contains one of the longest GA-STRs at 11-repeats. In the present study, we analyzed the evolutionary implications of this STR across species. We also studied this STR in a sample of 2,143 Iranian human subjects that encompassed a number of neuropsychiatric disorders and controls. We report that this GA repeat is functional and different lengths of the repeat result in significant alteration in gene expression activity. The 11-repeat allele was human specific and the sole allele detected in 110 unrelated Iranian individuals randomly selected and sequenced from our control pool. Remarkably, homozygosity for a 5-repeat allele was detected in a consanguineous, hospitalized case of schizophrenia, which significantly decreased gene expression activity (p < 5 × 10-6). The frequency of the 5-repeat allele in the Iranian population was calculated at <0.0001, putting this allele in the deleterious mutations category based on allele frequency. The 5-repeat allele is annotated in the Ensembl database in the heterozygous status (5/11) in one of four indigenous hunter-gatherer men sequenced from southern Africa (BUSHMAN KB1: rs113265205). The present findings indicate for the first time, selective advantage for a human-specific allele at an STR locus, and a phenomenon in which genotypes and alleles at the extreme length of STRs occur with disease only. This is a pilot study that warrants large-scale sequencing of the RIT2 core promoter STR in diseases and characteristics that are linked to the brain function.

Mutation screening of the Krüppel-like factor 1 gene using single-strand conformational polymorphism in a cohort of Iranian ß-thalassemia patients.

The Krüppel-like factor 1 (KLF1) is an essential erythroid-specific transcription factor. Mutations in the human KLF1 gene have different phenotypic effects, ranging from increased Hb F levels to the disruption of erythropoiesis. Here, we screened 227 Iranian ß-thalassemia (ß-thal) patients for the presence of KLF1 mutations by using the single-strand conformational polymorphism (SSCP) approach. Our aim was to assess the potential effect of these mutations on the ß-thal disease severity. After screening, two variants were found. One patient carried a potentially deleterious variant (Polyphen-2) in exon 2 (p.F182L). Another patient was homozygous for a previously unreported intronic variant (KLF1: c.911 + 84A > G). The patient with the p.F182L variant (KLF1: c.544T > C) had noticeably high Hb A2 levels (7.6%), consistent with the phenotypic effect of several previously characterized KLF1 mutations in the same exonic region. In addition, he had higher platelet counts (1,069,000/µL) compared to other patients in the cohort.

Core promoter short tandem repeats as evolutionary switch codes for primate speciation.

Alteration in gene expression levels underlies many of the phenotypic differences across species. Because of their highly mutable nature, proximity to the +1 transcription start site (TSS), and the emerging evidence of functional impact on gene expression, core promoter short tandem repeats (STRs) may be considered an ideal source of variation across species. In a genome-scale analysis of the entire Homo sapiens protein-coding genes, we have previously identified core promoters with at least one STR of ≥ 6-repeats, with possible selective advantage in this species. In the current study, we performed reverse analysis of the entire Homo sapiens orthologous genes in mouse in the Ensembl database, in order to identify conserved STRs that have shrunk as an evolutionary advantage to humans. Two protocols were used to minimize ascertainment bias. Firstly, two species sharing a more recent ancestor with Homo sapiens (i.e. Pan troglodytes and Gorilla gorilla gorilla) were also included in the study. Secondly, four non-primate species encompassing the major orders across Mammals, including Scandentia, Laurasiatheria, Afrotheria, and Xenarthra were analyzed as out-groups. We introduce STR evolutionary events specifically identical in primates (i.e. Homo sapiens, Pan troglodytes, and Gorilla gorilla gorilla) vs. non-primate out-groups. The average frequency of the identically shared STR motifs across those primates ranged between 0.00005 and 0.06. The identified genes are involved in important evolutionary and developmental processes, such as normal craniofacial development (TFAP2B), regulation of cell shape (PALMD), learning and long-term memory (RGS14), nervous system development (GFRA2), embryonic limb morphogenesis (PBX2), and forebrain development (APAF1). We provide evidence of core promoter STRs as evolutionary switch codes for primate speciation, and the first instance of identity-by-descent for those motifs at the interspecies level.

Utility of the multivariate approach in predicting ß-thalassemia intermedia or ß-thalassemia major types In Iranian patients.

Recently, five genetic modifiers [ß-globin mutations, coinheritance of α-thalassemia (α-thal), XmnI polymorphism and single nucleotide polymorphisms (SNPs) in the BCL11A and HBS1L-MYB loci] were used to predict the ß-thal major (ß-TM) or ß-thal intermedia (ß-TI) types in 106 French patients with 83.2% accuracy. The dichotomous grouping was based on the age when the patient received his/her first transfusion (4 years). Here, a similar study was conducted in a cohort of 306 Iranian ß-thal patients having distinct ß-globin mutations and minor allele frequencies of key SNPs in these loci. Multivariate regression analyses and a simple scoring system were used to predict the ß-TM/ß-TI types using three scenarios: 1) when considering only the severe ß-TM and the mild ß-TI cases, 2) using clinical parameters for ß-thal typing, and 3) using age at first transfusion as the basis for classification. Using these scenarios, the ß-thal types could be correctly predicted in 77.6, 75.5 and 68.0% of cases, respectively.