Gap-polymerase Chain Reaction Research Articles

Phenotypic Analysis of the HBA2: C.95 G > A Mutation in China

This study aimed to analyze the clinical phenotype of the HBA2: c.95G>A mutation in the Chinese population and to provide guidance for clinical diagnosis and genetic counseling. Peripheral blood samples were collected from 16 patients, including 6 newborns, 2 children, and 8 adults. Hematological parameters and hemoglobin electrophoresis were analyzed, and genotypes were identified using methods such as PCR combined with reverse dot blot (RDB), nested PCR, gap polymerase chain reaction (Gap-PCR), and DNA sequencing. The results showed that 10 patients had mild anemia, 2 had moderate anemia, and 12 exhibited microcytic hypochromic features with MCV values ranging from 53 to 74.7 fl and MCH values from 16.2 to 25.4 pg. Additionally, 3 cases displayed obvious HbH + HbBarts bands (>15%). Among the 16 cases, various combinations of the HBA2: c.95G>A mutation were observed: one case had –α3.7 combined with HBA2: c.95G>A, another had –α4.2 combined with HBA2: c.95G>A, and five had –SEA combined with HBA2: c.95G>A, while the remaining cases were HBA2: c.95G>A heterozygotes. The study concludes that the HBA2: c.95G>A mutation in the α2 globin gene causes α+ thalassemia. When this mutation is combined with the Southeast Asian deletion (–SEA), it results in HbH disease, characterized by moderate microcytic hypochromic anemia and the presence of HbH + HbBarts bands.

Analysis of Thalassemia Gene Mutation Types and Ethnic Distribution Characteristics in Hechi Area, Guangxi

To investigate the genotype, mutation type, and ethnic distribution characteristics of thalassemia in the population of Hechi area, Guangxi, and to provide a reference basis for prevention and control of thalassemia and eugenic counseling in the region. Gap-polymerase chain reaction (gap-PCR) and reverse dot blot (RDB) were used for genetic testing on suspected thalassemia persons, and the results were analyzed. Among 29 136 samples, a total of 17 016 (58.40%) positive samples for thalassemia genes were detected, with a higher detection rate in males than in females (χ2=49.917，P < 0.001). The detection rates of thalassemia genes were significant different among Zhuang, Han, Yao, Mulao, and Maonan ethnic groups (χ2=546.121, P < 0.001). The α-thalassemia genotypes were mainly --SEA /αα (16.67%), -α3.7/αα (8.90%), α CSα/αα (6.00%). Additionally, four rare genotypes were detected, including -- THAI/αα (47 cases), HKαα/αα (2 cases), --SEA /-α 21.9 (2 cases), and -- THAI/αCSα (1 case). The β-thalassemia genotypes were mainly β CD17/βN (7.49%), βCD41-42/βN (6.70%), βCD71-72/βN (0.44%). 108 cases of moderate and severe β-thalassemia were detected, of which 81 cases had a history of blood transfusion, the transfusion frequency of 60 cases was more than 10 times/year, and 10 cases received bone marrow transplantation. Thalassemia in Hechi area is predominantly deletion type --SEA /αα, the detection rate of thalassemia in ethnic minorities is higher than that in Han population. In this area, moderate and severe β-thalassemia have certain incidence, these patients mostly need regular blood transfusion and iron removal treatment, and very few patients have received bone marrow transplantation. This study provides a certain reference basis for prevention and control of thalassemia and eugenic counseling in the region.

Third-generation sequencing identified a novel complex variant in a patient with rare alpha-thalassemia

BackgroundThalassemias represent some of the most common monogenic diseases worldwide and are caused by variations in human hemoglobin genes which disrupt the balance of synthesis between the alpha and beta globin chains. Thalassemia gene detection technology is the gold standard to achieve accurate detection of thalassemia, but in clinical practice, most of the tests are only for common genotypes, which can easily lead to missing or misdiagnosis of rare thalassemia genotypes.Case presentationWe present the case of an 18-year-old Chinese female with abnormal values of routine hematological indices who was admitted for genetic screening for thalassemia. Genomic DNA was extracted and used for the genetic assays. Gap polymerase chain reaction and agarose gel electrophoresis were performed to detect HBA gene deletions, while PCR-reverse dot blot hybridization was used to detect point mutations in the HBA and HBB genes. Next-generation sequencing and third-generation sequencing (TGS) were used to identify known and potentially novel genotypes of thalassemia. We identified a novel complex variant αHb WestmeadαHb Westmeadαanti3.7/-α3.7 in a patient with rare alpha-thalassemia.ConclusionsOur study identified a novel complex variant that expands the thalassemia gene variants spectrum. Meanwhile, the study suggests that TGS could effectively improve the specificity of thalassemia gene detection, and has promising potential for the discovery of novel thalassemia genotypes, which could also improve the accuracy of genetic counseling. Couples who are thalassemia carriers have the opportunity to reduce their risk of having a child with thalassemia.

Screening and Diagnosis of Rare Thalassemia Variants: Is Third-Generation Sequencing Enough?

Rare thalassemia subtypes are often undiagnosed because conventional testing methods can only identify 23 common types of α- and β-thalassemia. To assess a comprehensive approach for the screening and diagnosis of rare thalassemia. The study cohort included 72 individuals with suspected rare thalassemia variants. Screening was conducted by next-generation sequencing (NGS) combined with third-generation sequencing (TGS) and chromosomal microarray analysis (CMA)/copy number variation sequencing. Of the 72 individuals with suspected rare thalassemia, 49 had rare α- or β-gene variants. NGS combined with gap polymerase chain reaction detected a total of 42 cases, resulting in a positive detection rate of 58.3%. Additionally, 4 α-globin genetic deletions were identified by TGS, which increased the variant detection rate by 5.6%. Two samples with a microdeletion of chromosome 16 or 11 were detected by CMA, which increased the detection rate by 2.8%. For one sample, reanalysis of the NGS and TGS data confirmed the presence of the β41-42/βN and βN/βN mosaic. The HBB:c.315 + 2delT mutation was initially reported in Guangdong Province, China. Two HBB gene mutations (HBB:c.315 + 5G>C and HBB:c.295G>A) and 4 rare HBA gene deletions (-11.1, -α27.6, -α2.4, and -α21.9) were initially identified in the Zhonshan region. The hematologic phenotypes of all rare cases in this study were clarified. Rare thalassemia variants are more common than previously thought. Despite advancements in TGS, there is still no foolproof method for detection of all types of thalassemia. Thus, a comprehensive approach is necessary for accurate screening and diagnosis of rare thalassemia variants.

Hematological Profile of Hb Adana Among High School Students in Northeast Peninsular Malaysia.

Background Hb Adana is a non-deletional alpha (α)-thalassaemia variant resulting from mutations in α1- or α2-globin codon 59 (αCD59), leading to the production of unstable α-globin. Clinical manifestations can vary from silent carrier status to dependence on blood transfusions, hepatosplenomegaly, skeletal deformities, and spinal cord compression. Despite the significance of Hb Adana inheritance, studying this variant poses challenges due to the scarcity of molecular tests and the potential for routine diagnoses to be overlooked. This study aims to investigate the prevalence of Hb Adana among local high school students and assess the hematological parameters and hemoglobin analysis of Hb Adana in Malaysia. Methodology This retrospective study analyzed 13,721 blood samples collected from high school students participating in Malaysia's National Thalassaemia Screening Program at Hospital Raja Perempuan Zainab II (HRPZ II). Deletional α-thalassaemia was detected using multiplex gap-polymerase chain reaction (PCR), while common non-deletional α-thalassaemia was identified using multiplex amplification refractory mutation system (ARMS) PCR. Data were extracted from the HRPZ II database for analysis. Results Among the participants, 2327 individuals were found to have either common deletional (n=1037, 44.6%) or non-deletional (n=1290, 55.4%) α-thalassaemia. Hb Constant Spring was the most prevalent non-deletional α-thalassaemia, accounting for 53.03% of cases. Thirty-one participants (1.33%) exhibited αCD59α/αα, and one (0.04%) had αCD59α/-α3.7. Among the 32 subjects with Hb Adana, 87.5% were Malay, and 12.5% were Orang Asli. Additionally, seven cases of HbE/Hb Adana co-inheritance were identified. Hemoglobin levels in heterozygous Hb Adana individuals ranged from mild anemia to normal, between 95 g/L and 153 g/L. Mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were approximately 73 fL and 23 pg, respectively. Conclusion This study delineates the distribution ofα-thalassaemia mutation patterns among high school students in Kelantan, Northeast Peninsular Malaysia. Our findings indicate that Hb Adana is rare in our region and co-inheritance with an α-gene deletion results in α+-thalassaemia and with HbE, α0-thalassaemia. All heterozygous Hb Adana individuals exhibited low MCVs and MCHs.

Misdiagnosis of β-Thalassemia Major Due to Chinese Gγ+(Aγδβ)0-Thalassemia Combined with β0-Thalassemia

δβ-thalassemia is a rare type of thalassemia characterized by increased Hb F levels, including mainly Chinese Gγ(Aγδβ)0-thalassemia, Yunnanese Gγ(Aγδβ)0-thalassemia, Cantonese Gγ(Aγδβ)0-thalassemia in China. Due to the low rate of δβ-thalassemia carriers, there are few reports of δβ-thalassemia combined with β-thalassemia causing β-thalassemia major. Herein, we described the combination of Chinese Gγ(Aγδβ)0-thalassemia and β-thalassemia leading to β-thalassemia major in a Chinese patient. Hemoglobin analysis was performed by capillary electrophoresis (CE). Routine genetic analysis was carried out by gap-polymerase chain reaction (Gap-PCR) and PCR and reverse dot blot (PCR-RDB). Multiple ligation-dependent probe amplification (MLPA) was used to detect the large deletion, and Gap-PCR confirmed the deletion. A CE result showed an elevated Hb F level of 98.7% and 11.7% in the proband and her mother, but the proband was diagnosed with βCD17M/βCD17M using routine genetic analysis. However, her father was heterozygous for CD17 in β-globin, and her mother was detected as SEA heterozygous. The further analysis presented that the proband had actually missed the diagnosis of Chinese Gγ(Aγδβ)0-thalassemia by MLPA and PCR-RDB. Finally, the genotype of the proband was corrected from βCD17M/βCD17M to βCD17M/βGγ(Aγδβ)0. This is the first report of Chinese Gγ(Aγδβ)0-thalassemia combined with β-thalassemia resulting in β-thalassemia major in China. Screening for δβ-thalassemia by Hb analysis could be an effective method.

CTNS Mutations Causing Autosomal Recessive Cystinosis in a Subset of Iranian Population: Report of Two New Variants

Abstract Background: Nephropathic cystinosis (NC) is an uncommon autosomal recessive disease with abnormality in lysosomal storage that appearances in patients with mutations in the CTNS gene encoding a lysosomal transporter cystinosin. Disrupted function of this transporter is followed by accumulation of cysteine crystals in cells of many various organs. This study aimed to investigate the mutations of the CTNS gene in 20 Iranian patients suffering from NC. Materials and Methods: Twenty Iranian cystinosis patients referring to Imam Hossein Hospital of Isfahan were employed in this case-series study. After extraction of genomic DNA, the promoter and entire coding regions of CTNS were analysed using sanger sequencing in all patients. Gap–Polymerase Chain Reaction was used to detect 57 kb deletion in the CTNS gene. In silico study was performed to analyse variants. Results: The large deletion was not seen in any NC patients. Molecular analysis which conducted to screen the CTNS gene of patients, identified eight different mutations, including two new mutations, c.971_972insC and c.956_956delA, which have not been reported before, and c.681G&gt;A mutation, which was identified as a frequently founded mutation in the Middle East and was observed in 35% of patients. In this study, five other mutations including c.1015G&gt;A, c.922G&gt;A, c.323_323delA, c.433C&gt;T, and c.18_21delGACT were also observed, which have been reported in previous studies. Conclusion: The mutational spectrum in the Iranian patients is the same as previously reported mutations except that two new mutations were found. The present findings will present suggestions for regular molecular diagnosis of cystinosis in Iran.

Identification of Hb Lepore, Hb anti-Lepore, and α-globin gene triplications by long-read single-molecule real-time sequencing.

Hemoglobin (Hb) Lepore and Hb anti-Lepore are infrequent fusion gene variants that result from nonhomologous crossovers during meiosis. Conventional molecular testing methods may face challenges in identifying these variants. During Hb analysis using capillary electrophoresis, we encountered 6 cases with unusual Hb variants. Our aim was to identify the alterations in their globin genes. Gap-polymerase chain reaction (PCR), reverse dot-blot assay (RDB), Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), and long-read single-molecule real-time (SMRT) sequencing were used to confirm the presence of globin gene alterations. The routine thalassemia gene test kit using the gap-PCR and RDB techniques did not detect common gene variations. Direct sequencing failed to identify any known or unknown globin gene alterations. The MLPA analysis, however, revealed the possible presence of α-globin gene triplications as well as 2 types of fusion gene alterations. Further analysis using long-read SMRT sequencing accurately identified 3 rare gene variations: αααanti-3.7, Hb Lepore-Boston-Washington, and Hb anti-Lepore P-India. Conventional methods may overlook rare thalassemias or Hb variants. Long-read SMRT sequencing has the potential to identify breakpoints in fusion genes, demonstrating that it is a promising technique for detecting rare thalassemias.

A comprehensive preimplantation genetic testing approach for SEA-type α-thalassemia by fluorescent gap-polymerase chain reaction combined with haplotype analysis

A comprehensive preimplantation genetic testing approach for SEA-type α-thalassemia by fluorescent gap-polymerase chain reaction combined with haplotype analysis

Long-Read Sequencing Identified a Large Novel δ/β-Globin Gene Deletion in a Chinese Family

Objective. Increasingly rare thalassemia has been identified with the advanced use of long-read sequencing based on long-read technology. Here, we aim to present a novel δ/β-globin gene deletion identified by long-read sequencing technology. Methods. Enrolled in this study was a family from the Quanzhou region of Southeast China. Routine blood analysis and hemoglobin (Hb) capillary electrophoresis were used for hematological screening. Genetic testing for common α- and β-thalassemia was carried out using the reverse dot blot hybridization technique. Long-read sequencing was performed to detect rare globin gene variants. Specific gap-polymerase chain reaction (gap-PCR) and/or Sanger sequencing were further used to verify the detected variants. Results. None of the common α- and β-thalassemia mutations or deletions were observed in the family. However, decreased levels of MCV, MCH, and abnormal Hb bands were observed in the family members, who were suspected as rare thalassemia carriers. Further, long-read sequencing demonstrated a large novel 7.414 kb deletion NG_000007.3:g.63511_70924del partially cover HBB and HBD globin genes causing delta-beta fusion gene in the proband. Parental verification indicated that the deletion was inherited from the proband’s father, while none of the globin gene variants were observed in the proband’s mother. In addition, the novel δ/β-globin gene deletion was further verified by gap-PCR and Sanger sequencing. Conclusion. In this study, we first present a large novel δ/β-globin gene deletion in a Chinese family using long-read sequencing, which may cause δβ-thalassemia. This study further enhances that long-read sequencing would be applied as a sharp tool for detecting rare and novel globin gene variants.

Association Analysis between Genotype and Phenotype of α,β-Thalassaemia Carriers in Huizhou Area of Guangdong Province

To analyze the prevalence, genotype distribution and hematological characteristics of α,β-thalassaemia carriers in Huizhou area of Guangdong Province. 10 809 carriers of simple β-thalassaemia and 1 757 carriers of α,β-thalassaemia were enrolled as our study cohort. The hematological parameters were detected by automated blood cell counters and automatic capillary electrophoresis. Suspension array technology, gap-polymerase chain reaction (gap-PCR) and PCR-reverse dot blot were used for the genotyping of thalassaemia carriers. The prevalence of α,β-thalassaemia in Huizhou area of Guangdong Province was 1.99%. A total of 62 genotypes were detected, and the most prevalent genotype was --SEA/ αα, βCD41-42/ βN (19.29%), the next was --SEA/ αα, βIVS-II-654/ βN (16.73%). Significant differences in mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were found between different genotype groups for simple β-thalassaemia and α,β-thalassaemia. Violin plots showed that carriers with co-inheritance of β-thalassaemia and mild α-thalassaemia expressed the lightest anemia, and carriers with co-inheritance of β-thalassaemia and hemoglobin H (Hb H) disease expressed the most severe anemia. There is a high prevalence of α,β-thalassaemia in Huizhou area of Guangdong Province. Because of the lack of specific hematological makers for diagnosis of α,β-thalassaemia, it is necessary to distinguish it from simple β-thalassaemia by genotyping of α- and β-thalassaemia in order to correctly guide genetic counseling and prenatal disgnosis.

Rare mutations in the beta-globin gene and their clinical phenotypes

Objectives: Beta-thalassemia is one of the most common inherited genetic disorders and the repertoire of mutations in the beta-globin gene is ever-expanding. Sequencing for beta-globin gene mutations, is done, where phenotype-high-performance liquid chromatography discrepancies exist or where amplification refractory mutation system – polymerase chain reaction (ARMS-PCR) cannot identify common mutations, and often leads to the discovery of rare and novel mutations. Materials and Methods: This is a retrospective data analysis of 160 patients of beta-thalassemia and other hemoglobinopathies where some patients were found to have unexplained clinical features. Comprehensive genetic diagnosis was done on these patients by ARMS-PCR, gap-polymerase chain reaction, and sequencing. Results: Out of the total, 124 cases were homozygous/compound heterozygous for beta-thalassemia; 26 cases had heterozygous beta mutations with coexistent alpha-triplications and four patients (with unique clinical features) were found to harbor five rare mutations. The mutations detected were hemoglobin (Hb) Monroe (co-occurring with beta nt-42 mutation), beta-globin mutation −90(C&gt;T), Hb Randwick, and Hb-M-Saskatoon (a variant hemoglobin causing methemoglobinemia and cyanosis). The spectrum of common mutations detected, in our study, was similar to that published in the literature. The unique clinical features of the patients were conclusively explained by the sequencing results. Conclusion: This study emphasizes the role of sequencing in the genetic diagnosis of beta-thalassemia. As next-generation sequencing increasingly finds use in routine diagnostics, newer clinically significant mutations will continue to be added to the large palette of mutations in beta-thalassemia.

Molecular analysis and prenatal diagnosis of seven Chinese families with genetic epilepsy.

Genetic epilepsy is a large group of clinically and genetically heterogeneous neurological disorders characterized by recurrent seizures, which have a clear association with genetic defects. In this study, we have recruited seven families from China with neurodevelopmental abnormalities in which epilepsy was a predominant manifestation, aiming to elucidate the underlying causes and make a precise diagnosis for the cases. Whole-exome sequencing (WES) combined with Sanger sequencing was used to identify the causative variants associated with the diseases in addition to essential imaging and biomedical examination. A gross intragenic deletion detected in MFSD8 was investigated via gap-polymerase chain reaction (PCR), real-time quantitative PCR (qPCR), and mRNA sequence analysis. We identified 11 variants in seven genes (ALDH7A1, CDKL5, PCDH19, QARS1, POLG, GRIN2A, and MFSD8) responsible for genetic epilepsy in the seven families, respectively. A total of six variants (c.1408T>G in ALDH7A1, c.1994_1997del in CDKL5, c.794G>A in QARS1, c.2453C>T in GRIN2A, and c.217dup and c.863+995_998+1480del in MFSD8) have not yet been reported to be associated with diseases and were all evaluated to be pathogenic or likely pathogenic according to the American College of Medical Genetics and Genomics (ACMG) guidelines. Based on the molecular findings, we have associated the intragenic deletion in MFSD8 with the mutagenesis mechanism of Alu-mediated genomic rearrangements for the first time and provided genetic counseling, medical suggestions, and prenatal diagnosis for the families. In conclusion, molecular diagnosis is crucial to obtain improved medical outcomes and recurrence risk evaluation for genetic epilepsy.

Ace gene insertion/deletion polymorphism is associated with glioblastoma in an Iranian population: a case-control study

Background. The insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene has recently been reported to be associated with the pathogenesis and development of human cancers.This study aimed to assess the potential association between ACE (I/D) polymorphism and glioblastoma in an Iranian population.Material and Methods. This case-control study was conducted on 80 patients with glioblastoma and 80 healthy blood donors as controls. Gap-polymerase chain reaction (Gap-PCR) was used to determine the ACE (I/D) genotypes. PCR products were separated and measured by electrophoresis on a 2 % agarose gel.Results. Analysis of demographic data showed a significant difference in the family history of cancer between the case and control groups (p=0.03). The distribution of ACE gene variants including II, ID, and DD genotypes was also calculated, and significant differences were seen in the DD genotype (p=0.03) and D allele (p=0.04) between the glioblastoma cases and controls.Conclusion. ACE gene polymorphism was associated with glioblastoma in the study population. Further studies are needed to approve this finding.

εγ-Thalassemia, a New Hemoglobinopathy Category.

Large β-globin gene cluster deletions (hereditary persistence of fetal hemoglobin [Hb] or β-, δβ-, γδβ-, and ϵγδβ-thalassemia), are associated with widely disparate phenotypes, including variable degrees of microcytic anemia and Hb F levels. When present, increased Hb A2 is used as a surrogate marker for β-thalassemia. Notably, ϵγδβ-thalassemias lack the essential regulatory locus control region (LCR) and cause severe transient perinatal anemia but normal newborn screen (NBS) results and Hb A2 levels. Herein, we report a novel deletion of the ϵ, Aγ, Gγ, and ψβ loci with intact LCR, δ-, and β-regions in 2 women and newborn twins. Capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), DNA sequencing, multiplex ligation-dependent probe amplification (MLPA), gap-polymerase chain reaction (gap-PCR), and long-read sequencing (LRS) were performed. NBS showed an Hb A > Hb F pattern for both twins. At 20 months, Hb A2 was increased similarly to that in the mother and an unrelated woman. Unexplained microcytosis was absent and the twins lacked severe neonatal anemia. MLPA, LRS, and gap-PCR confirmed a 32 599 base pair deletion of ϵ (HBE1) through ψβ (HBBP1) loci. This deletion represents a hemoglobinopathy category with a distinct phenotype that has not been previously described, an ϵγ-thalassemia. Both the NBS Hb A > F pattern and the subsequent increased Hb A2 without microcytosis are unusual. A similar deletion should be considered when this pattern is encountered and appropriate test methods selected for detection. Knowledge of the clinical impact of this new category will improve genetic counselling, with distinction from the severe transient anemia associated with ϵγδβ-thalassemia.

Molecular spectrum of α- and β-thalassemia among individuals of reproductive age in the Zhuhai region of southern China.

Thalassemia is the most common monogenic disease in South and Southeast Asia. An accurate assessment of the relative frequency and composition of thalassemia mutations is important for the design of appropriate strategies to prevent the disease. In this study, we aimed to decode the molecular characterization of thalassemia mutations in Zhuhai region of southern China. A total of 8048 individuals who were potential thalassemia carriers were enrolled. Gap-polymerase chain reaction (Gap-PCR) and reverse dot-blot (RDB) hybridization methods were employed to detect common deletional and non-deletional thalassemia mutations. Multiplex ligation dependent probe amplification (MLPA) and Sanger sequencing were used to analyze and verify rare and complex mutations. We diagnosed 3433 individuals as thalassemia carriers or patients. Of these, 2395 (69.76%) individuals with α-thalassemia harbored 13 α-globin gene mutations. The three most common α-thalassemia mutations were --SEA (60.08%), -α3.7 (20.62%) and -α4.2 (9.25%). We diagnosed 903 (26.30%) individuals with β-thalassemia and identified 20 β-globin gene mutations, of which the three most frequent were CD41/42 (-TCTT) (38.10%), IVS-II-654 (C>T) (23.69%) and TATAbox-28 (A>G) (15.18%). In addition, we identified 15 rare thalassemia variants. We also summarized the association between the thalassemia genotype and hematological parameters, which demonstrated the broad phenotypic heterogeneity caused by globin gene mutations. This is the first survey of thalassemia molecular epidemiology and hematological phenotype in Zhuhai region. It uncovered a high prevalence and complex molecular spectrum of thalassemia. These findings can be used as a basis for thalassemia diagnosis, counseling and prevention management.

Hb Q-Thailand heterozygosity unlinked with the (–α4.2/) α+-thalassemia deletion allele identified by long-read SMRT sequencing: hematological and molecular analyses

ABSTRACTObjectiveIn the present study, two unrelated cases of Hb Q-Thailand heterozygosity unlinked with the (–α4.2/) α+-thalassemia deletion allele were identified by long-read single molecule real-time (SMRT) sequencing in southern China. The aim of this study was to report the hematological and molecular features as well as diagnostic aspects of the rare manifestation.MethodsHematological parameters and hemoglobin analysis results were recorded. A suspension array system for routine thalassemia genetic analysis and long-read SMRT sequencing were applied in parallel for thalassemia genotyping. Traditional methods, including Sanger sequencing, multiplex gap-polymerase chain reaction (gap-PCR) and multiplex ligation-dependent probe amplification (MLPA), were used together to confirm the thalassemia variants.ResultsLong-read SMRT sequencing was used to diagnose two Hb Q-Thailand heterozygous patients for whom the hemoglobin variant was unlinked to the (–α4.2/) allele for the first time. The hitherto undescribed genotypes were verified by traditional methods. Hematological parameters were compared with those of Hb Q-Thailand heterozygosity linked with the (–α4.2/) deletion allele in our study. For the positive control samples, long-read SMRT sequencing revealed a linkage relationship between the Hb Q-Thailand allele and the (–α4.2/) deletion allele.ConclusionsIdentification of the two patients confirms that the linkage relationship between the Hb Q-Thailand allele and the (−α4.2/) deletion allele is a common possibility but not a certainty. Remarkably, as it is superior to traditional methods, SMRT technology may eventually serve as a more comprehensive and precise method that holds promising prospects in clinical practice, especially for rare variants.

Gene Mutation Spectrum among Alpha-Thalassaemia Patients in Northeast Peninsular Malaysia.

(1) Background: Alpha (α)-thalassaemia is a genetic disorder that affects 5% of the world population. Deletional or nondeletional mutations of one or both HBA1 and HBA2 on chromosome 16 will result in reduced production of α-globin chains, a component of haemoglobin (Hb) that is required for the formation of red blood cells (RBCs). This study aimed to determine the prevalence, haematological and molecular characterisations of α-thalassaemia. (2) Method: The parameters were based on full blood count, high-performance liquid chromatography and capillary electrophoresis. The molecular analysis involved gap-polymerase chain reaction (PCR), multiplex amplification refractory mutation system-PCR, multiplex ligation-dependent probe amplification and Sanger sequencing. (3) Results: With a total cohort of 131 patients, the prevalence of α-thalassaemia was 48.9%, leaving the remaining 51.1% with potentially undetected α gene mutations. The following genotypes were detected: -α3.7/αα (15.4%), -α4.2/αα (3.7%), --SEA/αα (7.4%), αCSα/αα (10.3%), αAdanaα/αα (0.7%), αQuong Szeα/αα (1.5%), -α3.7/-α3.7 (0.7%), αCSα/αCSα (0.7%), -α4.2/αCSα (0.7%), -SEA/αCSα (1.5%), -SEA/αQuong Szeα (0.7%), -α3.7/αAdanaα (0.7%), --SEA/-α3.7 (2.2%) and αCSα/αAdanaα (0.7%). Indicators such as Hb (p = 0.022), mean corpuscular volume (p = 0.009), mean corpuscular haemoglobin (p = 0.017), RBC (p = 0.038) and haematocrit (p = 0.058) showed significant changes among patients with deletional mutations, but not between patients with nondeletional mutations. (4) Conclusions: A wide range of haematological parameters was observed among patients, including those with the same genotype. Thus, a combination of molecular technologies and haematological parameters is necessary for the accurate detection of α-globin chain mutations.

Impact of age-dependent red blood cell parameters on α-globin gene genotyping in children.

When screening for α-thalassemia in children, adult cut-offs for mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) are generally applied to guide genetic evaluation. However, the normal ranges for MCV and MCH are lower in children than in adults, so we hypothesized that using age-matched cut-offs could lead to a more rational diagnostic strategy. The aim of this study was to evaluate if age-matched cut-offs could be applied advantageously. Data on children referred to a hemoglobin fractionation at the Department of Clinical Biochemistry, Aarhus University Hospital between 2016-2021 were identified in the laboratory information system. α-globin gene (HBA1/HBA2) genotyping was performed using multiplex gap-polymerase chain reaction. A total of 387 children were identified. HBA1/HBA2-genotyping was performed in 207 children (53%), and α-thalassemia was diagnosed in 47 children (23%) with -α3.7/αα being the predominant genotype (13%). We found that 23 children had MCV and MCH levels in the normal age-matched range, and two of these children (9%) were α+ thalassemia carriers with three functional α-globin genes. Using age-specific cut-off levels resulted in a reduction of 23 (11%) genotypes performed. In conclusion, applying age-matched cut-offs for MCV and MCH when screening children for α-thalassemia lead to 11% fewer genotypes performed while 9% carriers of α+ thalassemia (of the medically innocuous genotype -α3.7/αα) would have been overlooked.

Germline intergenic duplications at Xq26.1 underlie Bazex-Dupré-Christol basal cell carcinoma susceptibility syndrome.

Bazex-Dupré-Christol syndrome (BDCS; MIM301845) is a rare X-linked dominant genodermatosis characterized by follicular atrophoderma, congenital hypotrichosis and multiple basal cell carcinomas (BCCs). Previous studies have linked BDCS to an 11·4-Mb interval on chromosome Xq25-q27.1. However, the genetic mechanism of BDCS remains an open question. To investigate the genetic aetiology and molecular mechanisms underlying BDCS. We ascertained multiple individuals from eight unrelated families affected with BDCS (F1-F8). Whole-exome (F1 and F2) and genome sequencing (F3) were performed to identify putative disease-causing variants within the linkage region. Array comparative genomic hybridization and quantitative polymerase chain reaction (PCR) were used to explore copy number variations, followed by long-range gap PCR and Sanger sequencing to amplify the duplication junctions and to define the head-tail junctions. Hi-C was performed on dermal fibroblasts from two affected individuals with BDCS and one control. Public datasets and tools were used to identify regulatory elements and transcription factor binding sites within the minimal duplicated region. Immunofluorescence was performed in hair follicles, BCCs and trichoepitheliomas from patients with BDCS and sporadic BCCs. The ACTRT1 variant c.547dup (p.Met183Asnfs*17), previously proposed to cause BDCS, was evaluated with t allele frequency calculator. In eight families with BDCS, we identified overlapping 18-135-kb duplications (six inherited and two de novo) at Xq26.1, flanked by ARHGAP36 and IGSF1. Hi-C showed that the duplications did not affect the topologically associated domain, but may alter the interactions between flanking genes and putative enhancers located in the minimal duplicated region. We detected ARHGAP36 expression near the control hair follicular stem cell compartment, and found increased ARHGAP36 levels in hair follicles in telogen, in BCCs and in trichoepitheliomas from patients with BDCS. ARHGAP36 was also detected in sporadic BCCs from individuals without BDCS. Our modelling showed the predicted maximum tolerated minor allele frequency of ACTRT1 variants in control populations to be orders of magnitude higher than expected for a high-penetrant ultra-rare disorder, suggesting loss of function of ACTRT1 variants to be an unlikely cause for BDCS. Noncoding Xq26.1 duplications cause BDCS. The BDCS duplications most likely lead to dysregulation of ARHGAP36. ARHGAP36 is a potential therapeutic target for both inherited and sporadic BCCs. What is already known about this topic? Bazex-Dupré-Christol syndrome (BDCS) is a rare X-linked basal cell carcinoma susceptibility syndrome linked to an 11·4-Mb interval on chromosome Xq25-q27.1. Loss-of-function variants in ACTRT1 and its regulatory elements were suggested to cause BDCS. What does this study add? BDCS is caused by small tandem noncoding intergenic duplications at chromosome Xq26.1. The Xq26.1 BDCS duplications likely dysregulate ARHGAP36, the flanking centromeric gene. ACTRT1 loss-of-function variants are unlikely to cause BDCS. What is the translational message? This study provides the basis for accurate genetic testing for BDCS, which will aid precise diagnosis and appropriate surveillance and clinical management. ARHGAP36 may be a novel therapeutic target for all forms of sporadic basal cell carcinomas.