Multiplex Minisequencing of the HBB Gene: A Rapid Strategy to Confirm the Most Frequent β-Thalassemia Mutations in the Tunisian Population

Abstract

The β hemoglobinopathies [β-thalassemia (β-thal) and structural hemoglobin (Hb) variants such as Hb S (HBB: c.20A?>?T) and Hb E (HBB: c.79G?>?A)] are among the most common inherited diseases worldwide. In Tunisia, due to the high prevalence of consanguineous marriages, the recurrent risk of this disease is high. The average prevalence of hemoglobinopathies is 4.48%, reaching 12.50% in some focus regions. The molecular investigations on thalassemia contributed to establishing the spectrum of mutations in the Tunisian population. The total number of HBB gene mutations identified was 24. The two most frequent mutations, codon 39 (C?>?T) (HBB: c.118C?>?T) and IVS-I-110 (G?>?A) (HBB: c.93–21G?>?A) accounted for 70.0% of the total encountered β-thal cases. These two mutations together with IVS-I-2 (T?>?G) (HBB: c.92?+?2T?>?G) and the Hb S variant account for more than 90.0% of all HBB genetic variants in Tunisia. Thus, developing rapid, inexpensive and reliable mutation-specific molecular diagnostic assays targeting our Tunisian populations is our aim to facilitate routine detection of hemoglobinopathies. In this report, we describe the successful application of the multiplex minisequencing assay as an alternative strategy for genetic diagnosis of HBB gene disorders in Tunisia.     

Genetic testing of Mucopolysaccharidoses disease using multiplex PCR- based panels of STR markers: in silico analysis of novel mutations

The Mucopolysaccharidoses (MPS) are group of inherited metabolic diseases caused by the deficiency of enzymes required to degrade glycosaminoglycans (GAGs) in the lysosomes. GAGs are sulfated polysaccharides involving repeating disaccharides, uronic acid and hexosamines including chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS) and keratan sulfate (KS). Hyaluronan is excluded in terms of being non-sulfated in the GAG family. Different types of mutations have been identified as the causative agent in all types of MPS. Herein, we planned to investigate the pathogenic mutations in different types of MPS including type I (IDUA gene), IIIA (SGSH) and IIIB (NAGLU) in the eight Iranian patients. Autozygosity mapping was performed to identify the potential pathogenic variants in these 8 patients indirectly with the clinical diagnosis of MPSs. so three panels of STR (Short Tandem Repeat) markres flanking IDUA, SGSH and NAGLU genes were selected for multiplex PCR amplification. Then in each family candidate gene was sequenced to identify the pathogenic mutation. Our study showed two novel mutations c.469 T?>?C and c.903C?>?G in the IDUA gene, four recurrent mutations: c.1A?>?C in IDUA, c.220C?>?T, c.1298G?>?A in SGSH gene and c.457G?>?A in the NAGLU gene. The c.1A?>?C in IDUA was the most common mutation in our study. In silico analysis were performed as well to predict the pathogenicity of the novel variants.

     

Molecular Epidemiological Investigation of Thalassemia in the Chengdu Region,Sichuan Province,Southwest China

Abstract

Thalassemia is the most common inherited disease in southern China. However, this disorder is usually ignored by the health system in the Sichuan Province due to the lack of epidemiological data. To provide basic epidemiological data for thalassemia screening, genetic counseling, and prenatal diagnosis (PND) in the Chengdu region, a total of 3262 healthy subjects were assessed by complete blood count (CBC), reverse dot-blot gene chip, gap-polymerase chain reaction (gap-PCR), and PCR-DNA sequencing. A frequency of heterozygous thalassemia of 3.43% (112/3262) was found, of which 2.21% (72/3262) patients carried α-thalassemia (α-thal), 1.19% (39/3262) β-thalassemia (β-thal) and 0.3% (1/3262) hereditary persistence of fetal hemoglobin (Hb) (HPFH)/δβ-thalassemia (δβ-thal). Four types of α-thal mutations were found, the most prevalent being –?–^SEA (68.06%), followed by ?α^3.7 (rightward deletion, 25.0%), Hb Quong Sze (Hb QS; HBA2: c.377?T?>?C) (4.17%), and ?α^4.2 (leftward deletion, 2.78%). The seven β-thal mutations included: codons 41/42 (?TTCT), HBB: c.126_129delCTTT (13/39, 33.33%); codon 17 (A?>?T), HBB: c.52?A?>?T (11/39, 28.95%); IVS-II-654 (C?>?T), HBB: c.316-197?C?>?T (9/39, 23.68%); ?28 (A?>?G), HBB: c.–78?A?>?G (3/39, 7.69%); ?29 (A?>?G), HBB: c.–79?A?>?G (1/39, 2.56%); codons 27/28 (+C), HBB: c.84_85insC (1/39, 2.56%), and the rare IVS-II-850 (G?>?T), HBB: c.316-1?G?>?T (1/39, 2.56%). Only one case of the Southeast Asian HPFH deletion was found. This is the first detailed molecular epidemiological survey of thalassemia in the Chengdu region, Sichuan Province, People’s Republic of China (PRC).     

Molecular Basis of β-Thalassemia in the Population of the Aegean Region of Turkey: Identification of A Novel Deletion Mutation

Abstract

β-Thalassemia (β-thal) is the most common monogenic disorder in Turkey. The aim of this study was to investigate the spectrum of β-thal mutations in the Aegean region of Turkey. The data was derived from 1171 unrelated β-thal subjects, detected in a regional reference hospital between November 2004 and December 2013. Screening for the 22 common mutations was performed using the polymerase chain reaction (PCR)-reverse dot-blot method, and direct automated DNA sequencing for the unknown samples. Thirty-one different β-thal alleles were identified. Seven mutations, namely IVS-I-110 (G?>?A) (41.7%), IVS-I-1 (G?>?A) (8.9%), IVS-II-745 (C?>?G) (8.6%), codon 8 (–AA) (7.7%), IVS-II-1 (G?>?A) (7.2%), IVS-I-6 (T?>?C) (6.6%), codon 39 (C?>?T) (4.6%) accounted for 85.3% of the mutated alleles. Frequencies of the remaining 24 β-thal mutations were less than 2.2%; these included one novel mutation [HBB: c.206_212del (p.Leu69Profs*19)], and four others [–56 (G?>?C), codon 16 (–C), IVS-I (–3) (C?>?T) (codon 29), codon 76 (–C)] found in Turkey for the first time. The results will help to prevent severe β-thal through genetic counseling and prenatal diagnosis (PND) in the Aegean region of Turkey.     

Bacterial expression of mutant argininosuccinate lyase reveals imperfect correlation of in-vitro enzyme activity with clinical phenotype in argininosuccinic aciduria

Background

The urea cycle defect argininosuccinate lyase (ASL) deficiency has a large spectrum of presentations from highly severe to asymptomatic. Enzyme activity assays in red blood cells or fibroblasts, although diagnostic of the deficiency, fail to discriminate between severe, mild or asymptomatic cases. Mutation/phenotype correlation studies are needed to characterize the effects of individual mutations on the activity of the enzyme.

Methods

Bacterial in-vitro expression studies allowed the enzyme analysis of purified mutant ASL proteins p.I100T (c.299?T?>?C), p.V178M (c.532?G?>?A), p.E189G (c.566A?>?G), p.Q286R (c.857A?>?G), p.K315E (c.943A?>?G), p.R379C (c.1135?C?>?T) and p.R385C (c.1153?C?>?T) in comparison to the wildtype protein.

Results

In the bacterial in-vitro expression system, ASL wild-type protein was successfully expressed. The known classical p.Q286R, the novel classical p.K315E and the known mutations p.I100T, p.E189G and p.R385C, which all have been linked to a mild phenotype, showed no significant residual activity. There was some enzyme activity detected with the p.V178M (5 % of wild-type) and p.R379C (10 % of wild-type) mutations in which K_m values for argininosuccinic acid differed significantly from the wild-type ASL protein.

Conclusion

The bacterially expressed enzymes proved that the mutations found in patients and studied here indeed are detrimental. However, as in the case of red cell ASL activity assays, some mutations found in genetically homozygous patients with mild presentations resulted in virtual loss of enzyme activity in the bacterial system, suggesting a more protective environment for the mutant enzyme in the liver than in the heterologous expression system and/or in the highly dilute assays utilized here.     

Geographical distribution of β-globin gene mutations in Syria

ABSTRACT

Objectives: β-Thalassemia disease is caused by mutations in the β-globin gene. This is considered as one of the common genetic disorders in Syria. The aim of this study was to identify the geographical distribution of the β-thalassemia mutations in Syria.

Methods: β-Globin gene mutations were characterized in 636 affected patients and 94 unrelated carriers using the amplification refractory mutations system-polymerase chain reaction technique and DNA sequencing.

Results: The study has revealed the presence of 38 β-globin gene mutations responsible for β-thalassemia in Syria. Important differences in regional distribution were observed. IVS-I.110 [G?>?A] (22.2%), IVS-I.1 [G?>?A] (17.8%), Cd 39 [C?>?T] (8.2%), IVS-II.1 [G?>?A] (7.6%), IVS-I.6 [T?>?C] (7.1%), Cd 8 [?AA] (6%), Cd 5 [?CT] (5.6%) and IVS-I.5 [G?>?C] (4.1%) were the eight predominant mutations found in our study. The coastal region had higher relative frequencies (37.9 and 22%) than other regions. A clear drift in the distribution of the third common Cd 39 [C?>?T] mutation in the northeast region (34.8%) to the northwest region (2.5%) was noted, while the IVS-I.5 [G?>?C] mutation has the highest prevalence in north regions. The IVS-I.6 [T?>?C] mutation had a distinct frequency in the middle region. Ten mutations ?86 [C?>?G], ?31 [A?>?G], ?29 [A?>?G], 5′UTR; +22 [G?>?A], CAP?+?1 [A?>?C], Codon 5/6 [?TG], IVS-I (?3) or codon 29 [C?>?T], IVS-I.2 [T?>?A], IVS-I.128 [T?>?G] and IVS-II.705 [T?>?G] were found in Syria for the first time.

Conclusions: These data will significantly facilitate the population screening, genetic counseling and prenatal diagnosis in Syrian population.     

Molecular Scanning of β-Thalassemia in the Southern Region of Central Java,Indonesia; a Step Towards a Local Prevention Program

Abstract

Thalassemia is the most prevalent genetic blood disorder worldwide, and particularly prevalent in Indonesia. The purpose of this study was to determine the spectrum of β-thalassemia (β-thal) mutations found in the southern region of Central Java, Indonesia. The subjects of the study included 209 β-thal Javanese patients from Banyumas Residency, a southwest region of Central Java Province. DNA analysis was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), amplification refractory mutation system (ARMS), and the direct sequencing method. The results showed that 14 alleles were found in the following order: IVS-I-5 (G?>?C) (HBB: c.92?+?5G?>?C) 43.5%, codon 26 (Hb E; HBB: c.79G?>?A) 28.2%, IVS-I-1 (G?>?A) (HBB: c.92?+?1G?>?A) 5.0%, codon 15 (TGG?>?TAG) (HBB: c.47G?>?A) 3.8%, IVS-I-1 (G?>?T) (HBB: c.92?+?1G?>?T) 3.1%, codon 35 (–C) (HBB: c.110delC) 2.4%. The rest, including codons 41/42 (–TTCT) (HBB: c.126_129delCTTT), codons 8/9 (+G) (HBB: c.27_28insG), codon 19 (AAC?>?AGC) (HBB: c.59A?>?G), codon 17 (AAG?>?TAG) (HBB: c.52A?>?T), IVS-I-2 (T?>?C) (HBB: c.92?+?2T?>?C), codons 123/124/125 (–ACCCCACC) (HBB: c.370_378delACCCCACCA), codon 40 (–G) (HBB: c.123delG) and Cap +1 (A?>?C) (HBB: c.-50A?>?C), accounted for up to 1.0% each. The most prevalent alleles would be recommended to be used as part of β-thal screening for the Javanese, one of the major ethnic groups in the country.     

Characterization of Hb Lepore Variants in the UK Population

Abstract

A molecular study of Hb Lepore heterozygotes identified by the UK population screening program has revealed four out of the five known Lepore variants. The region of homologous δ- and β-globin gene sequence was determined in 58 unrelated Hb Lepore heterozygotes referred for confirmation of their carrier status by DNA analysis through the national thalassemia and sickle cell screening program over a period of 10 years. The most common variant found was Hb Lepore-Boston-Washington (Hb LBW, HBD: c.265?C?>?c.315?+?7?C) observed in 46 carriers (79.0%). Hb Lepore-Hollandia (HBD: c.69?A?>?c.92?+?16?A) was found in nine cases (16.0%); Hb Lepore-Baltimore (HBD: c.208?G?>?c.254?C) in two cases (4.0%) and Hb Lepore-ARUP (HBD: c.97?C?>?c.150?C) in one carrier (2.0%). Analysis of the hematological findings showed no significant differences between the four groups. The wide range of Hb Lepore variants observed in this study confirms the very diverse range of α- and β-globin gene mutations observed in the UK population by previous studies.     

Novel mutations of integrin αIIb and β3 genes in Turkish children with Glanzmann’s thrombasthenia

Glanzmann’s thrombasthenia (GT) is an inherited disorder of platelet aggregation, characterized by qualitative and quantitative defect on platelet αIIbβ3 integrin (GpIIb/IIIa), resulting in lifelong bleeding tendency due to defective platelet plug formation. The αIIb gene (ITGA2B) and β3 gene (ITGB3) are closely located at chromosome 17q21.31-32. ITGA2B consist of 30 exons and encoding α chain, whereas ITGB3 has 15 exons and encoding β chain. Until now, according to the Human Gene Mutation Database (HGMD), 138 mutations at ITGA2B gene and 101 mutations at ITGB3 gene have been identified. We aimed to determine whether there was any mutation in the ITGA2B and ITGB3 genes, and a correlation between clinical phenotype and genotype in Turkish GT patients. We examined 20 patients with GT followed at the Department of Pediatric Hematology, Meram Faculty of Medicine, for Clinical and Laboratory Findings and Molecular Genetic Analysis. Peripheral blood was collected from patients, and a written informed consent for genetic analysis was obtained from parents. DNA was isolated from by proteinase K and phenol/chloroform extraction. ITGA2B and ITGB3 genes were screened by polymerase chain reaction. There were 12 females and 8 males with a median age of 15.25 years. Major clinical presentations of these patients were mucocutaneous bleedings. The most common bleeding type was epistaxis (85%). Life-threatening bleedings were seen in five patients. Seven (35%) patients showed various mutations in the ITGA2B or ITGB3 genes. We detected four novel mutations in three different regions and two mutations defined previously within the ITGA2B gene. These changes are at exon 4; c.570 T?>?G alteration, at exon 13 c.1277 T?>?A, c.1291 T?>?G alterations, at exon 19 c.1921A?>?G alterations. And from the start point of exon 14, behind 107 bases, we detected a heterozygous alteration at Thymine to Guanine. According to PolyPhen Database Program and NCBI Multiple Alignment Tool Database, four transitions are conserved at evolutionary process, so we can say that these transitions are novel mutations. c. 468T?>?G alteration at exon 4 and c. 1378 T?>?A alteration at exon 13 were reported to HGMD previously. Screening the exons of the ITGB3 gene from the same patient groups, we reported a novel missense mutation at exon 5, at nucleotide 680. No correlation was found between clinical phenotype and genotype. These mutations were described for the first time in Turkish population, and all novel mutations are not defined previously. Furthermore, collaborative studies are needed for the population point of view.     

Clopidogrel Pharmacogenetics in Iranian Patients Undergoing Percutaneous Coronary Intervention

Clopidogrel is used in patients with coronary syndromes and at risk of thrombotic events or receiving percutaneous coronary intervention (PCI) for reducing heart attack and stroke. Here we present genotype and phenotype study of Iranian patients undergoing PCI treated with clopidogrel during a 6-month period of follow-up; common variants of CYP2C19, CYP3A5, CYP3A4, and ABCB1 genes were determined as well as the patients’ cardiovascular outcomes to find out the effect of these variants individually and in combination. 388 individuals receiving PCI were enrolled in this study. Different pretreatment doses of clopidogrel were prescribed under the interventional cardiologists’ guidance. The patients were followed for a duration of 1 month, and 6 months. Six SNPs were selected for genotyping including CYP2C19*2 (c.681G?>?A), CYP2C19*3 (c.636G?>?A), CYP2C19*17 allele (c.?806C?>?T), ABCB1 (c.3435C?>?T), CYP3A5 (c.6986A?>?G), and CYP3A4 (c.1026?+?12G?>?A). The mean loading dose was 600 mg/day in 267 (68.8%) individuals, 300 mg/day in 121 (31.2%). 8 patients had cardiovascular events such as thrombosis, unstable angina, and non-STEMI. The studied alleles and genotypes were in Hardy–Weinberg equilibrium. None of the SNPs individually were significantly associated with outcome events. Our results indicate that combinations of different alleles of genes are involved in pharmacokinetic variability and joint factors are important; this means that genotyping and analysis of an individual variant may not be as straightforward in risk assessment and pharmacogenetics. This highlights the importance of personalized medicine in risk assessment and treatment.     

Two new class III G6PD variants [G6PD Tunis (c.920A > C: p.307Gln > Pro) and G6PD Nefza (c.968T > C: p.323 Leu > Pro)] and overview of the spectrum of mutations in Tunisia

We screened 423 patients referred to our laboratory after hemolysis triggered by fava beans ingestion, neonatal jaundice or drug hemolysis. Others were asymptomatic but belonged to a family with a history of G6PD deficiency. The determination of enzymatic activity using spectrophotometric method, revealed 293 deficient (143 males and 150 females). The molecular analysis was performed by a combination of PCR-RFLP and DNA sequencing to characterize the mutations causing G6PD deficiency. 14 different genotypes have been identified : G6PD A^? (376A > G;202G > A) (46.07%) and G6PD Med (33.10%) were the most common variants followed by G6PD Santamaria (5.80%), G6PD Kaiping (3.75%), the association [c.1311T and IVS11 93c] (3.75%), G6PD Chatham (2.04%), G6PD Aures (1.70%), G6PD A^? Betica (0.68%), the association [ 376G;c.1311T;IVS11 93c] (0.68%), G6PD Malaga, G6PD Canton and G6PD Abeno respectively (0.34%). Two novel missense mutations were identified (c.920A > C: p.307Gln > Pro and c.968T > C: p.323 Leu > Pro). We designated these two class III variants as G6PD Tunis and G6PD Nefza. A mechanism which could account for the defective activity is discussed.     

Mutations in inherited fibrinogen disorders correlated with clinical features in the Chinese population

Two probands with unknown reasons for low fibrinogen activity were considered to investigate the association between mutations in inherited fibrinogen disorders (IFDs) and clinical features in the Chinese population. A routine coagulation test was conducted on a Sysmex CS5100 coagulation analyzer, and Sanger sequencing was employed to analyze mutations. A PubMed database search through May 2020 was performed to identify relevant studies regarding the congenital fibrinogen disorder epidemic in China. A common heterozygous missense mutation (c.1233G?>?A p.Arg35His), a novel heterozygous mutation (c.2014T?>?C p.Trp672Arg), and a novel homozygous mutation (c.16A?>?G p.Ile6Val) in the FGA gene were identified in two probands with dysfibrinogenemia. The global coagulation screening assay can distinguish four types of IFD, especially a ratio of Fib:Ag/Fib:C equal to 1.5, which can distinguish patients with hypofibrinogenemia from those with hypodysfibrinogenemia. A total of 81 mutations from 76 probands in 45 IFD families have been reported in the Chinese population. Arg35 in FGA and Arg301 in FGG were responsible for IFD in more than half of patients in the Chinese population. It is possible to distinguish four types of IFD by using a global coagulation screening assay. Mutations in FGA, FGB and FGG occur in different functional regions, and Arg changes account for more than 70% of patients with IFD in the Chinese population, especially Arg-Cys, which may be associated with severe clinical symptoms.

     

The Spectrum of β-Thalassemia Mutations in a Population from the Brazilian Amazon

The spectrum of β-thalassemia (β-thal) mutations was investigated for the first time in a cohort of 33 unrelated patients from the Brazilian Amazon attending the Center for Hemotherapy and Hematology of the Pará Foundation (HEMOPA), in Belém, the state capital of Pará, Northern Brazil. Identification of the β-thal mutations was made by direct genomic sequencing of the β-globin gene. Mutations were identified in all patients, corresponding to a spectrum of 10 different point mutations and a total of 37 alleles studied. HBB: c.92?+?5G?>?A [IVS-I-5 (G?>?A)], was the most common β-thal mutation, followed by HBB: c.118C?>?T [codon 39 (C?>?T)], HBB: c.?138C?>?T [?88 (C>T)], HBB: c.92?+?1G?>?A [IVS-I-1 (G?>?A)] and HBB: c.92?+?6T?>?C [IVS-I-6 (T?>?C)] mutations. These five mutations (four Mediterranean origin and one African origin) accounted for 86.5% of the β-thal alleles. The profile of β-thal mutations found in northern Brazil is different from those described in other regions of the country. In the southeast and south, the nonsense mutation HBB: c.118C?>?T is the most prevalent, followed by HBB: c.93-21G?>?A [IVS-I-110 (G?>?A)], whereas in the northeast, HBB: c.92?+?6T?>?C has been identified as the most common mutation, followed by HBB: c.92?+?1G?>?A. This heterogeneous geographical distribution is certainly related to the ancestry of Brazilian populations because they have similar genetic backgrounds (European, African and Amerindian), although with slightly different admixture proportions. Furthermore, the European contribution in the southeast and south was largely made up of immigrants of other nationalities, such as Italian and Spanish, in addition to Portuguese.     

Elevated Hb A2 Levels in a Patient with a Compound Heterozygosity for the (β+) −31 (A > G) and (β0) Codon 17 (A > T) Mutations Together with a Single α-Globin Gene

Abstract

We report the molecular and hematological feature of a Thai woman who had clinical diagnosis of β-thalassemia intermedia (β-TI). Hemoglobin (Hb) high performance liquid chromatography (HPLC) analysis identified Hb A (64.4%), Hb F (12.3%) and Hb A₂/E (15.9%) with small peaks of Hb Bart’s (γ4) and Hb H (β4). She was initially diagnosed as EA Bart’s disease, which occurs from combination of Hb H disease and Hb E (HBB: c.79G?>?A) trait. However, the Hb analysis using capillary electrophoresis (CE) demonstrated no Hb E, 68.5% Hb A, 15.5% Hb F and 16.0% Hb A₂. DNA analysis showed a compound heterozygosity for (β⁺) ?31 (A?>?G) (HBB: c.-81A?>?G) and (β⁰) codon 17 (A?>?T) (HBB: c.52A?>?T) mutations and deletional Hb H (–?–^SEA/–α^3.7). Thus, she was finally diagnosed with a combination of Hb H disease and compound heterozygosity of β⁺/β⁰-thalassemia (β⁺/β⁰-thal). The β-globin mutations could affect not only hematological parameters but also elevate the Hb A₂ levels. These effects could not be ameliorated by the coinheritance of Hb H disease. Therefore, a better understanding of the effects of this combination on hematological analysis data will be useful for providing accurate diagnosis, genetic counseling, prevention and control programs of β-thalassemia major (β-TM).     

Origin of the Frameshift Codons 41/42 (–TCTT) Mutation in the First Cases Described in the Spanish Population

Thalassemias are hereditary anemias. In β-thalassemia (β-thal), β-globin synthesis is either deficient or absent. A high incidence of β-thal is found in populations of Mediterranean and African origin. Smaller, but significant concentrations of β-thal are present throughout the Middle East, India, Pakistan and China, while sporadic cases have been reported in most ethnic groups. Over 200 β-thal mutations have been described so far. But each population group displays its own mutations. In Spain, as in other countries of the Mediterranean region, the most often seen mutations are codon 39 (C?>?T); IVS-I-1 (G?>?A); IVS-I-6 (T?>?C) and IVS-I-110 (G?>?A). However, a large number of rarer alleles have been observed both in Spain and other populations. The frameshift codons (FSC) 41/CD42 (–TCTT) mutation is a rather common allele in individuals of Chinese origin, but rare in the Mediterranean region, although, it has been recorded in East Asian populations.

We describe the first eight Spanish patients displaying the FSC 41/42 mutation. This mutation was initially detected with a real-time polymerase chain reaction (PCR) method on a LightCycler?, using a probe designed to detect mutations in codons 37 and 39, and subsequently specifically characterized by automatic sequencing. The haplotype found in our patients suggested that this mutation has not arisen independently in our population but must be taken into account when identifying most β-thal mutations.     

A polymorphism in transforming growth factor-β1 is associated with carotid plaques and increased carotid intima-media thickness in older Chinese men: the Guangzhou Biobank Cohort Study-Cardiovascular Disease Subcohort

The goal of this project was to identify families with autosomal dominant hypercholesterolemia (ADH) to facilitate early detection and treatment and to provide genetic counselling as well as to approximate the mutational diversity of ADH in Mexico. Mutational analysis of the LDLR and APOB genes in 62 index cases with a clinical and/or biochemical diagnosis of ADH was performed. Twenty-five mutations (24 LDLR, 1 APOB) were identified in 38 index cases. A total of 162 individuals with ADH were identified using familial segregation analysis performed in 269 relatives of the index cases. In addition, a novel PCSK9 mutation, c.1850 C > A (p.Ala617Asp), was detected. The LDLR mutations showed the following characteristics: (1) four mutations are novel: c.695 −1G > T, c.1034_1035insA, c.1586 G > A, c.2264_2273del; (2) the most common mutations were c.682 G > A (FH-Mexico), c.1055 G > A (FH-Mexico 2), and c.1090 T > C (FH-Mexico 3); (3) five mutations were identified in 3 or more apparently unrelated probands; (4) three mutations were observed in a true homozygous state; and (5) four index cases were compound heterozygous, and one was a carrier of two mutations in the same allele. These results suggest that, in Mexico, ADH exhibits allelic heterogeneity with 5 relatively common LDLR mutations and that mutations in the APOB gene are not a common cause of ADH. This knowledge is important for the genotype–phenotype correlation and for optimising both cholesterol lowering therapies and mutational analysis protocols. In addition, these data contribute to the understanding of the molecular basis of ADH in Mexico.     

Clinical,biochemical, neuroradiological and molecular characterization of Egyptian patients with glutaric acidemia type 1

Glutaric acidemia type 1 (GA1) is an inherited metabolic autosomal recessive disorder that is caused by a deficiency in glutaryl-CoA dehydrogenase (GCDH). Untreated patients suffer primarily from severe striatal damage. More than 250 variants in the GCDH gene have been reported with a variable frequency among different ethnic groups. In this study, we aimed to characterize 89 Egyptian patients with GA1 and identify the variants in the 41 patients who were available for genotyping. All of our patients demonstrated clinical, neuroradiological, and biochemical characteristics that are consistent with a diagnosis of GA1. All patients presented with variable degrees of developmental delay ranging from mild to severe. Most of the 89 patients presented with acute onset type (71.9%), followed by insidious (19%) and asymptomatic (9%). A delay in diagnosis was inversely associated with macrocephaly. The prevalence rate ratio (PR) for macrocephaly that was associated with each 6-month delay was 0.95 (95%CI 0.91–0.99). However, high body weight was associated with a higher likelihood of having macrocephaly (PR 1.16, 95%CI 1.06–1.26 per 1 SD increment of Z score weight). However, body weight was inversely associated with the morbidity score. Consanguinity level was 64% among our patient’s cohort and was positively associated with the C5DC level (β (95%CI) 1.06 (0.12–1.99)). Forty-one patients were available for genotyping and were sequenced for the GCDH gene. We identified a total of 25 variants, of which the following six novel variants were identified: three missense variants, c.320G?>?T (p.Gly107Val), c.481C?>?T (p.Arg161Trp) and c.572 T?>?G (p.Met191Arg); two deletions, c.78delG (p.Ala27Argfs34) and c.1035delG (p.Gly346Alafs*11); and one indel, c.272_331del (p.Val91_Lys111delinsGlu). All of the novel variants were absent in the 300 normal chromosomes. The most common variant, c.*165A?>?G, was detected in 42 alleles, and the most commonly detected missense variant, c.1204C?>?T (p.Arg402Trp), was identified in 29 mutated alleles in 15/41 (34.2%) of patients. Our findings suggest that GA1 is not uncommon organic acidemia disease in Egypt; therefore, there is a need for supporting neonatal screening programs in Egypt.

     

Clinical characteristics and mutation analysis of five Chinese patients with maple syrup urine disease

Maple syrup urine disease (MSUD) is an autosomal recessive disorder affecting branched-chain amino acids (BCAAs) metabolism and caused by a defect in the thiamine-dependent enzyme branched chain α-ketoacid dehydrogenase (BCKD) with subsequent accumulation of BCAAs and corresponding branched-chain keto acids (BCKAs) metabolites. Presently, at least 4 genes of BCKDHA, BCKDHB, DLD and DBT have been reported to cause MSUD. Furthermore, more than 265 mutations have been identified as the cause across different populations worldwide. Some studies have reported the data of gene mutations in Chinese people with MSUD. In this study, we present clinical characteristics and mutational analyses in five Chinese Han child with MSUD, which had been screened out by tandem mass spectrometry detection of amino acids in blood samples. High-throughput sequencing, Sanger sequence and real-time qualitative PCR were performed to detect and verify the genetic mutations. Six different novel genetic variants were validated in BCKDHB gene and BCKDHA gene, including c.523 T?>?C, c.659delA, c.550delT, c.863G?>?A and two gross deletions. Interestingly, 3 cases had identical mutation of BCKDHB gene (c.659delA). We predicted the pathogenicity and analyzed the clinical characteristics. The identification of these mutations in this study further expands the mutation spectrum of MSUD and contributes to prenatal molecular diagnosis of MSUD.     

A β-Thalassemia Trait with Two Mutations in Cis in a Chinese Family

Abstract

A female of Chinese origin carried the codon 43 (G>T) (HBB: c.130G?>?T) and codons 71/72 (+A) (HBB: c.216_217insA) mutations of the β-globin gene in cis, identified during prenatal thalassemia screening. The double in cis mutations were inherited from her mother. Both of the two carriers behave as a traditional heterozygote for β-thalassemia (β-thal) with microcytosis and a high Hb A₂ level. This case report indicates that the possibility of multiple mutations in cis in a fetus with thalassemia trait has to be considered in a prenatal screening program.     

Prevalence and Molecular Characterization of Structural Hemoglobin Variants in the Dongguan Region of Guangdong Province,Southern China

The aim of the present study was to find the most prevalent structural hemoglobin (Hb) variants in southern China and to present hematological and molecular data of abnormal Hbs in the population from southern China. The type and frequency of structural Hb variants and their hematological and molecular characteristics were identified in 131 individuals from 30,848 unrelated partners who were referred to the prenatal clinic of Dongguan Maternal &; Children Health Hospital, Dongguan, Guangdong, People’s Republic of China (PRC) from 2011 to 2013. α-Globin or β-globin chain variants were screened using a capillary electrophoresis (CE) system, and α-globin or β-globin gene mutations were confirmed using sequencing techniques. The gene frequency of Hb variants was 0.4% (131/30,848). The most common α-globin variants were Hb Constant Spring (Hb CS, HBA2: c.427T?>?C) (0.2%), followed by Hb Q-Thailand (HBA1: c.223G?>?C) and Hb G-Honolulu (HBA2: c.91G?>?C). The most common β-globin variant was Hb E (HBB: c.79G?>?A) (0.09%), followed by Hb New York (HBB: c.341T?>?A). Our results provide a detailed prevalence and molecular characterization of abnormal Hbs in the population of the Dongguan region. These findings have important implications for a region with a high frequency of α- and β-thalassemias.