Background Thalassemia, a prevalent monogenic inherited disorder, represents a significant public health concern in southern China. This comprehensive study retrospectively analysed prenatal diagnostic data for thalassemia over an 11-year period in Guangdong Province, a high-prevalence region in southern China.Methods Prenatal diagnosis (PND) of thalassemia was performed on 22,127 foetuses from at-risk pregnancies, following standardized protocols for invasive sampling, foetal sample collection and laboratory processing. Thalassemia mutations were identified using PCR-based molecular techniques.Results The analysis revealed that 16,812 cases (75.98%) were diagnosed with thalassemia, including 12,636 cases (57.11%) identified as α-thalassemia, 3961 cases (17.90%) as β-thalassemia and 215 cases (0.97%) as co-inheritance of α- and β-thalassemia. Among these, 5769 cases (26.08%) were diagnosed with intermedia or severe thalassemia genotypes, of which 2593 underwent pregnancy termination. Additionally, our study detected 275 cases of rare thalassemia variants. The retrospective analysis encompassed critical aspects including the timing of PND, genotype spectrum and rare variant identification, complemented by a synthesis of clinical practice insights.Conclusions This large-scale study provides substantial epidemiological data and clinical insights that significantly contribute to the enhancement of thalassemia prevention and control strategies in Guangdong Province.

Abnormal hemoglobin anti-Lepore Hong Kong is a rare βδ fusion variants resulting from non-homologous crossover during meiosis. Anti-Lepore Hong Kong is known to consistently exhibit significantly increased level of HbA2. In this study, we used multiplex ligation-dependent probe amplification (MLPA) and single molecular real-time (SMRT) sequencing, as well as Sanger sequencing, to identify variants in five unrelated families with abnormal elevated HbA2 level. All probands in these five families were found to be heterozygous for anti-Lepore Hong Kong. Among them, two families showed co-occurrence of β 0 -thalassemia and α-thalassemia (– SEA / or α CS α/). Heterozygotes for anti-Lepore Hong Kong displayed an average HbA2 level of 17.7% and behaved normal. However, when combined with β 0 -thalassemia and α-thalassemia, the probands exhibited higher HbA2 level (30.2–40.8%) and behaved with β-thalassemia trait. Furthermore, determination of the α/β-mRNA ratio revealed a slight downregulation of β-globin, similar to that of β-thalassemia minor. Our study is the first to identify compound heterozygotes for anti-Lepore Hong Kong, β 0 -thalassemia and α-thalassemia, provide valuable information for prenatal counseling.

BackgroundGenetic disorders often manifest as abnormal fetal or childhood development. Copy number variations (CNVs) represent a significant genetic mechanism underlying such disorders. Despite their importance, the effectiveness of clinical exome sequencing (CES) in detecting CNVs, particularly small ones, remains incompletely understood. We aimed to evaluate the detection of both large and small CNVs using CES in a substantial clinical cohort, including parent-offspring trios and proband only analysis.MethodsWe conducted a retrospective analysis of CES data from 2428 families, collected from 2018 to 2021. Detected CNV were categorized as large or small, and various validation techniques including chromosome microarray (CMA), Multiplex ligation-dependent probe amplification assay (MLPA), and/or PCR-based methods, were employed for cross-validation.ResultsOur CNV discovery pipeline identified 171 CNV events in 154 cases, resulting in an overall detection rate of 6.3%. Validation was performed on 113 CNVs from 103 cases to assess CES reliability. The overall concordance rate between CES and other validation methods was 88.49% (100/113). Specifically, CES demonstrated complete consistency in detecting large CNV. However, for small CNVs, consistency rates were 81.08% (30/37) for deletions and 73.91% (17/23) for duplications.ConclusionCES demonstrated high sensitivity and reliability in CNV detection. It emerges as an economical and dependable option for the clinical CNV detection in cases of developmental abnormalities, especially fetal structural abnormalities.

Background β-Thalassemia is mainly caused by point mutations in the β-globin gene cluster. With the rapid development of sequencing technic, more and more variants are being discovered. Results In this study, we found two novel deletion mutations in two unrelated families, HBB: c.180delG (termed β CD59 ) and HBB: c.382_402delCAGGCTGCCTATCAGAAAGTG (termed β CD128-134 ) in family A and B, respectively. Both the two novel mutations lead to β-thalassemia trait. However, when compounded with other β 0 -thalassemia, it may behave with β-thalassemia intermedia or β-thalassemia major. Conclusion Our study broadens the variants spectral of β-thalassemia in Chinese population and provides theoretical guidance for the prenatal diagnosis.

ABSTRACTObjective In 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.Methods Hematological 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.Results Long-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.Conclusions Identification 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.

Background At present, the methods generally used to detect α-thalassemia mutations are confined to detecting common mutations, which may lead to misdiagnosis or missed diagnosis. The single-molecule real-time (SMRT) sequencing enables long-read single-molecule sequencing with high detection accuracy, and long-length DNA chain reads in high-fidelity read mode. This study aimed to identify novel large deletions and complex variants in the α-globin locus in Chinese population. Methods We used SMRT sequencing to detect rare and complex variants in the α-globin locus in four individuals whose hematological data indicated microcytic hypochromic anemia. However, the conventional thalassemia detection result was negative. Multiplex ligation-dependent probe amplification and droplet digital polymerase chain reaction were used to confirm SMRT sequencing results. Results Four novel large deletions were observed ranging from 23 to 81 kb in the α-globin locus. One patient also had a duplication of upstream of HBZ in the deletional region, while another, with a 27.31-kb deletion on chromosome 16 (hg 38), had abnormal hemoglobin Siriraj (Hb Siriraj). Conclusion We first identified the four novel deletions in the α-globin locus using SMRT sequencing. Considering that the conventional methods might lead to misdiagnosis or missed diagnosis, SMRT sequencing proved to be an excellent method to discover rare and complex variants in thalassemia, especially in prenatal diagnosis.

Hemoglobin Santa Ana [β88(F4)Leu→Pro (CTG > CCG) HBB: c.266T > C] is an unstable hemoglobin variant characterized by a substitution of the amino acid leucine by proline at the 88th position of the β-globin chain. We for the first time identified this hemoglobin variant in a Chinese patient by capillary electrophoresis (CE). The proband was an 8-year-old boy with chronic anemia, brown urine and splenomegaly. He had been affected by moderate anemia, twice approaching a severe degree, that was attributed to infection. The CE result revealed an abnormal hemoglobin peak at electrophoretic zone 4 that correspond to the hemoglobin Santa Ana peak, and a CTG > CCG mutation at codon 88 of the β-globin gene was confirmed by DNA sequencing. To avoid misdiagnosis and genetic risks, a literature review of other unstable hemoglobins that migrate similarly to the hemoglobin Santa Ana was performed. Our findings indicate that hemoglobin Santa Ana can be clearly separated by CE, with accurate diagnosis depending on molecular analysis. This information will be useful for providing appropriate genetic counselling and for prenatal diagnosis.

目的 利用基因检测技术,调查广东地区育龄妇女叶酸代谢相关基因多态性分布,筛查出机体叶酸利用能力有缺陷的高风险个体,为其叶酸增补方案提供科学理论指导.方法 选取5386例育龄妇女作为研究样本,利用DNA提取以及多重连接探针扩增(multiplex ligation-dependent probe amplification,MLPA)检测技术,检测亚甲基四氢叶酸还原酶(methyltetrahydrofolate reductase,MTHFR)叶酸代谢相关基因多态性分布.结果 MTHFR基因677位点CC、CT、TT基因型频率分别为53.5％、37.4％、9.1％;MTHFR基因1298位点AA、AC、CC基因型的频率分别为59.7％,35.1％,5.2％.结论 MTHFR基因相关位点的检测可成功筛查出叶酸利用欠缺的高风险育龄妇女,为其叶酸增补方案提供科学量化指导.

Objectives Here we report two rare alpha-globin chain variants in two unrelated families: Hb Val de Marne [alpha 133(H16) Ser > Arg (AGC > CGC); HBA2: c.400A > C] and Hb Dongguan [alpha 52(E6) Ser > Cys (TCT > TGT); HBA1: c.158C > G]. Notably, HBA2: c.400A > C is an unreported new variant in the third exon of the alpha(2) gene, and simple heterozygous unstable Hb Dongguan haematological characteristics are proposed for the first time. Methods Hb analysis was performed by using capillary electrophoresis (CE). Twenty-three common mutations were detected using a suspension array system. Mutations were identified by DNA sequencing. Results The CE results showed an abnormal peak with incomplete separation from Hb A at zone 8 in two members of Family 1. DNA sequencing confirmed the presence of Hb Val de Marne [alpha 133(H16) Ser > Arg (AGC > CGC); HBA2: c.400A > C]. Five members of Family 2 exhibited an abnormal peak at zone 11, and DNA sequencing confirmed the presence of Hb Dongguan [alpha 52(E6) Ser > Cys (TCT > TGT); HBA1: c.158C > G]. Conclusions The discovery of HBA2: C.400A > C expands the existing spectrum of alpha-globin variants. The carriers of simple heterozygous Hb Dongguan generally do not have obvious clinical symptoms. The information in this study will help clinicians understand the screening, molecular diagnosis and clinical significance of Hb variants.

Abstract β-Thalassemia (β-thal), a highly prevalent disease in tropical and subtropical regions of Southern China, is caused mainly by point mutations in the β-globin gene cluster. However, large deletions have also been found to contribute to some types of β-thal. We identified a novel 5 kb deletion in the β-globin cluster in a Chinese patient using multiplex ligation-dependent probe amplification (MLPA), and characterized it with single molecule real-time (SMRT) sequencing, gap-polymerase chain reaction (gap-PCR) and Sanger sequencing. The deletion was located between positions 5226189 and 5231091 on chromosome 11 (GRCh38), extending from 4 kb upstream of the 5' untranslated region (5'UTR) to the second intron of the β-globin gene. The patient with this deletion presented with microcytosis and hypochromic red cells, as well as relatively high Hb F and Hb A2 levels. Our research indicated that SMRT sequencing is a useful tool for accurate detection of large deletions. Our study broadens the spectrum of deletional β-thalassemias and provides a perspective for further study of the function of the β-globin cluster.

Objective: The 3.7 kb deletion (-alpha(3.7)) in the alpha-globin cluster, which characterizes alpha(+)-thalassemia, has been reported to have a carrier rate of 4.78% in southern China. Three -alpha(3.7) subtypes have been identified worldwide. However, the -alpha(3.7 III) subtype has not previously been identified in China. Herein, we reported identification of the -alpha(3.7 III) subtype in a Chinese patient. Methods: We used gap-PCR and a liquid chip system to detect alpha-thalassemia mutations. Multiple ligation-dependent probe amplification was performed to detect the large deletion. We finally used Sanger sequencing and single molecule real-time sequencing to characterize and confirm the genotype. Results: The proband, characterized as -alpha(3.7 III) heterozygous, showed microcytosis and hypochromic red cells, with a mean corpuscular volume of 78 fL and mean corpuscular hemoglobin of 25.4 pg. The proband's mutation was inherited from her father, who had normal blood parameters. Conclusion: We first identified the -alpha(3.7 III) subtype in China. Consequently, all -alpha(3.7) subtypes have now been identified in the Chinese population. Therefore, attention should be paid to -alpha(3.7 III) in clinical prenatal diagnosis, given that commonly used methods such as gap-PCR may lead to misdiagnosis or missed diagnosis.

Objective To investigate whether β-globin gene 3'UTR+101G>C (HBB:c.*233G>C) variant has genetic effect and provide basis for gene diagnosis and genetic counseling. Method Whole blood cell analysis and capillary zone electrophoresis (CZE) were used to analyze the hematological indexes. The most frequent 23 mutations in southern Chinese individuals were routinely measured by PCR-flow fluorenscence immunmicrobeads assay. Sanger sequencing was used to detect the other variants of β-globin gene (HBB). Results In 463 cases, a total of 7 cases with HBB:c.*233G>C variant were detected, among them 4 cases carried other pathogenic variants of HBB gene (2 cases were in trans, 2 cases were in cis), who had typical hematological characteristics of mild β-thalassemia, and 3 cases also carried abnormal hemoglobin variation, but did not have hematological characteristics of β-thalassemia. Conclusion The study shows that HBB:c.*233G > C variant has no obvious genetic effect and should be a benign polymorphism.

AbstractIntroductionAlthough over 1000 hemoglobin (Hb) variants were identified so far, Hb Port Phillip compound with α‐thalassemia deletion had no reported before.MethodsTwo patients and the associated families from Guangdong province in China were recruited. Hematological parameters were determined by blood routine examination and hemoglobin electrophoresis. Genotyping was performed by Gap‐PCR and Sanger sequencing.ResultsOne patient was diagnosed as Hb Port Phillip, while her daughter was compounded with ‐α4.2 deletion, with normal Hb level (150 g/L), mean corpuscular volume (MCV) 108.4 fl and mean corpuscular hemoglobin (MCH) (30.5 pg). Another patient was diagnosed as compound Hb Port Phillip and ‐‐SEA deletion. This proband presented with more severe α‐thalassemia trait than the patient compounded with ‐α4.2 deletion, with hemoglobin 80 g/L, MCV 61.7 fl, and MCH 18.7 pg.ConclusionHere we first time identified two patients compound with Hb Port Phillip and ‐α4.2 and ‐‐SEA deletions, respectively, which had never been reported. Our study widens the genotypes of hemoglobinopathy and provides reference for genetic counselling and prenatal diagnosis in this population.

We report two unrelated cases of compound heterozygosity for hemoglobin (Hb) variant Broomhill and the Southeast Asian (- - SEA/) α-thalassemia deletion, whose clinical features and laboratory findings have never been reported. Hematological analyses revealed abnormal values for both cases as α-thalassemia traits, and capillary electrophoresis suggested an abnormal peak that was incompletely separated from the Hb A peak. A suspension array system and Sanger sequencing were used to characterize the genotypes. Sanger sequencing confirmed the presence of Hb Broomhill [α114(GH2)Pro→Ala; HBA1: c.343C>G]. Eventually, both cases were accurately diagnosed as compound heterozygotes for Hb Broomhill and the (- - SEA/) α-thalassemia deletion, which is the first known report of these variants. This information will be useful when providing appropriate genetic counselling and prenatal diagnosis.

目的 评价纯合和复合杂合点突变型(地中海贫血的血液学特征,为临床产前咨询提供依据.方法 对明确诊断的11例纯合和复合杂合点突变型α地中海贫血进行回顾性血液学指标分析.结果 11例纯合型和复合杂合点突变型α地中海贫血病例中共检出纯合点突变型α地中海贫血5例,血红蛋白96?137g/L.复合杂合点突变型α地中海贫血5例,血红蛋白93?139g/L.纯合点突变型α地中海贫血同时合并β地中海贫血1例,血红蛋白106g/L.结论 纯合型和复合杂合点突变型α地中海贫血血液学参数及血红蛋白电泳结果存在一定的相关性,同时也存在一定的差异.纯合和复合杂合点突变型α地中海贫血结果分析应该结合血液学参数及血红蛋白电泳结果进行分析.

目的 对2005年5月至2020年4月来广东省妇幼保健院就诊且血红蛋白电泳和基因诊断确诊的血红蛋白H病(HbH病)合并β地贫及中重型β地贫合并α地贫患者进行血液学特征分析,为临床遗传咨询及产前诊断提供指导意义.方法 采用血常规检测及血红蛋白电泳方法分别获得HbH病合并β地贫及中重型β地贫合并α地贫患者的红细胞参数及各类型血红蛋白的含量,使用聚合酶链式反应(polymerase chain reaction,PCR)结合反向点杂交(reverse dot blot,RDB)、gap-PCR等方法进行地中海贫血基因检测.结果 在近5年来就诊于本院的患者中,HbH病合并β地贫的患者有19例(男性7例、女性12例),其血红蛋白(hemoglobin,Hb)的平均水平为(103.32士16.66) g/L,男性为(120.57士9.82)g/L,女性为(93.25士10.41)g/L;男性与女性的Hb平均水平比较,差别有统计学意义(P＜0.01).HbA2的平均水平为(4.55±0.94)％;男性为(4.33士1.32)％,女性为(4.68士0.58)％,男性与女性的HbA2平均水平比较,差别没有统计学意义(P＞0.05).中重型β地贫合并α地贫的患者有19例(男性8例,女性11 例),Hb 的平均水平为(91.89士16.44)g/L,男性为(97.88±12.85)g/L,女性为(87.55士17.37)g/L;HbA2的平均水平为(4.94±2.62)％,男性为(3.85士1.84)％,女性为(5.74士2.80)％,男性与女性的Hb及HbA2平均水平比较,差别均没有统计学意义(P＞0.05).结论 在38例病例中,患者的血常规结果及临床表现均显示为轻中度贫血.

目的 分析少见突变α2珠蛋白基因CD30 (delGAG)病例的表型特征,并对携带此突变的家系进行产前基因诊断.方法 采集所有病例外周血进行红细胞参数和血红蛋白电泳分析,收集羊水用于产前诊断.PCR-流式荧光杂交法和Sanger测序的方法被应用于珠蛋白基因突变检测.结果 在5例CD30 (delGAG)突变病例中,4例是此突变的杂合子,1例复合东南亚缺失型α地贫突变(--SEA/αCD30α).所有杂合子均有小细胞低色素的血液学表现,但血红蛋白值均正常.__SEA/αCD30α病例除表现小细胞低色素特征以外,还有轻度贫血且血红蛋白电泳分析显示Hb H(35.8％)和Hb Bart's(0.6％)明显升高.进行产前诊断的4个家系中,检测到1例胎儿为αCD30α/αα、2例胎儿为--SEA/αCD30α和1例胎儿为--SEA/-α4.2.结论 α2珠蛋白基因CD30 (delGAG)变杂合子携带者无贫血症状,复合其他α地中海贫血突变时会加重相关血液学表现.由于该突变不在临床地中海贫血常见基因突变类型检测范围内,因此应用其他分子方法精确诊断此突变对产前诊断具有重要价值.

目的 分析Hb Westmead(Hb WS)携带者的血液学特征,探讨携带Hb WS突变的不同基因型的表型特点.方法 回顾性分析在广东省妇幼保健院行地中海贫血(地贫)基因检测后诊断为Hb WS携带者的179例样本,收集其血常规和血红蛋白电泳相关参数结果,运用SPSS软件进行统计分析.结果 在检出的179例样本中,Hb WS杂合子血液学指标基本正常,与静止型α-地中海贫血临床表型相吻合;Hb WS杂合子合并(-α37/)或(-α4.2/)时,平均红细胞血红蛋白量(mean corpuscular hemoglobin,MCH)较单纯的Hb WS下降得明显,但其血液学各项指标变化较东南亚缺失型杂合子轻微;Hb WS杂合子合并东南亚缺失型α-地贫时,表型类似轻型α-地贫;Hb WS杂合子合并轻型β-地贫时,表型类似轻型β-地贫.结论 Hb WS无论以杂合子单独存在还是合并α-或β-地贫时,对血液学表型的影响均较小.

目的 分析多种分子检测方法在精确诊断罕见α-珠蛋白生成障碍性贫血中的应用,探讨其对预防珠蛋白生成障碍性贫血出生缺陷的重要性.方法 对2例外周血标本进行血红蛋白毛细管电泳和红细胞参数的分析,再应用跨越断裂点PCR、PCR结合反向点杂交、多重连接依赖性探针扩增、珠蛋白基因测序及染色体微阵列等多种方法鉴定此病例的α-珠蛋白生成障碍性贫血基因突变类型.结果 检测到2例病例均为罕见的缺失型α-珠蛋白生成障碍性贫血.1例为α-珠蛋白基因簇调控位点HS-40区域至ζ珠蛋白基因上游区域杂合缺失,导致2个α-珠蛋白基因无法正常表达;另1例缺失大小为27.6 kb合并常见的东南亚型的双重缺失突变.结论 此类罕见缺失型的α-珠蛋白生成障碍性贫血合并最常见东南亚缺失类型时,可引起中重型α-珠蛋白生成障碍性贫血患儿的出生.对罕见α-珠蛋白生成障碍性贫血的精确分子诊断可为遗传咨询和产前诊断提供重要的指导意义,对预防中重型珠蛋白生成障碍性贫血出生缺陷至关重要.