Carrier testing and prenatal diagnosis of haemophilia B by SSCP in an Iranian family

Haemophilia B is an X-linked recessive bleeding disorder caused by multiple molecular defects in factor IX gene. During our genotyping programme of haemophilia B patients, a family from the west of Iran was referred to us. We first detected the mutation in the index case of the family by single-strand conformation polymorphism (SSCP) technique. This technique was then applied in carrier detection and prenatal diagnosis for females of this family. Sequencing later revealed the mutation as being G to A at 20519.     

Genomics‐based treatment in a patient with two overlapping heritable skin disorders: Epidermolysis bullosa and acrodermatitis enteropathica

Next‐generation sequencing (NGS) is helpful in diagnosing complex genetic disorders and phenotypes, particularly when more than one overlapping condition is present. From a large cohort of 362 families with clinical manifestations of skin and mucosal fragility, referred by several major medical centers, one patient was found by NGS to have two overlapping heritable skin diseases, recessive dystrophic epidermolysis bullosa (RDEB; COL7A1 mutations) and acrodermatitis enteropathica (AE; SLC39A4 mutations). The pathogenicity of the variants was studied at gene expression as well as ultrastructural and tissue levels. Although there is no specific treatment for RDEB except avoiding trauma, supplementation with oral zinc (3 mg·kg^?1·day^?1) for the AE resulted in rapid amelioration of the skin findings. This case demonstrates the power of NGS in identifying two genetically unlinked diseases that led to effective treatment with major clinical benefits as an example of genomics‐guided treatment.     

Type II protein C deficiency: identification and molecular modelling of two natural mutants with low anticoagulant and normal amidolytic activity

Two mutations in exons 3 and 9 of the protein C gene were identified by amplification and sequencing from symptomatic probands referred for venous thromboembolism and thrombophilia screening. The phenotype associated with the mutations is a type II protein C deficiency with normal amidolytic activity. In one family, the mutation in exon 3 (G3545-->A), which predicts an R9 to H substitution in the Gla domain, was identified. A mutation in exon 9 (G10899-->A), which predicts an R352 to W substitution in the catalytic site, was identified in the second family and has been reported previously in association with type II deficiency with low amidolytic activity. Western blotting of the purified proteins from the probands' plasma did not show any abnormal migratory pattern. Molecular modelling suggested a possible impairment in the recently described Na+ binding pocket for the R352-->W mutant. No conclusions could be drawn relative to the R9-->H mutant.     

Beta-thalassemia intermedia from southern Iran: IVS-II-1 (G-->A) is the prevalent thalassemia intermedia allele

The preliminary results of a pilot study are reported, intended as an initiation of a research plan, focused on the prevention of beta-thalassemia in Iran. The aims of this study are: (i) to improve the knowledge of the molecular background of beta-thalassemia intermedia in Southern Iran; (ii) to verify the role of the -158 (G)gamma (C-->T) (Xmn I) polymorphism as a modulating factor in thalassemia intermedia; (iii) to test the validity of the multiplex and single mutation specific amplification refractory mutation system in analyzing the molecular defects causing beta-thalassemia in multiethnic populations; and (iv) to develop suitable strategies for the application of prevention protocols in Iran. To accomplish the task we have selected 87 beta-thalassemia intermedia patients and adapted the DNA methodology to detect the following 11 frequent mutations in Iran: codon 5 (-CT); frameshift codons (FSC) 8/9 (+G); codon 30 (G-->C); IVS-I-1 (G-->A); IVS-I-5 (G-->C); IVS-I-6 (T-->C); IVS-I-110 (G-->A); codons 36/37 (-T); codon 44 (-C); IVS-II-1 (G-->A); IVS-II-745 (C-->G). Because of the multiethnicity of the population we have also included the Indian IVS-I (25 bp deletion) and the Mediterranean IVS-I-130 (G-->C) and codon 39 (C-->T) mutations. Forty-eight patients were randomly studied for the Xmn I polymorphism together with 50 healthy volunteers as a control group. The molecular analysis conducted in Iran, identified only 31% of the alleles that were presumed to be thalassemic, revealing either a strategic or a technical insufficiency of the chosen method. However, the mutations with the highest prevalence in the country (IVS-II-1, IVS-I-110, IVS-I-1 and FSC 8/9) were found. As expected the IVS-II-1 defect, being the most frequent in south Iran, was present at the highest rate (24%). The Xmn I polymorphism was found in association with this prevalent mutation and was detected in the homozygous state in 87.5% of the patients homozygous for the IVS-II-1 (G-->A) mutation. The overall positivity for Xmn I was found in 40.6% of the thalassemic alleles vs. 14% in the non-thalassemic, confirming the hypothesis of an older event, antecedent to the IVS-II-1 mutation. In trying to assess a more suitable molecular detection method we intend to continue this study in collaboration with the European centers involved, applying more effective technologies and better defining the molecular spectrum of beta-thalassemia in the sub-populations. We also intend to verify the effect of alpha-thalassemia in the genotype/phenotype correlation of beta-thalassemia intermedia.     

Efficacy of hepatic T2* MRI values and serum ferritin concentration in predicting thalassemia major classification for hematopoietic stem cell transplantation

Liver biopsy has been performed for many decades for classifying the patients with TM. Meanwhile, using non‐invasive methods such as T2* MRI technique has been recently much more considered to determine the hepatic iron overload. Ninety‐three pediatric HSCT candidates with TM who underwent liver biopsy were included in this study. Hepatic T2* MRI values and serum ferritin concentrations were assessed to investigate and determine the useful method in detection of patients with TM class III whom received different conditioning regimens, in comparison with class I and II. Twenty (21.5%) patients were categorized as class III. Hepatic T2* MRI could detect TM class III patients with 60% sensitivity and 87.67% specificity (LR+: 4.867, accuracy: 81.72%), while predictive feature of ferritin values for distinguishing patients with TM class III was not statistically significant (p‐value >0.01). Combination of T2*MRI with age (T2*‐age) could detect TM class III with 85% sensitivity and 72.6% specificity (LR+: 3.1, accuracy: 75.27%).T2*‐age may be considered as an alternative and non‐invasive method to liver biopsy for differentiation and classification of patients with TM before transplantation.     

Platelet kinetic studies in patients with idiopathic thrombocytopenic purpura

PURPOSE: To determine the value in diagnosis and treatment of mean platelet life, platelet production, and major sites of platelet destruction in patients with idiopathic thrombocytopenic purpura (ITP). PATIENTS AND METHODS: Sternal or posterior superior iliac spine bone marrow aspiration was performed in 141 patients. Platelet kinetic studies with Indium-111 tropolonate labeled autologous platelets were utilized to determine platelet production. RESULTS: Two subgroups of patients could be defined. The first group (n = 81, 58%) had normal or increased platelet production and increased peripheral platelet destruction. These patients fulfilled the conventional criteria for ITP, including reduced platelet survival time (mean +/- SD, 1.6 +/- 1.4 days). Forty-eight (59%) of these patients had increased splenic sequestration and 30 (88%) of the 34 patients who underwent splenectomy had a complete or partial remission. The second group (n = 60, 42%) had decreased platelet production, with significantly greater platelet survival times (3.6 +/- 2 days, P <0.0001). In this group, the proportion of patients with complete or partial response to splenectomy (62%) was somewhat lower (P = 0.09). These patients mainly had ineffective platelet production in the bone marrow. CONCLUSIONS: Platelet kinetic studies suggest that ITP is a heterogeneous disease that comprises two subgroups. Further studies are needed to validate these findings and to determine their effect on the choice and outcome of therapy.     

A modified conformation sensitive gel electrophoresis (CSGE) method for rapid and accurate detection of low density lipoprotein (LDL) receptor gene mutations in Familial Hypercholesterolemia

OBJECTIVES: A conformation sensitive gel electrophoresis (CSGE) method was modified for detection of LDL receptor gene mutations. DESIGN AND METHODS: Usage of a temperature gradient against running time was tested with 33 identified mutations in the LDL receptor gene. Most of the mutations were missense. RESULTS: 32 mutations were detected using this method (sensitivity: 97%). The duration required for running the test for each of the exons was reduced to 3-4 h. CONCLUSIONS: This modified CSGE method may be used as a screening procedure in genetic diseases where a variety of mutations may cause illness.