RESUMEN

Haemoglobin (Hb) Lepore is a variant Hb consisting of two α-globin and two δβ-globin chains. In a heterozygote, it is associated with clinical findings of thalassaemia minor, but interactions with other haemoglobinopathies can lead to various clinical phenotypes and pose diagnostic challenges. We reported a pair of siblings from a Malay family, who presented with pallor and hepatosplenomegaly at the ages of 21 months and 14 months old. The red cell indices and peripheral blood smears of both patients showed features of thalassaemia intermedia. Other laboratory investigations of the patients showed conflicting results. However, laboratory investigation results of the parents had led to a presumptive diagnosis of compound heterozygote Hb Lepore/β-thalassaemia and co-inheritance α+-thalassaemia (-α3.7). Hb Lepore has rarely been detected in Southeast Asian countries, particularly in Malaysia. These two cases highlight the importance of family studies for accurate diagnosis, hence appropriate clinical management and genetic counseling.

RESUMEN

Alpha (Α) thalassaemia is the most common inherited disorder in Malaysia. The clinical severity is dependant on the number of Α genes involved. Full blood count (FBC) and haemoglobin (Hb) analysis using either gel electrophoresis, high performance liquid chromatography (HPLC) or capillary zone electrophoresis (CE) are unable to detect definitively alpha thalassaemia carriers. Definitive diagnosis of Α-thalassaemias requires molecular analysis and methods of detecting both common deletional and non-deletional molecular abnormailities are easily performed in any laboratory involved in molecular diagnostics. We carried out a retrospective analysis of 1623 cases referred to our laboratory in Universiti Kebangsaan Malaysia Medical Centre (UKMMC) for the diagnosis of Α-thalassaemia during the period October 2001 to December 2012. We examined the frequency of different types of alpha gene abnormalities and their haematologic features. Molecular diagnosis was made using a combination of multiplex polymerase reaction (PCR) and real time PCR to detect deletional and non-deletional alpha genes relevant to southeast Asian population. Genetic analysis confirmed the diagnosis of Α-thalassaemias in 736 cases. Majority of the cases were Chinese (53.1%) followed by Malays (44.2%), and Indians (2.7%). The most common gene abnormality was ΑΑ/--(SEA) (64.0%) followed by ΑΑ/-Α(3.7) (19.8%), -Α(3.7) /--(SEA) (6.9%), ΑΑ/ΑΑCS (3.0%), --(SEA)/--(SEA) (1.2%), -Α(3.7)/-Α(3.7) (1.1%), ΑΑ/-Α(4.2) (0.7%), -Α(4.2)/--(SEA (0.7%), -Α(3.7)/-Α(4.2) (0.5%), ΑΑ(CS)/-- SEA) (0.4%), ΑΑ(CS)/ΑΑ(Cd59) (0.4%), ΑΑ(CS)/ΑΑ(CS) (0.4%), -Α(3.7)/ΑΑ(Cd59) (0.3%), ΑΑ/ΑΑ(Cd59) (0.1%), ΑΑ(Cd59)/ ΑΑ(IVS I-1) (0.1%), -Α(3.7)/ΑΑ(CS) (0.1%) and --(SEA) /ΑΑ(Cd59) (0.1%). This data indicates that the molecular abnormalities of Α-thalassaemia in the Malaysian population is heterogenous. Although Α-gene deletion is the most common cause, non-deletional Α-gene abnormalities are not uncommon and at least 3 different mutations exist. Establishment of rapid and easy molecular techniques is important for definitive diagnosis of alpha thalassaemia, an important prerequisite for genetic counselling to prevent its deleterious complications.

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RESUMEN

Alpha (α) thalassaemia is the most common inherited disorder in Malaysia. The clinical severity is dependant on the number of α genes involved. Full blood count (FBC) and haemoglobin (Hb) analysis using either gel electrophoresis, high performance liquid chromatography (HPLC) or capillary zone electrophoresis (CE) are unable to detect definitively alpha thalassaemia carriers. Definitive diagnosis of α-thalassaemias requires molecular analysis and methods of detecting both common deletional and non-deletional molecular abnormailities are easily performed in any laboratory involved in molecular diagnostics. We carried out a retrospective analysis of 1623 cases referred to our laboratory in Universiti Kebangsaan Malaysia Medical Centre (UKMMC) for the diagnosis of α-thalassaemia during the period October 2001 to December 2012. We examined the frequency of different types of alpha gene abnormalities and their haematologic features. Molecular diagnosis was made using a combination of multiplex polymerase reaction (PCR) and real time PCR to detect deletional and non-deletional alpha genes relevant to southeast Asian population. Genetic analysis confirmed the diagnosis of α-thalassaemias in 736 cases. Majority of the cases were Chinese (53.1%) followed by Malays (44.2%), and Indians (2.7%). The most common gene abnormality was αα/--SEA (64.0%) followed by αα/-α3.7 (19.8%), -α3.7 /--SEA (6.9%), αα/ααCS (3.0%), --SEA/--SEA (1.2%), -α3.7/-α3.7 (1.1%), αα/-α4.2 (0.7%), -α4.2/--SEA (0.7%), -α3.7/-α4.2 (0.5%), ααCS/-- SEA (0.4%), ααCS/ααCd59 (0.4%), ααCS/ααCS (0.4%), -α3.7/ααCd59 (0.3%), αα/ααCd59 (0.1%), αα Cd59/ ααIVS I-1 (0.1%), -α3.7/ααCS (0.1%) and --SEA /ααCd59 (0.1%). This data indicates that the molecular abnormalities of α-thalassaemia in the Malaysian population is heterogenous. Although α-gene deletion is the most common cause, non-deletional α-gene abnormalities are not uncommon and at least 3 different mutations exist. Establishment of rapid and easy molecular techniques is important for definitive diagnosis of alpha thalassaemia, an important prerequisite for genetic counselling to prevent its deleterious complications.

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RESUMEN

Haemoglobin Constant Spring (Hb CS) mutation and single gene deletions are common underlying genetic abnormalities for alpha thalassaemias. Co-inheritance of deletional and non-deletional alpha (α) thalassaemias may result in various thalassaemia syndromes. Concomitant co-inheritance with beta (β) and delta (δ) gene abnormalities would result in improved clinical phenotype. We report here a 33-year-old male patient who was admitted with dengue haemorrhagic fever, with a background history of Grave’s disease, incidentally noted to have mild hypochromic microcytic red cell indices. Physical examination revealed no thalassaemic features or hepatosplenomegaly. His full blood picture showed hypochromic microcytic red cells with normal haemoglobin (Hb) level. Quantitation of Hb using high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) revealed raised Hb F, normal Hb A2 and Hb A levels. There was also small peak of Hb CS noted in CE. H inclusions was negative. Kleihauer test was positive with heterocellular distribution of Hb F among the red cells. DNA analysis for α globin gene mutations showed a single -α-3.7 deletion and Hb CS mutation. These fi ndings were suggestive of compound heterozygosity of Hb CS and a single -α-3.7 deletion with a concomitant heterozygous δβ thalassaemia. Co-inheritance of Hb CS and a single -α-3.7 deletion is expected to result at the very least in a clinical phenotype similar to that of two alpha genes deletion. However we demonstrate here a phenotypic modifi cation of α thalassemia presumptively as a result of co-inheritance with δβ chain abnormality as suggested by the high Hb F level.

RESUMEN

Sickle cell disease (SCD) is an inherited red cell disorder, characterized by the tendency of haemoglobin S or sickle haemoglobin to polymerize and assume a characteristic sickle shape. Molecular analysis has been the mainstay of detection method when confirmation is required. Previously a polymerase chain reaction (PCR)-based restriction enzyme analysis was used for this purpose. A simple bidirectional allele-specific amplification, recently described by Waterfall in 2001 was used to detect the GAG --> GTG mutation on codon 6 of the beta globin gene. Two sets of primers for the mutant and the wild type alleles were used in a single PCR reaction to amplify the regions of interest. The resultant PCR products will produce two fragments at 517 and 267 base pair (bp) respectively. This report highlights the investigations for SCD in the family of a 16-year old girl with recurrent painful crisis affecting the lower limbs whereby the family members are asymptomatic for the disease. Her haemoglobin electrophoresis at an alkaline pH showed dense bands at the HbS and HbF regions, while her father and two sisters had bands at HbS, HbF and HbA. The PCR analysis showed that she was homozygous for the mutation by the presence of only one band at 267 bp fragment, while the father and her sisters were heterozygotes, with the presence of two bands at 267 as well as 517 bp fragments. DNA sequencing of the sample confirmed the mutation. In conclusion, this case report highlighted the simple and cheap yet practical method for molecular confirmation of the presence of HbS gene in subjects with homozygous or heterozygous state of the condition.

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RESUMEN

Sickle cell disease (SCD) is an inherited red cell disorder, characterized by the tendency of haemoglobin S or sickle haemoglobin to polymerize and assume a characteristic sickle shape. Molecular analysis has been the mainstay of detection method when confirmation is required. Previously a polymerase chain reaction (PCR)-based restriction enzyme analysis was used for this purpose. A simple bidirectional allele-specific amplification, recently described by Waterfall in 2001 was used to detect the GAG --> GTG mutation on codon 6 of the beta globin gene. Two sets of primers for the mutant and the wild type alleles were used in a single PCR reaction to amplify the regions of interest. The resultant PCR products will produce two fragments at 517 and 267 base pair (bp) respectively. This report highlights the investigations for SCD in the family of a 16-year old girl with recurrent painful crisis affecting the lower limbs whereby the family members are asymptomatic for the disease. Her haemoglobin electrophoresis at an alkaline pH showed dense bands at the HbS and HbF regions, while her father and two sisters had bands at HbS, HbF and HbA. The PCR analysis showed that she was homozygous for the mutation by the presence of only one band at 267 bp fragment, while the father and her sisters were heterozygotes, with the presence of two bands at 267 as well as 517 bp fragments. DNA sequencing of the sample confirmed the mutation. In conclusion, this case report highlighted the simple and cheap yet practical method for molecular confirmation of the presence of HbS gene in subjects with homozygous or heterozygous state of the condition.

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RESUMEN

OBJECTIVES: This study aimed to determine the prevalence of four variants of organic anion transporter polypeptide 2 (OATP2) gene, and their association with severe hyperbilirubinemia. DESIGN: Observational study. SETTING: A tertiary university unit. PATIENTS: Term infants of Chinese descent. METHODS: 175 infants, consisting of 65 admitted for treatment of severe hyperbilirubinemia (with serum bilirubin levels > 250 mmol/L at age 1-2 days or > 300 micromol/L at age > or = 3 days) and 110 randomly selected inborn infants without severe hyperbilirubinemia during their first month of life, were recruited. Their blood samples were subjected to sequencing analysis of exon 4 and exon 5 of OATP2 gene for detection of c.388A > G, c.521T > C, c.571T > C and c.597C > T variants. RESULTS: The c.388A > G variant was the most common, and the c.521 T > C was least common, being present in 90.9% and 26.9% of the infants, respectively. Forward logistic regression analysis showed that the only significant risk factors associated with severe hyperbilirubinemia among these Chinese infants were: exclusive breast feeding (adjusted odds ratio (OR) = 12.5, 95% C.I.: 2.9, 53.4; p = 0.001), infants with homozygous 211 variant of the UDPG 1A1 gene (adjusted OR = 37.7, 95% C.I.: 4.4, 324.1; p = 0.001), and G6PD enzyme level < 8.5 IU/g Hb (adjusted OR = 7.3, 95% C.I.: 3.1, 17.5; p < 0.00001). Gestational age, G6PD mutation status, actual G6PD enzyme level, and the 4 variants of the OATP2 gene mutation were not significant risk factors. CONCLUSION: Variants of OATP2 gene were not significant risk factors associated with severe hyperbilirubinemia in Malaysian Chinese infants.

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RESUMEN

Objectives: This study aimed to determine the prevalence of four variants of organic anion transporter polypeptide 2 (OATP2) gene, and their association with severe hyperbilirubinemia. Design: Observational study. Setting: A tertiary university unit. Patients: Term infants of Chinese descent. Methods: 175 infants, consisting of 65 admitted for treatment of severe hyperbilirubinemia (with serum bilirubin levels > 250 mmol/L at age 1-2 days or > 300 μmol/L at age ≥ 3 days) and 110 randomly selected inborn infants without severe hyperbilirubinemia during their fi rst month of life, were recruited. Their blood samples were subjected to sequencing analysis of exon 4 and exon 5 of OATP2 gene for detection of c.388A>G, c.521T>C, c.571T>C and c.597C>T variants. Results: The c.388A>G variant was the most common, and the c.521 T>C was least common, being present in 90.9% and 26.9% of the infants, respectively. Forward logistic regression analysis showed that the only signifi cant risk factors associated with severe hyperbilirubinemia among these Chinese infants were: exclusive breast feeding (adjusted odds ratio (OR) = 12.5, 95% C.I.: 2.9, 53.4; p=0.001), infants with homozygous 211 variant of the UDPG 1A1 gene (adjusted OR = 37.7, 95% C.I.: 4.4, 324.1; p=0.001), and G6PD enzyme level <8.5 IU/g Hb (adjusted OR = 7.3, 95% C.I.: 3.1, 17.5; p<0.00001). Gestational age, G6PD mutation status, actual G6PD enzyme level, and the 4 variants of the OATP2 gene mutation were not signifi cant risk factors. Conclusion: Variants of OATP2 gene were not signifi cant risk factors associated with severe hyperbilirubinemia in Malaysian Chinese infants.

RESUMEN

We performed DNA analysis on cord blood samples of 128 Chinese male neonates diagnosed as G6PD deficiency in Hospital Universiti Kebangsaan Malaysia by a combination PCR-restriction enzyme digest technique, Single Stranded Conformation Polymorphism analysis and DNA sequencing. We found 10 different G6PD-deficient mutations exist. The two commonest alleles were G6PD Canton 1376 G>T (42.3%) and Kaiping 1388 G>A (39.4%) followed by G6PD Gaohe 592 G>A (7.0%), Chinese-5 1024 C>T, Nankang 517 T>C (1.5%), Mahidol 487 G>A (1.6%), Chatham 1003 G>T (0.8%), Union 1360 C>T (0.8%), Viangchan 871 G>A (0.8%) and Quing Yang 392 G>T (0.8%). Sixty eight percent (88/125) neonates in this study had neonatal jaundice and 29.7% developed hyperbilirubinemia >250 micromol/l. The incidence of hyperbilirubinemia >250 micromol/l was higher in G6PD Kaiping (43.8%) than G6PD Canton (22%) (p< 0.05). There was no significant difference in the incidence of neonatal jaundice, mean serum bilirubin, mean age for peak serum bilirubin, percentage of babies requiring phototherapy and mean duration of phototherapy between the two major variants. None of the 88 neonates required exchange transfusion. In conclusion we have completely characterized the molecular defects of a group of Chinese G6PD deficiency in Malaysia. The mutation distribution reflects the original genetic pool and limited ethnic admixture with indigenous Malays.

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