novel variant in the PAH gene causing phenylketonuria in an Iranian pedigree

Background: Phenylalanine hydroxylase [PAH] gene is the well-known causative gene for classic Phenylketonuria [PKU] [OMIM#261600] disease, with more than 500 reported mutations. Through this study, a novel mutation in the PAH gene in an Iranian pedigree with phenylketonuria was introduced

Methods: A consanguineous family with a 10-year old affected girl was referred for genetic analysis. Mutation screening of all exons and exon-intron boundaries was performed by Sanger sequencing, and mini haplotype analysis was carried out by genotyping of Short Tandem Repeat [STR] and Variable Number Tandem Repeat [VNTR] alleles

Results: Mutation analysis revealed a novel homozygous insertion of a single adenine nucleotide at position 335 in exon 3 of the PAH gene. Based on the American College of Medical Genetics and Genomics [ACMG] guidelines, the change is interpreted as a pathogenic mutation which produces a premature termination signal [TAA] at codon 113 according to in silico assessments. The mini haplotype analysis showed that this mutation was linked to STR [15] -VNTR [3]

Conclusion: In this study, a novel mutation was reported in a patient who had PKU symptoms without any previously reported mutations in the PAH gene [NM_000277.1: p.Asp112Glufs*2] that can be responsible for the classical PKU phenotype in the Iranian population. Detection of novel mutations indicates notable allelic heterogeneity of the PAH locus among this population

efficient trio-based mini-haplotyping method for genetic diagnosis of phenylketonuria

Objective: The phenylalanine hydroxylase [PAH] locus has high linkage disequilibrium. Haplotypes related to this locus may thus be considered sufficiently informative for genetic diagnosis and carrier screening using multi-allelic markers. In this study, we present an efficient method for haplotype analysis of PAH locus using multiplexing dyes. In addition, we explain how to resolve the dye shift challenge in multiplex short tandem repeat [STR] genotyping

Materials and Methods: One hundred family trios were included in this descriptive study. The forward primer of a tetra-nucleotide STR and the reverse primer of a variable number tandem repeat [VNTR] were labeled with three different non-overlapping dyes 5-carboxyfluorescein [FAM], 6-carboxy-N,N,N',N'-tetramethylrhodamine [HEX] and 6-carboxy-N,N,N',N'-tetramethylrhodamine [TAMRA]. The polymerase chain reaction [PCR] products from each family trio were multiplexed for capillary electrophoresis and results were analyzed using Peak Scanner software

Results: Multiplexing trio products decreased the cost significantly. The TAMRA labeled products had a significant predictable shift [migrated at a slower electrophoretic rate] relative to the HEX and FAM labeled products. Through our methodology we achieve, the less inter-dye shift than intra-dye shift variance. Correcting the dye shift in the labeled products, according to the reference allele size, significantly decreased the inter-dye variability [P<0.001]

Conclusion: Multiplexing trio products helps to detect and resolve the dye shift accurately in each family, which otherwise would result in diagnostic error. The dye system of FAM, HEX and TAMRA is more feasible and cheaper than other dye systems

Functional analysis of a novel splicing mutation in the mutase gene of two unrelated pedigrees

Objective: methylmalonic acidura [MMA] is a rare autosomal recessive inborn error of metabolism. In this study we present a novel nucleotide change in the mutase [MUT] gene of two unrelated Iranian pedigrees and introduce the methods used for its functional analysis

Materials and Methods: two probands with definite diagnosis of MMA and a common novel variant in the MUT were included in a descriptive study. Bioinformatic prediction of the splicing variant was done with different prediction servers. Reverse transcription- polymerase chain reaction [RT-PCR] was done for splicing analysis and the products were analyzed by sequencing

Results: the included index patients showed elevated levels of propionylcarnitine [C3]. Urine organic acid analysis confirmed the diagnosis of MMA, and screening for mutations in the MUT revealed a novel C to G variation at the 3' splice acceptor site in intron 12. In silico analysis suggested the change as a mutation in a conserved sequence. The splicing analysis showed that the C to G nucleotide change at position -3 in the acceptor splice site can lead to retention of the intron 12 sequence

Conclusion: this is the first report of a mutation at the position -3 in the MUT intron 12 [c.2125-3C>G]. The results suggest that the identified variation can be associated with the typical clinical manifestations of MMA

Whole exome sequencing reveals a BSCL2 mutation causing progressive encephalopathy with lipodystrophy [PELD] in an Iranian pediatric patient

Background: Progressive encephalopathy with or without lipodystrophy is a rare autosomal recessive childhoodonset seipin-associated neurodegenerative syndrome, leading to developmental regression of motor and cognitive skills. In this study, we introduce a patient with developmental regression and autism. The causative mutation was found by exome sequencing

Methods: The proband showed a generalized hypertonia and regression of all developmental milestones. Based on the advantages of next-generation sequencing [NGS], whole exome sequencing [WES] was requested. The functional significance of variants was evaluated by NGS-specific prediction servers. Sanger sequencing was used for segregation analysis in the family

Results: There was no specific sign in the clinical and paraclinical investigations of the patient to establish a conclusive clinical diagnosis. WES detected a known homozygous nonsense mutation in BSCL2 [NM_001122955.3:c.985C>T; p.Arg329*]. The variant is segregating in the pedigree with an autosomal recessive pattern

Conclusion: Exome sequencing is a robust method for identifying the candidate gene variants in Mendelian traits

effect of Setarud [IMOD[TM]] on angiogenesis in transplanted human ovarian tissue to nude mice

One of the promising methods in fertility preservation among women with cancer is cryopreservation of ovarian cortex but there are many drawbacks such as apoptosis and considerable reduction of follicular density in the transplanted ovary. One solution to reduce ischemic damage is enhancing angiogenesis after transplantation of ovarian cortex tissue. The aim of this study was to investigate the effect of Setarud, on angiogenesis in transplanted human ovarian tissue. In this case control study, twenty four nude mice were implanted subcutaneously, with human ovarian tissues, from four women. The mice were randomly divided into two groups [n=12]: the experimental group was treated with Setarud, while control group received only vehicle. Each group was divided into three subgroups [n=4] based on the graft recovery days post transplantation [PT]. The transplanted fragments were removed on days 2, 7, and 30 PT and the expression of Angiopoietin-1, Angiopoietin-2, and Vascular endothelial growth factor at both gene and protein levels and vascular density were studied in the grafted ovarian tissues. On the 2[nd] and 7[th] day PT, the level of Angiopoietin-1 gene expression in case group was significantly lower than that in control group, while the opposite results were obtained for Angiopoietin-2 and Vascular endothelial growth factor. These results were also confirmed at the protein level. The density of vessels in Setarud group elevated significantly on day 7 PT compared to pre-treatment state. Our results showed that administration of Setarud may stimulates angiogenesis in transplanted human ovarian tissues, although further researches are needed before a clear judgment is made

Investigation on estrogen receptor alpha gene polymorphisms in Iranian women with recurrent pregnancy loss

Recurrent pregnancy loss [RPL] is a multifactorial disorder. Environmental factors and genetics can affect pregnancy outcomes. Conflicting data suggest an association between estrogen receptor alpha [ESR1] gene polymorphisms and RPL. In this study, such association was investigated in Iranian women with RPL. In this case control study, blood samples were collected from 244 women with a history of three or more consecutive pregnancy losses and 104 healthy women with at least two live births. Using polymerase chain reaction- restriction fragment length polymorphism [PCR-RFLP], we studied -397C/T and -351A/G polymorphisms on ESR1 gene in case and control subjects. The genotypic frequencies of -397C/T and -351A/G polymorphisms on ESR1were not significantly different between RPL and control groups [p=0.20 and p=0.09, respectively]. A significantly negative correlation was observed between -397C/T and -351A/G [r=-0.852, p<0.001] in RPL women and complete linkage disequilibrium between the investigated polymorphisms was found [D': 0.959; r-square= 0.758, p<0.001]. This investigation suggests that the analyzed polymorphisms on ESR1gene are not associated with an increased risk of RPL in the studied population

effect of sortilin silencing on ovarian carcinoma cells

Our preliminary data on the protein expression of SORT1 in ovarian carcinoma tissues showed that sortilin was overexpressed in ovarian carcinoma patients and cell lines, while non-malignant ovaries expressed comparably lower amount of this protein. In spite of diverse ligands and also different putative functions of sortilin [NTR3], the function of overexpressed sortilin in ovarian carcinoma cells is an intriguing subject of inquiry. The aim of this study was, therefore, to investigate the functional role of sortilin in survival of ovarian carcinoma cell line. Expression of sortilin was knocked down using RNAi technology in the ovarian carcinoma cell line, Caov-4. Silencing of SORT1 expression was assessed using real-time qPCR and Western blot analyses. Apoptosis induction was evaluated using flow cytometry by considering annexin-V FITC binding. [3H]-thymidine incorporation assay was also used to evaluate cell proliferation capacity. Real-time qPCR and Western blot analyses showed that expression of sortilin was reduced by nearly 70-80% in the siRNA transfected cells. Knocking down of sortilin expression resulted in increased apoptosis [27.5 +/- 0.48%] in siRNA-treated ovarian carcinoma cell line. Sortilin silencing led to significant inhibition of proliferation [40.1%] in siRNA-transfected Caov-4 cells as compared to mock control-transfected counterpart [p<0.05]. As it was suspected from overexpression of sortilin in ovarian tumor cells, a cell survival role for sortilin can be deduced from these results. In conclusion, the potency of apoptosis induction via silencing of sortilin expression in tumor cells may introduce sortilin as a potential candidate for developing a novel targeted therapy in patients with ovarian carcinoma

Predisposing factors for nephrolithiasis and nephrocalcinosis in cystic fibrosis

Cystic fibrosis [CF] is characterized by chronic pulmonary disease, insufficient pancreatic and digestive function, and abnormal sweat concentration. There is controversy about predisposing factors of nephrolithiasis and nephrocalcinosis in patients with cystic fibrosis. We assessed the results of metabolic evaluation in patients with cystic fibrosis and its correlation with nephrocalcinosis. Forty five CF patients, mean age 47.1 months, were enrolled in the study. No one had past history of nephrolithiasis and/or nephrocalcinosis. The records were reviewed for clinical characteristics and all patients underwent metabolic evaluation including serum electrolyte measurements and spot urine analysis. Ultrasonography was performed in all patients to detect nephrocalcinosis and urolithiasis. Nephrocalcinosis was found in 5 [11%] patients. No patient had clinical symptoms of nephrolithiasis and/or micro/macroscopic hematuria. Metabolic evaluation of the CF patients versus normal reference values showed decreased serum uric acid in 48.8%, elevated serum phosphate in 24.4%, and urine oxalate excretion in 51%. Metabolic evaluation of the nephrocalcinosis positive patients versus nephrocalcinosis negative group showed no statistical difference in serum electrolytes. The mean value of urine calcium excretion was lower in patients with nephrocalcinosis [P=0.001]. Despite lack of any significant correlation, higher numerical hyperoxaluria was observed in patients with severe steatorrhea. There was no statistical correlation between steatorrhea and urine calcium as well as oxalate excretion. Hypocalciuria in the nephrocalcinotic CF patients may be seen. It can be hypothesized that hypocalciuria may be due to a primary defect in renal calcium metabolism in CF patients

[Contributing factors to the attitudes toward using informationand communication technology [ICT] among students of shiraz university of medical sciences, school of management]

Background and Objective: The main audience of information technology is human resources and in the case of lack of positive attitude toward information technology, the desired results can not be achieved. The main purpose of this study was to evaluate factors related to the attitudes toward the use of ICT among the students of Shiraz University of Medical Sciences, School of Management

Methods: The present correlation study was performed on the students of School of Management, Shiraz University of Medical Sciences. Data collection instrument was a standard questionnaire and data were analyzed by path analysis and Lisrel software

Results: Variables of computer self-efficacy [0.23], perceived ease of using technology [0.37] and perceived usefulness of technology [0.28] had significant direct relationship with attitude toward use of ICT. Computer experience [0.14], computer self-efficacy [0.20] and perceived ease of using technology [0.18] showed significant direct effect on perceived usefulness of technology. It is also observed that computer experience [0.27] and computer self-efficacy [0.18] have significant direct effect on perceived ease of using technology and finally variable of computer experience [0.29] showed significant direct effect on computer self-efficacy

Conclusion: Among the studied variables, perceived ease of using technology had the greatest impact on the attitude toward ICT use and also the model showed appropriate and acceptable fitness with data?

Comparative molecular and microbiologic diagnosis of vaginal colonization by group B streptococcus in pregnant women during labor

Rapid tests for detection of Streptococcus agalactiae or Group B Streptococci [CGS] at the onset of labor are needed to permit early intrapartum antibiotic prophylaxis. This study aimed to evaluate the PCR assays targeting the 16S ribosomal RNA gene [16S rDNA] for detection of the GBS in comparison with a specific culture method. Two swabs were used to obtain vaginal specimens from the 330 pregnant women attended delivery room at Hedayat hospital, Tehran, Iran. One swab was analyzed by direct plating onto selective GBS agar medium [ISLAM] and the other swab was used for a PCR assay, which amplified the 16S rDNA of S.agalactiae. Comparative study between the selective culture and the PCR assay was done among the 330 tested women. The GBS colonization rate based on the culture results was 20.6% [68/330]. Both culture and PCR methods were positive for 56 and negative for 253 women. The culture method was positive and PCR was negative in 12 women. The culture was negative and the PCR positive for 9 women. Sensitivity of the PCR assay was 82.3% and specificity was 96.5%. The positive predictive value was 86.15% and negative predictive value was 95.4%. ISLAM diagnostic procedure and PCR are rapid and reliable analyzing methods, which might be useful for accurate diagnosis of GBS colonization in pregnant women at the time of delivery

Producing recombinant mTEX101; a murine testis specific protein

Production of antibodies against specific proteins of testis germ cells is of great significance for the investigation of processes involved in spermatogenesis, study of infertility problems and determination of the probable role of these proteins as cancer-testis antigens. Murine Testis Specific Recombinant Protein 101 [mTEX101] is a 38kDa, GPI-anchored protein which is expressed in testis germ cells of adult mice but it seems to be absent in other tissues. The structure and function of mTEX101 is not completely understood yet, but it is speculated that it may transduce biochemical signals into the cytoplasm since mTEX101 does not have an intracellular domain but the precise mechanisms are still ambiguous. RNA was extracted from three adult mice testis. The RNA was used in RT-PCR, employing a pair of specific primers for mTEX101 ORF region. TA-cloning technique was performed by the insertion of mTEX101 into a pGEM-T Easy Vector, followed by its subcloning into a His-tagged expression vector, pET-28a [+]. The recombinant mTEX101 was then produced by transfection of the expression vector into BL 21 [DE3] E. coli strain. A recombinant protein, weighing 27kDa, was produced upon IPTG-induction of the bacterial host. The presence of mTEX101 protein was detected through Western blot analysis by anti-mTEX101 peptide antibodies. We produced mTEX101 recombinant protein that could be used for the production of mono and polyclonal antibodies

Acute Rheumatic Fever in the North East of Iran: a Study of 80 Cases

To evaluate the frequency, clinical presentation and cardiac involvement of children with RF in the North-East of Iran. A case series analysis was conducted on 80 patients with acute rheumatic fever [ARF], who were hospitalized at Ghaem hospital in Mashad between 1994 and 2000, were studied. Laboratory tests and results from echocardiographic examinations, and clinical findings were analyzed. All patients received standard care for children with ARF. The X[2] test was used for comparison of binary data. When compared to similar studies from developed countries, our study demonstrates a decreased frequency of RF in North-East Iran over the past few years. However, it is still a major health problem and the most common cause of acquired heart disease in childhood. The distribution of the major modified Jones criteria in our study is slightly different from that described in the literature, with a higher incidence of carditis. It appears that carditis is endemic in this region. Considering the high morbidity and complications involved in this disease, there is an immediate need for effective preventive programs for the initiating cause streptococcal infections, especially since it is treatable

High-dose oral pyridoxin for treatment of pediatric recurrent intractable seizures

Intractable epilepsy is a common clinical problem in pediatrics and approximately 13% of children with epilepsy experience intractable seizures. To determine the efficacy of pyridoxine in treating seizures, 30 infants and children with recurrent seizures were enrolled in the present study. All of them were treated with high-dose oral pyridoxine [40 mg/kg/day], as an adjunct to antiepileptic drugs. Clinical efficacy criteria were based on the daily frequency of seizures after therapy was initiated during the following three weeks. The results indicated that the mean frequency of seizures decreased significantly from the first [16.2 +/- 11] to the fourth visit [7 +/- 6.2] [p<0.001, t=4]. Three patients became completely seizure free. No adverse effects of pyridoxine were apparent during the observation period. We conclude that pyridoxine is a safe, effective, and well-tolerated adjunct to routine antiepileptic drugs for the treatment of recurrent intractable seizures in children