Complex Interaction of Hb Q-Thailand with α and β Thalassemia in a Hakka Family

Background HbQ-Thailand is an α-globin chain variant that results from a point mutation at codon 74 of the α1-globin gene on chromosome 16p. It commonly appears with a leftward single α-globin gene deletion (-α 4.2 ). There have been few reports regarding the interaction between HbQ-Thailand and other globin gene disorders. Here we found and diagnosed it in the Hakka population of the Fujian Province, China. The study provides an important reference for the clinic diagnose and genetic counseling of thalassemia and hemoglobin diseases. Methods Fresh peripheral blood samples were collected from the proband and her family members testing hematological parameters, hemoglobin components, thalassemia gene, and hemoglobin variants. Results The proband (II1) and her sister (II5) manifested in the obvious microcytic hypochromic anaemia. The CE electropherogram of II1 showed an abnormal band in the migration time at 185 s, which was confirmed as HbQ-Thailand. Another exception band appeared at 250 s of migration time and was proved to be HbE by sequence analysis method. The CE electropherogram of I1 and II3 showed an anomalous band HbE. The mother of the proband (I2) and the III4 and III5 of the family members showed a HbQ-Thailand. The gene results showed that the father (I1) also carried α- and β-thalassemia genes. His genotype was -- SEA and β codons26 ; -- SEA was inherited to II1, II 3, II5, III 1, and III2, and β codons26 was inherited to II1 and II3. The mother (I2) carries the -α 4.2 gene, which was inherited to II1, II5, III4, and III5. Conclusion It was complex to diagnose when the thalassemia combined with several abnormal haemoglobin disorders, and we may use various methods to mutual confirmation. Here we found and diagnosed a rare hemoglobin disease in the Hakka population of the Fujian Province. The study provides an important reference for the clinic diagnose and genetic counseling.

Unusual genomic rearrangements in introns 1 and 22 of the F8 gene

SummaryIntron 1 and intron 22 inversions, two large rearrangements of the factor VIII gene, are generally associated with a severe phenotype of haemophilia A and a high risk of inhibitor formation. In several haemophiliacs, diagnostic analyses for detection of these inversions revealed unusual band patterns. Upon further examination, different copy number variations were detected in the factor VIII gene of these patients by multiplex ligationdependent probe amplification (MLPA). Since these duplications or deletions alone could not sufficiently explain the abnormal band patterns of the first analyses, we assumed a combination of intron 1 or intron 22 inversions together with a copy number variation. Result We could confirm this hypothesis by specific long range PCRs but a detailed characterization of the breakpoints and the mechanisms for these complex rearrangements have yet to be elucidated.

Clinical Features for Heterozygosity of the Common β Globin Gene Frameshift 41/42 Mutation: Identification and Properties of Hb Salouël (β[Phe41Leu;Val59X])

Coding region amino acid substitutions within the globin genes can cause thalassemia, any of a wide variety of pathologic variants or entirely harmless variant haemoglobin. Which occurs depends upon the nature and location of the mutation. Both structural and thalassemia-producing mutations can occur for a unique mutation depending on the mutant sequence produced. Homozygotes for frameshift β 41/42 and compound β 41/42 and beta0-thalassemia produced heterozygotes for frameshift severe symptoms and have a thalassemia major phenotype. Severe phenotype has not been reported in the β 41/42 frameshift heterozygosity and the resulting abnormal hemoglobin has never been studied. We report the β c.126–129del CTTT frameshift mutation (exon1) resulting in a truncated β chain (58 AA) in a heterozygous Vietnamese adult patient who was referred to the hospital for anemia and renal failure The haematological indices were as follows: Hb 9.0g/dL, RBC 4.38×1012L, MCH 19.8 pg, MCV 62 fL, and reticulocytes 100 000/mm3 (4.4%). Serum creatinine was 221 mmol/L and creatinine clearance was low (22 ml/mn/1.73m2). Both kidneys were small (L=64mm;l=22mm). Renal biopsy was not performed for this reason. Hemoglobin electrophoresis at alkaline pH did not show any abnormal band while electrophoresis at acidic pH on citrate agar plate demonstrated an abnormal band less cathodal than Hb F. Hb A2 was 5.7%. The elution profile obtained from VARIANT ™ HPLC system was normal when using the β-thalassemia short program (Bio-Rad Laboratories, Hercules, CA, USA), while an abnormal peak representing 10% of the total amount of Hb was identified in the D/E area when using the HbA1c elution program. Both the isopropanol precipitation and heat stability tests showed a relevant molecular instability of the variant Hb. Heinz body test was positive. The Hb variant was isolated from the unstable component. The seven common α-thalassemia deletion defects as well as triplication were excluded using gap-PCR. Except for Hb Indianapolis, no clear cases of renal failure have ever been reported in association with the heterozygous state for unstable β chain. We have studied the truncated β globin chain in order to understand the instability of the abnormal hemoglobin. We have built a 3D model of the abnormal variant. Using biochemistry techniques and molecular modelling we show that: the synthetic 58 amino acids truncated protein was unable to fold in physiological conditions, sequence from AA 41 to AA 53 is structured as a primary helix but fully buried and unable to be exposed to the solvent, the Phe42Ser substitution is responsible for the major structural defect, key residues in position β 42, 44, 45, 48, 49, 51, 54, 55 and 57 are very sensitive for stability of hemoglobin as most of the substitutions for these residues disturb subunit packing in flexible joint probably leading to dissociation at β globin gene with this 4 bp deletion encodes the α1-β1 interface. The a truncated translational product which is unstable in the erythrocyte β–globin chains. Our data leading to the unbalancing of the α and suggest that this thalassemic hemoglobinopathy may have a variable phenotypic pattern in heterozygotes and may sometimes be associated with haemolytic crisis responsible for late renal failure.

Use of CSGE, TspRI- RFLP and Western Blot in Carrier Detection in an Indian Family with Type I Glanzmann Thrombasthenia.

Glanzmann Thrombasthenia (GT) is an inherited, autosomal recessive, bleeding disorder which is characterized by absent/reduced platelet Glycoprotein IIb/IIIa. The sub classification of GT into Types I, II and III is based on the levels of GPIIb/IIIa by flow cytometry. Type I is the most severe form of GT and is found to be most common in north Indian population. Since not much study is available on carrier detection based on western blot analysis, it is suggested to confirm the defect in carriers by molecular diagnosis. Here we present a carrier status using TspRI in a family with Glanzmann Thrombasthenia patient. Glanzmann Thrombasthenia was diagnosed in a patient with bleeding manifestations accompanied by absent platelet aggregation, secondary to ADP, ADR, Arachidonic acid and collagen. The patient was sub typed as Type I based on flow cytometry analysis as he had absent GPIIb/IIIa. Patient’s DNA was analyzed for mutation in both the gpIIb and gpIIIa genes by CSGE, followed by sequencing. The patient was found to have mutation, CTG>CCG at exon 12 of GPIIb gene. The mutation caused amino acid change from Leu to Pro in the GPIIb protein. The same mutation was looked for in all the family members (Both parents and two siblings) using CSGE and by TspRI- RFLP analysis. Both the parents and siblings were heterozygous for this mutation, where as patient was homozygous (Fig 1). As this mutation is not present in the normal individuals and is not reported earlier, this considers being a novel mutation. Presence of abnormal protein in the family members was revealed by western blot analysis for GPIIb (Fig 2). The same mutation is being looked for in more number of patients with Glanzmann Thrombasthenia using TspRI- RFLP. So far, a total of two out of 25 GT patients found to carry this mutation. It is possible that abnormal GPIIb protein by western blot in family members may reflect their carrier status. It is also postulated that western blot and CSGE of GPIIb and IIIa in parents/siblings may detect carrier status in Glanzmann Thrombasthenia. Fig 1: Carrier detection by restriction digestion using TspRI Fig 1:. Carrier detection by restriction digestion using TspRI Fig 2: Immunoblot followed by chemiluminescent detection shows absent/reduced protein in patient and abnormal band pattern in the family members Fig 2:. Immunoblot followed by chemiluminescent detection shows absent/reduced protein in patient and abnormal band pattern in the family members