Identification of a SLC19A2 nonsense mutation in Persian families with thiamine-responsive megaloblastic anemia

This is an Open Access article. A definitive version was published in Gene, Vol.519, issue 2, (2013) doi: 10.1016/j.gene.2013.02.008.Thiamine-responsive megaloblastic anemia (TRMA) is an autosomal recessive syndrome characterized by early-onset anemia, diabetes, and hearing loss caused by mutations in the SLC19A2 gene. We studied the genetic cause and clinical features of this condition in patients from the Persian population. A clinical and molecular investigation was performed in four patients from three families and their healthy family members. All had the typical diagnostic criteria. The onset of hearing loss in three patients was at birth and one patient also had a stroke and seizure disorder. Thiamine treatment effectively corrected the anemia in all of our patients but did not prevent hearing loss. Diabetes was improved in one patient who presented at the age of 8months with anemia and diabetes after 2months of starting thiamine. The coding regions of SLC19A2 were sequenced in all patients. The identified mutation was tested in all members of the families. Molecular analyses identified a homozygous nonsense mutation c.697C>T (p.Gln233*) as the cause of the disease in all families. This mutation was previously reported in a Turkish patient with TRMA and is likely to be a founder mutation in the Persian population

Female Sex as a Risk Factor for Glycemic Control and Complications in Iranian Patients with Type One Diabetes Mellitus

Objective: The aim of this study was to evaluate the influence of sex on glycemic control, diabetes complications and associated abnormalities in patients with type one diabetes mellitus. Methods: In a cross-sectional study in 309 patients (156 females and 153 males within the age range of 3-16 years) with type one diabetes mellitus referred to endocrinology clinic in Children's Medical Center in Tehran from March 2005 to March 2007 gender differences in diabetes control were analyzed. Findings: Mean glycosylated hemoglobin (HbA1c), was significantly higher in females (9.25 vs. 8.01). Insulin dose per kilogram of body weight was significantly more in girls (0.91±0.31 vs. 0.74±0.37, P<0.001) self monitoring of blood glucose was performed significantly more in boys. Frequency of Diabetic ketoacidosis, height growth problems and dyslipidemia were significantly higher in girls. 1.20±0.86 vs. 0.93±0.55, P=0.004), (-0.05±1.20 vs. -0.41±1.17, P=0.015), (134.60±44.43 vs. 110.56±20.72, P=<0.001) respectively. Conclusion: Female sex is a risk factor in glycemic control and complications of diabetes type I and females should be managed more seriously regarding self monitoring of blood glucose, nutritional and psychological factors and puberty issues

Tetrahydrobiopterin responsiveness in a series of 53 cases of phenylketonuria and hyperphenylalaninemia in Iran

To determine the prevalence of 6R-Tetrahydrobiopterin (BH4) responsive phenylketonuria (PKU) in 53 cases of patients with various classification of hyperphenylalaninemia and PKU Excluding the BH4 deficient type referring to children's medical center in Iran (phenylalanine 360–2420 μmol/L), the single dose of 20 mg/kg (Kuvan®) and duration of 24 h was used. Results: Among the 4 different categories of mild hyperphenylalaninemia requiring treatment, mild, moderate and classic PKU, the BH4 responders were 90%, 35.7%, 5.6% and 0% respectively after 24 h. Conclusion: BH4 responsiveness is more prevalent in mild hyperphenylalaninemia and mild PKU patients in Iran

p.Gln318X and p.Val281Leu as the Major Variants of CYP21A2 Gene in Children with Idiopathic Premature Pubarche

Premature pubarche (PP) is the appearance of sexual hair in children before puberty. The PP phenotype may attribute to nonclassic congenital adrenal hyperplasia (NC-CAH). In this study, we investigated the role of CYP21A2 gene variants in patients with PP in the Iranian population. Forty patients (13 males and 27 females), clinically diagnosed with PP, were analyzed for molecular testing of CYP21A2 gene variants. Direct sequencing was performed for the samples. Also, gene dosage analysis was performed for the cases. Fourteen patients (35%) had a mutation of p.Gln318X and p.Val281Leu, out of which 10% had regulatory variants. Approximately 10% of the patients were homozygous (NC-CAH). 78.5% (11/14) of patients had trimodular RCCX of which 5 patients had two copies of CYP21A1P pseudogene. The prevalence of p.Val281Leu was higher than p.Gln318X in PP patients. In conclusion, CYP21A2 variant detection has implications in the genetic diagnosis of PP phenotype. The genetic characterization of the CYP21A2 gene is important for characterizing the variable phenotype of carriers and genetic counseling of PP and NC-CAH patients

An 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

Diagnosing mucopolysaccharidosis type IV a by the fluorometric assay of N-Acetylgalactosamine-6-sulfate sulfatase activity

Background: Mucopolysaccharidosis type IVA, also known as Morquio A or MPS IV A, is an autosomal recessive disease caused by the deficiency of the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS). The loss of GALNS activity leads to the impaired breakdown of glycosaminoglycans (GAGs) keratan sulfate and chondroitin- 6-sulfate. The accumulation of GAGs results in multiple organ damage. The accurate and early diagnosis of this disorder helps enhance the effectiveness of the treatment. The present study uses a pre-designed protocol for testing GALNS activity in the leukocytes of Iranian patients with MPS IV A and their parents and compares it with healthy controls. Methods: Patients with MPS IVA previously diagnosed through the measurement of enzyme activity or genetic analysis entered the study. Leukocytes were obtained from the heparinized blood of the participants. The GALNS activity was measured by a fluorometric method using 4-methylumbelliferyl-β-D-galactoside-6-sulfate (4MU-G6S) as the substrate and proper buffer solutions and calibrators. Results: The GALNS activity (nmol/17 h/mg protein) was reported as 0–7.4 in the MPSIV A patients, as 19.85–93.7 in their parents and as 38.4–164 in the healthy controls. Statistically significant differences were observed between the three groups in terms of enzyme activity. There were no significant differences in enzyme activity by age. The female subjects in both the patient and parents groups showed lower enzyme activity compared to the male subjects. Conclusion: The fluorometric method was validated for the measurement of GALNS activity in leukocyte samples and identifying Iranian patients with MPS IV A

Four years of diagnostic challenges with tetrahydrobiopterin deficiencies in Iranian patients

Hyperphenylalaninemia (HPA) is a condition caused by tetrahydrobiopterin (BH4) and phenylalanine hydroxylase (PAH) deficiencies. It is essential that differential diagnosis be conducted to distinguish these two causes of HPA, because BH4 deficiency is a more severe disease involving progressive neurologic deterioration. Based on the biological findings, HPA is defined as a plasma phenylalanine level of >2.0 mg/dl (>120 μmol/l). The National Biochemistry Reference Laboratory at the Pasteur Institute of Iran initiated BH4 deficiency screening tests for the first time during the implementation of a nationwide phenylketonuria (PKU) screening program. Measurement of blood phenylalanine and urinary neopterin and biopterin was conducted by high-performance liquid chromatography in 617 patients with HPA. Dihydropteridine reductase (DHPR) activity was measured in all patients by kinetic spectrophotometry. Differential diagnosis was conducted for PKU, transient HPA, and BH4 deficiencies.Our results indicated that out of 76 cases involving BH4 deficiencies, 37 had 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency, 35 had DHPR deficiency, 1 case had pterin-4a-carbinolamine dehydratase (PCD) deficiency, and 3 cases had GTP cyclohydrolase I (GTPCH) deficiency. In this study, 1 novel deletion mutation and 18 novel missense mutations were reported in addition to mutations that had previously been identified and registered in BIOMDB. At present, the screening program for PKU in Iran includes tests that detect different forms of BH4 deficiency presenting with HPA. Newborns that are BH4-deficient benefit from the availability of the tests because they can receive necessary care before being clinically affected

Discriminative Features in Three Autosomal Recessive Cutis Laxa Syndromes: Cutis Laxa IIA, Cutis Laxa IIB, and Geroderma Osteoplastica

Cutis laxa is a heterogeneous condition characterized by redundant, sagging, inelastic, and wrinkled skin. The inherited forms of this disease are rare and can have autosomal dominant, autosomal recessive, or X-linked inheritance. Three of the autosomal recessive cutis laxa syndromes, namely cutis laxa IIA (ARCL2A), cutis laxa IIB (ARCL2B), and geroderma osteodysplastica (GO), have very similar clinical features, complicating accurate diagnosis. Individuals with these conditions often present with cutis laxa, progeroid features, and hyperextensible joints. These conditions also share additional features, such as short stature, hypotonia, and congenital hip dislocation, but the severity and frequency of these findings are variable in each of these cutis laxa syndromes. The characteristic features for ARCL2A are abnormal isoelectric focusing and facial features, including downslanting palpebral fissures and a long philtrum. Rather, the clinical phenotype of ARCL2B includes severe wrinkling of the dorsum of the hands and feet, wormian bones, athetoid movements, lipodystrophy, cataract and corneal clouding, a thin triangular face, and a pinched nose. Normal cognition and osteopenia leading to pathological fractures, maxillary hypoplasia, and oblique furrowing from the outer canthus to the lateral border of the supraorbital ridge are discriminative features for GO. Here we present 10 Iranian patients who were initially diagnosed clinically using the respective features of each cutis laxa syndrome. Each patient's clinical diagnosis was then confirmed with molecular investigation of the responsible gene. Review of the clinical features from the cases reported from the literature also supports our conclusions.status: publishe

Identification of homozygous WFS1 mutations (p.Asp211Asn, p.Gln486*) causing severe Wolfram syndrome and first report of male fertility

Wolfram syndrome (WFS) is a neurodegenerative genetic condition characterized by juvenile-onset of diabetes mellitus and optic atrophy. We studied clinical features and the molecular basis of severe WFS (neurodegenerative complications) in two consanguineous families from Iran. A clinical and molecular genetic investigation was performed in the affected and healthy members of two families. The clinical diagnosis of WFS was confirmed by the existence of diabetes mellitus and optic atrophy in the affected patients, who in addition had severe neurodegenerative complications. Sequencing of WFS1 was undertaken in one affected member from each family. Targeted mutations were tested in all members of relevant families. Patients had most of the reported features of WFS. Two affected males in the first family had fathered unaffected children. We identified two homozygous mutations previously reported with apparently milder phenotypes: family 1: c.631G>A (p.Asp211Asn) in exon 5, and family 2: c.1456C>T (p.Gln486*) in exon 8. Heterozygous carriers were unaffected. This is the first report of male Wolfram patients who have successfully fathered children. Surprisingly, they also had almost all the complications associated with WFS. Our report has implications for genetic counseling and family planning advice for other affected families