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Two novel mutations in CYP11B1 and modeling the consequent alterations of the translated protein in classic congenital adrenal hyperplasia patients

Abbaszadegan MR, Hassani S, Vakili R, Saberi MR, Baradaran-Heravi A, Arabi A, Keify F, et al.
Endocrine. 2013;44(1):212-9.Original article

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Abstract: Mutations in the 11β-hydroxylase (CYP11B1) gene are the second leading cause of congenital adrenal hyperplasia (CAH), an autosomal recessive disorder characterized by adrenal insufficiency, virilization of female external genitalia, and hypertension with or without hypokalemic alkalosis. Molecular analysis of CYP11B1 gene in CAH patients with 11β-hydroxylase deficiency was performed in this study. Cycle sequencing of 9 exons in CYP11B1 was performed in 5 unrelated families with 11β-hydroxylase deficient children. Three-dimensional models for the normal and mutant proteins and their affinity to their known substrates were examined. Analysis of the CYP11B1 gene revealed two novel mutations, a small insertion in exon 7 (InsAG393) and a small deletion in exon 2 (DelG766), and three previously known missense mutations (T318M, Q356X, and R427H). According to docking results, the affinity of the protein to its substrates is highly reduced by these novel mutations. DelG766 has more negative impact on the protein in comparison to InsAG393. The novel mutations, InsAG393 and DelG766, change the folding of the protein and disrupt the enzyme's active site as it was measured in the protein modeling and substrate binding analysis. Molecular modeling and sequence conservation were predictive of clinical severity of the disease and correlated with the clinical diagnosis of the patients.

Pattern of chromosomal aberrations in patients from north East iran.

Ghazaey S, Mirzaei F, Ahadian M, Keifi F, Semiramis T, Abbaszadegan MR
Cell Journal (Yakhteh). 2013;15(3):258. Original article

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OBJECTIVE: Chromosomal aberrations are common causes of multiple anomaly syndromes. Recurrent chromosomal aberrations have been identified by conventional cytogenetic methods used widely as one of the most important clinical diagnostic techniques.

MATERIALS AND METHODS: In this retrospective study, the incidences of chromosomal aberrations were evaluated in a six year period from 2005 to 2011 in Pardis Clinical and Genetics Laboratory on patients referred to from Mashhad and other cities in Khorasan province. Karyotyping was performed on 3728 patients suspected of having chromosomal abnormalities.

RESULTS:The frequencies of the different types of chromosomal abnormalities were determined, and the relative frequencies were calculated in each group. Among these patients, 83.3% had normal karyotypes with no aberrations. The overall incidences of chromosomal abnormalities were 16.7% including sex and autosomal chromosomal anomalies. Of those, 75.1 % showed autosomal chromosomal aberrations. Down syndrome (DS) was the most prevalent autosomal aberration in the patients (77.1%). Pericentric inversion of chromosome 9 was seen in 5% of patients. This inversion was prevalent in patients with recurrent spontaneous abortion (RSA). Sex chromosomal aberrations were observed in 24.9% of abnormal patients of which 61% had Turner's syndrome and 33.5% had Klinefelter's syndrome.

CONCLUSION: According to the current study, the pattern of chromosomal aberrations in North East of Iran demonstrates the importance of cytogenetic evaluation in patients who show clinical abnormalities. These findings provide a reason for preparing a local cytogenetic data bank to enhance genetic counseling of families who require this service.

KEYWORDS: Chromosomal Aberrations; Cytogenetic Analysis; North East Iran

Inherited genetic markers for thrombophilia in northeastern Iran (a clinical-based report).

Keify F, Azimi-Nezhad M, Zhiyan-abed N, Nasseri M, Abbaszadegan MR
Reports of biochemistry & molecular biology. 2014;2(2):76. Original article

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BACKGROUND: Thrombophilia is a main predisposition to thrombosis due to a procoagulant state. Several point mutations play key roles in blood-clotting disorders, which are grouped under the term thrombophilia. These thrombophilic mutations are methylenetetrahydrofolate reductase (MTHFR, C677T, and A1298C), factor V Leiden (G1691A), prothrombin gene mutation (factor II, G20210A), and plasminogen activator inhibitor (PAI). In the present study, we assessed the prevalence of the above thrombophilia markers in patients with recurrent pregnancy loss or first and second trimester abortions, infertility, and failed in vitro fertilization (IVF).

METHODS: This study was conducted among 457 cases those were referred to detect the inherited genetic markers for thrombophilia. Markers for MTHFR, Factor II, and Factor V were assessed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), and PAI was assessed by Amplification Refractory Mutation System (ARMS-PCR).

RESULTS: Two hundred sixty cases (56.89%) were diagnosed as having at least one thrombophilia marker, whereas 197 cases (43.11%) had no thrombophilia markers and were normal.

CONCLUSION: According to the current study, the pattern of abnormal genetic markers for thrombophilia in northeastern Iran demonstrates the importance of genetic evaluations in patients who show clinical abnormalities with recurrent spontaneous abortion (RSA) or other serious obstetric complications.

KEYWORDS: Factor II; Factor V; MTHFR; PAI; Thrombophilia; Thrombophilic markers

Chromosomal Analysis of Couples with Repeated Spontaneous Abortions in Northeastern Iran.

Ghazaey S, Keify F, Mirzaei F, Maleki M, Tootian S, Ahadian M, et al.
International Journal of Fertility & Sterility. 2015;9(1):47-54. Original article

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Background: Cytogenetic study of reproductive wastage is an important aspect in determining the genetic background of early embryogenesis. Approximately 15 to 20% of all pregnancies in humans are terminated as recurrent spontaneous abortions (RSAs). The aim of this study was to detect chromosome abnormalities in couples with RSAs and to compare our results with those reported previously.

Materials and Methods: In this retrospective study, the pattern of chromosomal aberrations was evaluated during a six-year period from 2005 to 2011. The population under study was 728 couples who attended genetic counseling services for their RSAs at Pardis Clinical and Genetics Laboratory, Mashhad, Iran.

Results: In this study, about 11.7% of couples were carriers of chromosomal aberrations. The majority of abnormalities were found in couples with history of abortion, without stillbirth or livebirth. Balanced reciprocal translocations, Robertsonian translocations, inversions and sex chromosome aneuploidy were seen in these cases. Balanced reciprocal translocations were the most frequent chromosomal anomalies (62.7%) detected in current study.

Conclusion: These findings suggest that chromosomal abnormalities can be one of the important causes of RSAs. In addition, cytogenetic study of families who experienced RSAs may prevent unnecessary treatment if RSA are caused by chromosomal abnormalities. The results of cytogenetic studies of RSA cases will provide a standard protocol for the genetic counselors in order to follow up and to help these families.

Keywords: Chromosomal Abnormalities, Abortions, Cytogenetic Analysis

Molecular, biochemical, and structural analysis of a novel mutation in patients with methylmalonyl-CoA mutase deficiency

Keify F, Sankian M, Moghaddassian M, Rolfs A, Varasteh A
Gene. 2016; 576(1): 208-13. Original article

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BACKGROUND: Methylmalonic aciduria (MMA) is an inborn error of metabolism resulting from genetic defects in methylmalonyl-CoA mutase (MCM). This enzyme is encoded by the MUT gene and is required for the degradation of odd-chain fatty acids, the amino acids valine, isoleucine, methionine, and threonine, and cholesterol.

METHOD: Three unrelated affected patients with isolated MMA and their parents were studied. The MUT gene was analyzed by PCR and sequencing of its entire coding region and the highly conserved exon-intron splice junctions. The homology modeling of the novel mutation found in the MUT gene was performed using the online Swiss-Prot server for automated modeling and then analyzed with special bioinformatics software to better study the structural effects caused by the mutation.

RESULT: We found one homozygous nucleotide change in intron 12 of the MUT gene (c.2125-3 C>G). The variant is located near the highly conserved acceptor splice site of intron 12. A region at the C-terminus of the protein from ASP709 to GLN748 has been deleted by the alteration of c.2125-3 C>G in intron 12 of the MUT gene. Further studies of the novel mutation in the MUT gene by means of homology modeling revealed abnormalities in the protein's structure, which causes the protein to act malfunctioning and also the mRNA expression analysis of MUT gene confirmed these results.

CONCLUSION: We report this novel mutation, including its clinical and biochemical features and genetic defects, in the MUT gene of three patients affected with isolated MMA. Structural analyses of the mutated protein identified changes in the energy and stereochemical features of the protein that unfortunately altered the protein's functionalities. Therefore, we demonstrate that a novel splice site mutation in intron 12 of the MUT gene is a potential highly pathogenic allele via inhibition of alternative splicing.

KEYWORDS: Biochemical analysis; MUT gene; Methylmalonic aciduria; Mutation analysis; Structural analysis

Identification of a novel deletion in the MMAA gene in two Iranian siblings with vitamin B12-responsive methylmalonic acidemia.

Keify F, Abbaszadegan MR, Rolfs A, Orolicki S, Moghaddassian M and Varasteh AR.
CMBL. 2016. 21: 4.Original article

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Background: Adenosylcobalamin (vitamin B12) is a coenzyme required for the activity of methylmalonyl-CoA mutase. Defects in this enzyme are a cause of methylmalonic acidemia (MMA). Methylmalonic acidemia, cblA type, is an inborn error of vitamin B12 metabolism that occurs due to mutations in the MMAA gene. MMAA encodes the enzyme which is involved in translocation of cobalamin into the mitochondria.

Methods: One family with two MMA-affected children, one unaffected child, and their parents were studied. The two affected children were diagnosed by urine organic acid analysis using gas chromatography-mass spectrometry. MMAA was analyzed by PCR and sequencing of its coding region.

Results: A homozygous deletion in exon 4 of MMAA, c.674delA, was found in both affected children. This deletion causes a nucleotide frame shift resulting in a change from asparagine to methionine at amino acid 225 (p.N225M) and a truncated protein which loses the ArgK conserved domain site. mRNA expression analysis of MMAA confirmed these results.

Conclusion: We demonstrate that the deletion in exon 4 of the MMAA gene (c.674 delA) is a pathogenic allele via a nucleotide frame shift resulting in a stop codon and termination of protein synthesis 38 nucleotides (12 amino acids) downstream of the deletion.

KEYWORDS: Mutation analysis, MMAA, gene, Biochemical analysis, Methylmalonic acidemia, Vitamin B12, Novel deletion, Cobalamin, Structural analysis

Methylmalonic Acidemia Diagnosis by Laboratory Methods.

Keify F, Talebi S, Varasteh A-R.
Reports of biochemistry & molecular biology. 2016; 5(1):1-14. Review article

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Methylmalonic acidemia (MMA) is usually caused by a deficiency of the enzyme methylmalonyl-CoA mutase (MCM), a defect in the transport or synthesis of its cofactor, adenosyl-cobalamin (cblA, cblB, cblC, cblF, cblD, and cblX), or deficiency of the enzyme methylmalonyl-CoA epimerase. A comprehensive diagnostic approach involves investigations of metabolites with tandem mass spectrometry, organic acid analysis with gas chromatography, enzymatic studies with fibroblast cell culture, and finally, mutation analysis. With biochemical techniques and enzymatic assay the reliable characterization of patients with isolated MMA for mutation analysis can be achieved. Reliable classification of these patients is essential for ongoing and prospective studies on treatments, outcomes, and prenatal diagnoses. This article reviews the diagnostic techniques used to characterize patients with MMA.

Key Words: Diagnostic techniques, Enzyme assay, Methylmalonic acidemia, Mutation analysis, Organic acid analysis, Tandem mass spectrometry

Development and Validation of a GC-FID Method for Diagnosis of Methylmalonic Acidemia.

Keify F, Varasteh A.
Reports of biochemistry & molecular biology. 2016; 4(2):104-9. Original article

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Background: Urinary organic acids are water-soluble intermediates and end products of the metabolism of amino acids, carbohydrates, lipids, and a number of other metabolic processes. In the hereditary diseases known as organic acidurias, an enzyme or co-factor defect in a metabolic pathway leads to the accumulation and increased excretion of one or more of these acidic metabolites. Gas chromatography is the most commonly-used technology to separate and identify these metabolites. In this report the analytical conditions for the determination of methylmalonic acid using a gas chromatography/flame ionization detector (GC-FID) are studied with the aim to establish a method to analyze organic acids in human urine.

Methods: Studies included the GC-FID method development, the conditions of the derivatization (trimethylsilylation) reaction, and the stability of the methylmalonic acid standard solution and trimethylsilyl derivatives during storage. Also, a systematic comparison between GC-FID and gas chromatography/mass spectrometry (GC-MS) was performed.

Results: The highest resolution and sensitivity were obtained at 60 �C with a 30 min reaction time. Standard solutions and derivatized samples were stable for 7 days at 4-8 �C. Relative standard deviations of within-day and day-to-day assay results were less than 5%. Methylmalonic acid was detected in thirty human urine samples by the proposed GC-FID, and the results were compared with gold standard technique GC-MS. The correlation coefficient between GC-MS and GC-FID was obtained with R2= 0.997.

Conclusion: The developed method was applied to the quantitative analysis of methylmalonic acid in urine from hospitalized children with methylmalonic acidemia. With this method we aim to support pediatric clinics in Iran and assist in clinical diagnostics.

Key Words: Gas chromatography/Flame Ionization Detector (GC-FID), Method development, Methylmalonic acidemia disorder, Urine organic acid analysis

Clinical, Biochemical and Genetic Analysis of Biotinidase Deficiency in Iranian Population.

Asgari A, Rouhi Dehnabeh S, Zargari M, Khani S, Mozafari H, Varasteh A, Keify F, et al.
Archives of Iranian medicine. 2016; 19(11):774-8. Original article

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BACKGROUND: Biotinidase deficiency (BTD) is an autosomal recessive disorder of biotin metabolism. Biotin is a coenzyme that enhances the action of the four enzymes that play an important role in carbohydrates, amino acid, and fatty acid metabolism. Defects in these pathways cause severe metabolic disorder in the body. In general, biotinidase deficiency can be classified into two levels: partial and profound. The incidence of BTD is 1:40,000 to 1:60,000 births in the world, even though no convincing statistical data on the prevalence of this disorder exist in Iran. In this study, we aimed to set up a test for determining biotinidase activity among the Iranian population and report BTD mutations.

PATIENTS AND METHODS: The quantitative method for the determination of biotinidase activity was set up in the National Biochemistry Reference Laboratory (NBRL) of Pasteur Institute of Iran in Tehran. To detect mutations in BTD, polymerase chain reaction (PCR) was performed followed by DNA sequencing.

RESULTS: The biotinidase activity range values were 3.81 - 8.25 nmol/min/mL. We identified 8 BTD patients out of 47 cases with neurologic signs. We detected two mutations, c.98-104del7ins3 and p.Arg79Cys, in 5 patients with profound BTD, and one p.Asp444His mutation in 3 patients with partial BTD.

CONCLUSION: Infants suffering from BTD seem healthy during their first months of life. At present, the screening program for metabolic disorders such as BTD is in progress. The patients that are BTD deficient benefit from the availability of the tests, and consequently receive the Biotin supplements before being clinically affected.

A Description of Reference Ranges for Organic Acids in Urine Samples from A Pediatric Population in Iran.

Keify F, Lukacs Z, Varasteh A.
Reports of biochemistry & molecular biology. 2017; 6(1):40-50. Original article

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BACKGROUND: Organic acids refer to a family of compounds that are intermediates in a variety of metabolic pathways. Many organic acids are present in urine from clinically normal individuals. Elevated levels of urine organic acids cause to the organic acidurias, disorders in which some metabolic pathways in organic acid metabolism are blocked. The present work identified major and minor urinary acidic metabolites in normal subjects, and their quantitative ranges in a pediatric population of Iran.

METHODS: Two hundred and fifty-one healthy subjects, including 132 males and 119 females, from 2 days to 15 years of age were enrolled. Urinary organic acids were extracted from urine with organic solvents and identified and quantified by gas chromatography-mass spectrometry.

RESULTS: The results provide a foundation on which to check results for patients with potentially abnormal organic acidurias. By this method 98 organic acids were identified in a pediatric population of Iran.

CONCLUSION: The present work identifies and quantifies major and minor urinary metabolites excreted by normal subjects. We also analyzed urine from 30 patients with organic acid metabolism abnormalities and compared the concentrations of specific organic acids with those from urines of normal individuals.

KEYWORDS: Gas chromatography/mass spectrometry; Iran population; Normal individuals; Urine organic acid analysis; Urine organic acids range

Frequency of Inborn Errors of Metabolism in a Northeastern Iranian Sample with High Consanguinity Rates

Keify F, Nasseri M, Nayerabadi S, Alaei A, Mokhtariye A and Varasteh AR.
Human Heredity.2018. Original article

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OBJECTIVE: Inborn errors of metabolism (IEMs) are disorders with various manifestations that occur mainly in the pediatric population. In countries where consanguineous marriage is common, the association between consanguinity and IEMs is highly important. No studies have been conducted in Iran examining the impact of consanguinity on IEMs.

METHODS: In this retrospective study, the incidences of metabolic disorders were evaluated for the years 2006 through 2016 in the North East Iran Regional Diagnostic Laboratory (Pardis Clinical and Genetic Laboratory). A total of 13,327 infants with clinical symptoms were referred and investigated for IEMs. Newborn screening was performed on samples from all patients suspected of having IEMs.

RESULTS: Of 13,327 infants examined, 60 different IEMs were diagnosed in 1,118. The most frequent disorders among our patients were glucose-6-phosphate dehydrogenase deficiency (G6PDD) (14.04%), methylmalonic and propionic acidurias (MMA/PA) (9.12%), phenylketonuria (PKU) (8%), and isovaleric acidemia (IVA) (6.98%). A significant difference was found in the prevalence of amino acid disorders between the offspring of consanguineous and those of non-consanguineous parents. No statistically significant differences were found between the 2 groups for organic or fatty acids, carnitine or urine cycles, or lysosomal storage disorders. A total of 707 of the 1,118 infants with metabolic diseases (63.24%) were children of consanguineous parents. These findings show that consanguinity can be an important factor in the inheritance of recessive mutations in a homozygous state.

CONCLUSION: This study found a greater frequency of metabolic diseases in offspring of consanguineous parents than in those of non-consanguineous parents in a population with a high rate of consanguinity.

KEYWORDS: Childhood; Consanguinity; Inborn error of metabolism; Iranian population; Risk of disease

Diagnostic methods for Lysosomal Storage Disease

Mokhtariye A, Hagh-Nazari L,Varasteh AR, Keify F
Human Heredity.2018. Original article

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Lysosomal storage disorders (LSD) are a class of metabolic disturbance in which manifested by the accumulation of large molecules (complex lipids, glycoproteins, glycosaminoglycans, etc.) in lysosomes. LSDs have a wide range of clinical symptoms that may contain organ dysfunction, neurological and skeletal disorders. The first stage of diagnosis is clinically suspected by a physician. Next stage is enzyme activity assays including Fluorometry and MS/MS methods. These methods usually placed in newborn program screening. The second laboratory diagnostic stage is molecular examination (RFLP-PCR and ARMS-PCR, Mutations Scanning Methods, DNA sequencing, MLPA and NGS methods) that is confirmation of the enzyme assays. In this article, routine diagnostic methods for LSDs were discussed. The gold standard for enzyme activity assay and molecular diagnosis is TMS and NGS, respectively

Keywords: Diagnostic methods, Enzyme activity, Lysosomal storage disease, Molecular assay.

The effects of pre-analytical variables on the diagnosis of Inborn Errors of Metabolism

Mokhtariye A, Varasteh AR, Marzban S, Keify F
Journal of North Khorasan, University of Medical Sciences. Accepted. * Corresponding author.2018. Review article

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Background: Delaying plasma separation after phlebotomy (processing delay) can cause perturbations of numerous small molecule analytes. This poses a major challenge to the clinical application of metabolomics analyses. In this study, we further define the analyte changes that occur during processing delays and generate a model for the post hoc detection of this preanalytical error.

Methods: Using an untargeted metabolomics platform we analyzed EDTA-preserved plasma specimens harvested after processing delays lasting from minutes to days. Identified biomarkers were tested on (i) a test-set of samples exposed to either minimal (n=28) or long delays (n=40) and (ii) samples collected in a clinical setting for metabolomics analysis (n= 141).

Results: A total of 149 of 803 plasma analytes changed significantly during processing delays lasting 0-20 h. Biomarkers related to erythrocyte metabolism, e.g., 5-oxoproline, lactate, and an ornithine/arginine ratio, were the strongest predictors of plasma separation delays, providing 100% diagnostic accuracy in the test set. Together these biomarkers could accurately predict processing delays >2 h in a pilot study and we found evidence of sample mishandling in 4 of 141 clinically derived specimens.

Conclusions: Our study highlights the widespread effects of processing delays and proposes that erythrocyte metabolism creates a reproducible signal that can identify mishandled specimens in metabolomics studies.

Keywords: Preanalytical error, Clinical metabolomics, Quality control, Whole blood stability, Phlebotomy

The relationship between MTHFR polymorphisms and abortion in Iranian women

Keify F, Ebrahimzadeh-Vesal R, Zhiyan N, Nayebi M, Nasseri M, Abbaszadegan MR
Gene Reports. 2018;13:130-3 Original article

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Background: Recurrent spontaneous abortion (RSA) is a significant obstetrical complication during pregnancy. Two common polymorphisms (C677T and A1298C) of the methylenetetrahydrofolate reductase (MTHFR) gene are risk factors for RSA. This study was performed to determine the effect of C677T and A1298C polymorphisms in RSA. The true relationship between these two mutations and risk of recurrent spontaneous abortion is unknown, and it is unclear if women with these mutations should be given anticoagulants during pregnancy.

Methods: In this prospective and experimental study, one hundred and fifty three individuals were referred with clinical findings that included recurrent spontaneous abortions minimum of 2 and maximum of 5. Total genomic DNA was isolated from peripheral blood samples. To determine the frequency of the two common C677T and A1298C MTHFR gene polymorphisms in the patients, we used PCR-restriction fragment length polymorphism method.

Results: The frequency of Heterozygous and homozygous C677T polymorphisms were 18.13% and 13.1% respectively. The frequency of Heterozygous and homozygous A1298C polymorphisms were 18.3% and 15.7% respectively. The frequency of Heterozygous C677T and A1298C polymorphisms were 26.8% and wild-type individuals were 7.8%. All female subjects who had heterozygous or homozygous C677T and A1298C polymorphisms with recurrent RSA were followed and those who received appropriate anti-coagulant therapy resulted in successful pregnancy.

Conclusions: In present study, we provided witness to support the relationship between MTHFR C677T and A1298C polymorphisms and recurrent spontaneous abortion. Also, we suggest that anticoagulation therapy of these patients during pregnancy could lead to a successful pregnancy outcome.

Keywords: MTHFR, C677T polymorphism, A1298C polymorphism, Pregnancy loss

Evaluation of 25-OH vitamin D by high performance liquid chromatography: validation and comparison with electrochemiluminescence.

Keify F, Nahid S, Mokhtariye A, Nayerabadi S, Alaei A, Varasteh A-R.
Journal of Analytical Science and Technology. 2018;9(1):25.Original article

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Background: Vitamin D is a fat-soluble steroid hormone precursor that is mainly produced in the skin by exposure to sunlight. It is also supplied in the diet and plays a pivotal role in calcium homeostasis and skeletal metabolism throughout life.

Methods: To assess its analytical performance, we used the RECIPE HPLC Complete Kit and an HPLC-UV instrument. Our HPLC results were compared with a validated electrochemiluminescence method.

Results: The method was linear for the lower limit of quantification from 3 ng/l up to at least 200 ng/l for 25(OH)D3, with the following equation for the regression line: y=0.172 X+2.45 (R2 = 0.989). Intra-assay precision was determined by extracting and quantifying 10 serum replicates from one patient. The mean was 37.875 ng/ml, the standard deviation was 0.22, and the coefficient of variation was 0.58%. Comparisons of results demonstrated good agreement between HPLC and ECL methods (R2=0.883).

Conclusions: The HPLC assay demonstrates excellent linearity, acceptable accuracy and precision, and good agreement with a validated ECL method. The simple sample preparation and ease of use make it practical for the routine clinical laboratory.

Keywords: 25-OH vitamin D, Validation, High-performance liquid chromatography, Electrochemiluminescence

Mutation analysis of genes related to methylmalonic acidemia: identification of eight novel mutations

Keify F, Abbaszadegan MR, Sankian M, Rolfs A, Orolicki S, Pournasrollah M
Molecular biology reportsOriginal article

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Methylmalonic acidemia (MMA), an inherited metabolic disease, results from genetic defects in methylmalonyl-CoA mutase or any of the proteins involved in adenosylcobalamin synthesis. This enzyme is classified into several complementation groups and genotypic classes. In this work we explain the biochemical, structural and genetic analysis of 25 MMA patients, from Iran. The diagnosis was established by the measurement of propionylcarnitine in blood using tandem mass spectrometry and confirmed using a gas chromatography–flame ionization detector. Using clinical, biochemical, structural and molecular analyses we identified 15 mut MMA, three cblA, one cblB, and four cblC-deficient patients. Among mutations identified in the MUT gene (MUT) only one, the c.1874A>C (p.D625A) variant, is likely a mut⁻ mutation. The remaining mutations are probably mut⁰. Here, we present the first molecular analysis of MMA in Iranian patients and have identified eight novel mutations. Four novel mutations (p.D625A, p.R326G, p.V157F, p.F379L) were seen exclusively in patients from northern Iran. One novel splice site mutation (c.2125-3C>G) in MUT and two novel mutation (p.N225M and p.A99P) in the MMAA gene were associated with patients from eastern Iran. The rs184829210 SNP was recognized only in patients with the novel c.958G>A (p.A320T) mutation. This study confirms pathogenesis of deficient enzyme activity in MUT, MMAA, MMAB, and MMACHC as previous observations. These results could act as a basis for the performance of pharmacological therapies for increasing the activity of proteins derived from these mutations.

Mutation analysis MUT MMAA MMAB MMACHC Biochemical analysis Novel mutation Methylmalonic acidemia Iranian population

Evaluation of hemolysis effect on hemoglobin measurement by capillary electrophoresis.

22. Mokhtariye A, Varasteh A-R, Alaei A, Marzban S, Keify F
Journal of Analytical Science and Technology. 2019;10(1):6.Original article

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Background: Hemoglobin (Hb) is a hemeprotein with two linked pairs of globin chains. Each chain is connected to a heme residue in its center. Hemoglobinopathies are divided into quantitative and qualitative defects in globin synthesis. Hemolysis is a pre-analytical problem that reduces quality of sample for measurement of many analytes.

Methods: Blood samples from 311 female and 189 male subjects with normal and abnormal Hb electrophoresis patterns were studied. Three milliliters of whole blood was obtained from all subjects and transferred into EDTA tubes. To analyze hemolysis, 1.5 ml of blood from each tube was aliquoted and frozen, and the remaining blood was stored at 4 °C for 24 h. Hemoglobin was measured in both hemolyzed and non-hemolyzed samples by capillary electrophoresis.

Results: Data was analyzed with linear regression. The results were linear for the lower limit of detection of 9, 0.5, and 0.1% up to at least 99.5, 6.6, and 99.4% for HbA, HbA2, and HbF, respectively. Method comparison demonstrated good agreement between non-hemolyzed and hemolyzed conditions for hemoglobin measurement.

Conclusions: Use of hemolyzed samples had no effected on hemoglobin measurements.

Keywords: Hemoglobin, Pre-analytic, Capillary electrophoresis, Hemolysis