An Unexpected Finding of Klinefelter Syndrome during Glucose-6-Phosphate Dehydrogenase Deficiency (G6PD) Genetic Analysis.

BACKGROUND: Klinefelter syndrome is a common sex chromosome abnormality in males, characterized by an extra X chromosome compared to normal males. Glucose-6-phosphate dehydrogenase deficiency (G6PD) is an X-linked incomplete dominant defect disorder. In this study, we reported the unexpected detection of Klinefelter syndrome in a patient with G6PD. METHODS: G6PD enzyme activity was measured by immunoenzyme assay, and genetic analysis was performed using a fluorescent PCR melting curve method (PCR-melting curve). Sex chromosome number abnormalities were detected by multiplex ligation-dependent probe amplification (MLPA). The patient also underwent peripheral blood chromosome karyotype analysis. RESULTS: The patient's G6PD and 6PGD enzyme activities were 21.34 U/L and 22.85 U/L, respectively, and their ratio was below the reference range (0.93). The PCR-melting curve displayed a c.1388 heterozygous mutation in this boy, and the Sanger sequencing provided the same results. MLPA results suggested the presence of approxi-mately two copies of the X-chromosome in the boy. Finally, chromosome karyotype analysis confirmed that the boy had Klinefelter syndrome with a karyotype of 47, XXY. CONCLUSIONS: Klinefelter syndrome was accidentally detected during G6PD genetic analysis in a male. X-chromosomes can interfere with the results of G6PD genetic analysis and should be noted.

Hb Guigang [α90 (FG2)Lys→Asn; HBA1:c.273G˃T]: a Novel α-Globin Chain Variant.

BACKGROUND: New hemoglobin (Hb) variants are constantly being updated as assays are developed and the testing population expands. Here, we report a novel Hb variant, named Hb Guigang. METHODS: Hemoglobin (Hb) analysis was analyzed by capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). Glycated hemoglobin was performed by CE and HPLC. Routine genetic analysis was done with Gap-PCR and PCR-reverse dot-blot hybridization. The hemoglobin variant was identified by Sanger sequencing. RESULTS: CE of three cases showed the presence of Hb variants in Zone 5 and Zone 12, respectively. HPLC indicated an elevated P3 peak, suggesting the possible presence of the Hb variant. Hb A1c was measured by CE and HPLC, and the results were 6.7% and 4.76%, respectively. Sanger sequencing confirmed an AAG˃AAT mutation at codon 90 of the HBA1 gene. This mutation was reported for the first time, and we named it Hb Guigang based on the proband's place of residence. CONCLUSIONS: Hb Guigang with normal hematological parameters was separated and quantified by CE, whereas HPLC suggested that Hb Guigang co-eluted with the P3 peaks and could not be quantified.

Hb Hebei [α20 (B1) His→Leu; HBA2:C.62A > T]: A Novel Hemoglobin Variant Found during Measurement of Glycated Hemoglobin.

We report a novel hemoglobin (Hb) variant found in a 34-year-old Chinese male during a routine measurement of glycated hemoglobin. The variant resulted in a P3 peak of 27.5% of the total Hb on high performance liquid chromatography (HPLC) with a glycated hemoglobin mode. However, no abnormal Hb peaks were observed in capillary electrophoresis (CE) with 3.1% Hb A2 and 96.9% Hb A. The amino acid substitution was determined by Sanger sequencing as α20 (B1) His→Leu; the corresponding DNA mutation was identified as CAC > CTC at the first position of codon 20 of the α-chain. This is the first description of the mutation, and we have named it Hb Hebei for the region of origin of the proband.

Misdiagnosis of ß-Thalassemia Major Due to Chinese Gγ+(Aγδß)0-Thalassemia Combined with ß0-Thalassemia.

δß-thalassemia is a rare type of thalassemia characterized by increased Hb F levels, including mainly Chinese Gγ(Aγδß)0-thalassemia, Yunnanese Gγ(Aγδß)0-thalassemia, Cantonese Gγ(Aγδß)0-thalassemia in China. Due to the low rate of δß-thalassemia carriers, there are few reports of δß-thalassemia combined with ß-thalassemia causing ß-thalassemia major. Herein, we described the combination of Chinese Gγ(Aγδß)0-thalassemia and ß-thalassemia leading to ß-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 ßCD17M/ßCD17M using routine genetic analysis. However, her father was heterozygous for CD17 in ß-globin, and her mother was detected as SEA heterozygous. The further analysis presented that the proband had actually missed the diagnosis of Chinese Gγ(Aγδß)0-thalassemia by MLPA and PCR-RDB. Finally, the genotype of the proband was corrected from ßCD17M/ßCD17M to ßCD17M/ßGγ(Aγδß)0. This is the first report of Chinese Gγ(Aγδß)0-thalassemia combined with ß-thalassemia resulting in ß-thalassemia major in China. Screening for δß-thalassemia by Hb analysis could be an effective method.

A New δ-Globin Gene Variant: Hb A2-Yulin [δ46(CD5)Gly→Arg,HBD: C.139G > A].

We report a new δ-chain hemoglobin (Hb) variant observed in a 5-year-old female living in Yulin, Guangxi, China. Capillary electrophoresis revealed splitting of the Hb A2 peak into two fractions (Hb A2 and Hb A2 variant), and the Hb A2 variant was also detected by high-performance liquid chromatography. However, it could not be detected using matrix-assisted laser desorption lonization-time of flight mass spectrometry. CD41-42 (-TCTT) heterozygosity was observed on the HBB gene by PCR and reverse dot-blot hybridization. Sanger sequencing showed a new transition (G > A) at codon 46 of the HBD gene, resulting in glycine changing to arginine. Based on the patient's place of residence, the new variant was named Hb A2-Yulin [δ46(CD5)Gly→Arg,HBD:c.139G > A].

Evaluating the Clinical Utility of a Long-Read Sequencing-Based Approach in Prenatal Diagnosis of Thalassemia.

BACKGROUND: The aim is to evaluate the clinical utility of a long-read sequencing-based approach termed comprehensive analysis of thalassemia alleles (CATSA) in prenatal diagnosis of thalassemia. METHODS: A total of 278 fetuses from at-risk pregnancies identified in thalassemia carrier screening by PCR-based methods were recruited from 9 hospitals, and PCR-based methods were employed for prenatal diagnosis. CATSA was performed retrospectively and blindly for all 278 fetuses. RESULTS: Among the 278 fetuses, 263 (94.6%) had concordant results and 15 (5.4%) had discordant results between the 2 methods. Of the 15 fetuses, 4 had discordant thalassemia variants within the PCR detection range and 11 had additional variants identified by CATSA. Independent PCR and Sanger sequencing confirmed the CATSA results. In total, CATSA and PCR-based methods correctly detected 206 and 191 fetuses with variants, respectively. Thus, CATSA yielded a 7.9% (15 of 191) increment as compared with PCR-based methods. CATSA also corrected the predicted phenotype in 8 fetuses. Specifically, a PCR-based method showed one fetus had homozygous HBB c.52A > T variants, while CATSA determined the variant was heterozygous, which corrected the predicted phenotype from ß-thalassemia major to trait, potentially impacting the pregnancy outcome. CATSA additionally identified α-globin triplicates in 2 fetuses with the heterozygous HBB c.316-197C > T variant, which corrected the predicted phenotype from ß-thalassemia trait to intermedia and changed the disease prognosis. CONCLUSIONS: CATSA represents a more comprehensive and accurate approach that potentially enables more informed genetic counseling and improved clinical outcomes compared to PCR-based methods.

Hb Qinzhou [α1 78 (EF7) Asn→Lys (AAC>AAA); HBA1:c.237C>A]: a Novel α-Globin Variant in a Chinese Family.

BACKGROUND: Many new variants are constantly detected by capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). Here, we described a novel α-globin gene mutation. METHODS: The proband was a 46-year-old male who came to the hospital with his wife for pre-conception thalassemia screening. Hematological parameters were obtained from a complete blood count. Hb analysis was performed by CE and HPLC. Routine genetic analysis was carried out by Gap-polymerase chain reaction (Gap-PCR) and PCR and reverse dot-blot (PCR-RDB). Sanger sequencing was used to identify the hemoglobin variant. RESULTS: An abnormal Hb variant was observed at electrophoretic zone 5 and zone 1 on the CE program. HPLC showed a peak of abnormal Hb in the S window. No mutations were detected by Gap-PCR and PCR-RDB. Sanger sequencing revealed an AAC>AAA mutation at codon 78 of the α-globin gene [α1 78 (EF7) Asn→Lys (AAC> AAA); HBA1:c.237C>A]. The pedigree study demonstrated that the Hb variant was inherited from his mother. CONCLUSIONS: It is the first report about the variant, so we named it Hb Qinzhou for the place of origin of the proband. Hb Qinzhou presents a normal hematological phenotype.

The Unexpected Detection of a Novel Mutation in a Patient with Hb G-Taipei During Glycated Hemoglobin Test.

BACKGROUND: There are occasional unexpected detections in HbA1c tests. Here, we described a novel ß-globin gene mutation and its hematological phenotype. METHODS: The proband is a 60-year-old woman who was admitted to the hospital for two weeks due to chest pain. Complete blood count, fasting blood glucose, and glycated hemoglobin tests were performed before admission. High-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) were used to detect HbA1c. The hemoglobin variant was verified by Sanger sequencing. RESULTS: An abnormal peak was observed on HPLC and CE, but the value of HbA1c was normal. Sanger sequencing revealed a GAA>GGA mutation at codon 22 (corresponding to Hb G-Taipei) and a deletion (-GCAATA) at position 659_664 of the second intron of the ß-globin gene. The proband and her son, who inherited this new mutation, have no hematological phenotype changes. CONCLUSIONS: This is the first report of this mutation, named IVS II-659_664 (-GCAATA). It has a normal phenotype and does not cause thalassemia. IVS II-659_664 (-GCAATA) compounded Hb G-Taipei did not affect the detection of HbA1c.

False HbA1cValue due to a Rare Variant of Hemoglobin J-Cubujuqui.

BACKGROUND: Hemoglobin (Hb) J-Cubujuqui is a rare Hb variant, and reports about it are very limited. There are no descriptions that it affects the results of glycated Hb. METHODS: In this study, we describe a rare variant discovered during newborn screening. Both high-performance liquid chromatography (HPLC) and capillary electrophoresis for hemoglobin analysis displayed abnormal peaks. The Hb variant was confirmed by Sanger sequencing. RESULTS: The pedigree study shows the variant was inherited from the newborn's father. His fasting blood glucose (FBG) level was 5.5 mmol/L. HbA1c measured by HPLC was falsely low in her father (2.41%), whereas that measured by immunoassay was normal (5.11%). Sanger sequencing revealed a heterozygous mutation (CGT˃AGT) at amino acid position 141 of the α1 gene, corresponding to Hb J-Cubujuqui [α1 141(HC3) Arg→Ser (CGT˃AGT); HBA1:c.424C˃A (or HBA2)]. CONCLUSIONS: This is the first report that Hb J-Cubujuqui interferes with the measurement of HbA1cand prompts clinicians to pay attention to the accuracy of glycated Hb results.

[Hemoglobin C Variant Affecting Glycated Hemoglobin Test Results: A Rare Case Report].

Hemoglobin (Hb) variants are common factors that affect the results of glycosylated hemoglobin (A1C) tests. Hemoglobin variants react differently to different testing methods. Herein, we presented the first ever report of the effect of hemoglobin C (Hb C) on the test results of A1C in the Chinese population. High performance liquid chromatography (HPLC) and capillary electrophoresis were performed to measure A1C. Hemoglobin electrophoresis was conducted to identify the hemoglobin variants. Hb sequencing was performed to determine the mutation sites on the ß chain. HPLC showed decreased A1C results, which could be corrected by electrophoresis, but the electrophoresis graph still showed abnormal peaks. The hemoglobin electrophoresis results suggested that there were hemoglobin variants, which hemoglobin sequencing results revealed to be Hb C. Uncommon variations in a specific population tend to be overlooked. To avoid clinical decision-making being affected by the results of a single test, we recommend that an explanatory reporting model be routinely adopted for A1C tests so that all reports always contain explanatory notes for the testing methodology and analysis of the graphs.

Detection of hemoglobin H disease by long molecule sequencing.

BACKGROUND: Hemoglobin H (Hb H) disease is a moderate-to-severe form of α-thalassemia (α-thal), and parts of patients may require intermittent transfusion therapy, especially during intercurrent illness. However, rare Hb H diseases remain undetected using routine methods being outside of the testing scope. In this study, we present an approach to detecting Hb H disease by long molecule sequencing (LMS). METHODS: A total of 206 known genotype samples were collected and carried to blind detected by LMS on the PacBio Sequel platform. Circular consensus sequencing reads were aligned to the hg19 reference genome using Free-Bayes finished LMS. LMS accuracy would be compared with routine methods, including Gap-PCR and PCR-Reverse dot blot hybridization (PCR-RDB). RESULTS: The assay could detect carriers of both deletion and point mutations. It had an overall accuracy of 100% when compared with routine methods. In addition, LMS detected six mutations based on routine methods and corrected three case results. Hb H diseases were identified using LMS, whether a common or rare genotype, a deletion or non-deletion genotype. However, two cases of Hb H disease were misdiagnosed using routine methods. CONCLUSIONS: Long molecule sequencing can be suggested as a rapid and reliable assay to detect probable carriers of hemoglobinopathies. LMS accurately identified the common and rare genotypes of Hb H disease.

Three-dimensional imaging for the localization of related anatomical structures during surgery on the internal auditory canal.

BACKGROUND: The Fisch infra-temporal fossa approach (Fisch's method), first proposed in 1970, is commonly used during internal auditory canal (IAC) surgery with an approach that advances through the middle cranial fossa. This study was designed to address the technical difficulties encountered in recognizing and localizing the arcuate eminence with respect to the superior semicircular canal (SSC). METHODS: Forty men and 40 women (18-57 years of age) without space-occupying lesions in the petrous part of the temporal bone were selected for the study. In total, 160 samples were obtained from both sides of the temporal bone. The temporal bone in these 160 samples was scanned using computed tomography, and a three-dimensional coordinate system was established to measure the three-dimensional coordinate values of structures adjacent to the arcuate eminence, the SSC, and the IAC. RESULTS: The results showed that the shape of the arcuate eminence is highly variable. Approximately 23.12% of samples had no obvious arcuate eminence, which prevented the use of Fisch's method to localize the SSC. The arcuate eminence was difficult to identify in 37 samples. CONCLUSIONS: Analysis samples showed that the SSC was located in a fan ring centered at the midpoint of the upper edge of the petrous portion of the temporal bone. The arcuate eminence did not correspond directly with the SSC, as the former was located posterolateral to the latter in 85.83% of samples. The angle between the SSC and the IAC ranged from 0° to 60° degrees, as reported previously by Fisch. However, the angle typically ranged from 10-30° in our study.

Detection of Hb Yulin [ß13(A10)Ala→Val, HBB: c.41C>T] by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry.

We report a rare hemoglobin (Hb) variant on the ß-globin gene, which was detected in a female from Yulin City, Guangxi Autonomous Region, People's Republic of China (PRC), during routine thalassemia screening. The Hb variant remained unnoticed using capillary electrophoresis (CE) and high performance liquid chromatography (HPLC), while an additional peak was observed by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS). DNA sequencing revealed the GCC>GTC substitution at codon 13 on the ß-globin gene, causing a substitution of alanine to valine. The mutation is only described in the ITHANET database but no Hb variant name and other information, so we named it Hb Yulin after the place of origin of the proband in this study. Hb Yulin is clinically silent and easily leads to misdiagnosis during hemoglobinopathies screening based on the common methods of HPLC and CE.

Hb Laibin [ß96(FG3)Leu→Arg; HBB: c.290T>G]: A Novel Hemoglobin Variant Described in a Chinese Family.

We report a novel ß chain hemoglobin (Hb) variant found in a Chinese family. A high level of Hb F was observed on capillary electrophoresis (CE). However, high performance liquid chromatography (HPLC) showed a high level of Hb A2. DNA sequencing revealed a single base substitution (T>G) at codon 96 of exon 2 of the ß-globin gene. This alters the normally encoded leucine to arginine [ß96(FG3)Leu→Arg; HBB: c.290T>G] that we propose to name Hb Laibin for the region of origin of the proband. The pedigree study showed that it was inherited from his mother.

Hb Huadu [α124(H7)Ser→Thr (TCC>ACC), HBA2: c.373T>A]: A Novel Variant of the α-Globin Gene.

Here, we report a novel α chain hemoglobin (Hb) variant found during routine thalassemia screening. This Hb variant can be detected by capillary electrophoresis (CE) but cannot be recognized by high performance liquid chromatography (HPLC). Sanger sequencing revealed a heterozygous missense substitution at nucleotide 373 on the HBA2 gene, which results in the replacement of serine by threonine at codon 124 [α124(H7)Ser→Thr (TCC>ACC), HBA2: c.373T>A]. It is the first report of this variant, named Hb Huadu for the birthplace of the proband. In addition, the proband coinherited the heterozygous codons 41/42 (-TTCT) (HBB: c126_129delCTTT) on the ß-globin gene.

Hb Hezhou [ß64(E8)Gly→Ser; HBB: c.193G>A]: A Novel Variant on the ß-Globin Gene.

We report a novel mutation on the ß-globin gene, Hb Hezhou [ß64(E8)Gly→Ser; HBB: c.193G>A] that was detected in two unrelated Chinese individuals. Patient 1 also carried an α+-thalassemia (α+-thal) -α4.2 (leftward) deletion, but hematological analyses showed no clinical consequences. Patient 2 was heterozygous for Hb Hezhou. Hemoglobin (Hb) analysis was performed using capillary electrophoresis (CE) and high performance liquid chromatography (HPLC). The Hb variant remained undetected using HPLC, while an additional peak was detected by CE. The finding of Hb Hezhou indicates that the possibilities of rare Hb variants should be alerted in the thalassemia screening program and precisely diagnosed depending on the Hb separation technique used.

First Detection of Hb Cenxi [ß46(CD5)Gly→Arg (GGG>CGG), HBB: c.139G>C] by Capillary Electrophoresis.

We report a novel mutation on the ß-globin gene in a female of the Chinese population. This mutation produces a ß-globin variant that can be detected by the capillary electrophoresis (CE) method, but coelutes with Hb A2 by high performance liquid chromatography (HPLC). DNA sequencing showed a mutation of codon 46 and it was named Hb Cenxi [ß46(CD5)Gly→Arg (GGG>CGG), HBB: c.139G>C] for the city of birth of the proband. She presented normal hematological parameters.

Hb Dahua [ß59(E3)Lys→Met; HBB: c.179A>T] a Novel Variant on the ß-Globin Gene.

We report a novel hemoglobin (Hb) variant found in a Chinese individual from the Guangxi Zhuang Autonomous Region of the People's Republic of China. The proband was a 6-month-old boy who came from Dahua county, Hechi City. Capillary zone electrophoresis (CZE) showed an abnormal peak (51.2%) in zone 13. However, capillary isoelectric focusing electrophoresis (CIFE) presented an unknown peak in zone Bart's (51.4%). High performance liquid chromatography (HPLC) displayed an unknown peak (42.1%) at retention time 2.44 min., eluting before Hb A0. Direct DNA sequencing of the ß-globin gene revealed heterozygosity for a missense mutation at codon 59 (AAG>ATG), causing a lysine to methionine substitution [ß59(E3)Lys→Met; HBB: c.179A>T]. We decided to name the variant Hb Dahua for the place of origin of the proband.

Electrophoresis features and genotypes of Hb bart's hydrops fetalis.

Hydrops fetalis syndrome (Hb Bart's disease) is one of the common fetal development abnormalities and the most severe form of α-thalassemia. It causes fetal death during the third trimester or shortly after birth. The aim of this study is to investigate the feature of Hb Bart's disease by Capillary electrophoresis (CE) and its genotypes. A total of 38 cases of suspected fetal Hb Bart's disease were analyzed by B-ultrasound and CE, including 17 with 24-28 weeks of pregnancy, 9 with 29-32 weeks of pregnancy, and 12 with 33-38 weeks of pregnancy. All cases were then identified by DNA analysis. The minimum concentration of Hb Bart's was 72.2% and the highest 90.9%. Hb Bart's fraction increased while that of Hb Portland decreased with the duration of pregnancy. In order to avoid serious maternal morbidity, it is necessary to diagnose Hb Bart's disease as soon as possible and CE can be used as a method of rapid diagnosis.

Detection of a Hb A2 -Melbourne (HBD: c.130G>A) combined with ß-thalassemia in a Chinese individual.

BACKGROUND: Thalassemia is common in Southeast Asian countries, including China. Hb A2 -Melbourne is a rare hemoglobin variant and has never been reported in China. Here, we report a Hb A2 -Melbourne combined with ß-thalassemia in Chinese individuals which is the second case described in the published reports. METHODS: Complete blood counts (CBC) of a 28-year-old female showed signs of thalassemia during a routine screening. Hemoglobin analysis was subsequently performed using capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). Four common deletional α-thalassemia detection was carried out using a gap-polymerase chain reaction (PCR). PCR and reverse dot-blot were used to detect three non-deletional α-thalassemia and 17 types of point mutations in ß-thalassemia. Finally, it was identified by Sanger sequencing. Her husband also had CBC, hemoglobin analysis, and genetic diagnosis. RESULTS: CBC of the couple showed Hb 103 and 139 g/L, mean corpuscular volume 58 and 63.1 fL, mean corpuscular hemoglobin 19.7 and 20.4 pg, respectively. Hemoglobin analysis revealed Hb X 2.4%, Hb A2 2.8% by CE and Hb X 2.9%, Hb A2 2.4% by HPLC in the female. The results of her husband were Hb A93.5%, Hb A2 5.7%, Hb F 0.8% by CE. Genetic analysis of both spouses detected the same CD 41/42 mutations in ß-globin gene. Sanger sequencing of female identified a mutation of the δ-globin gene (HBD:c.130G>A), corresponding to Hb A2 -Melbourne. CONCLUSION: Hb A2 -Melbourne can lead to misdiagnosis of ß-thalassemia. δ-globin gene mutation must be carefully examined in routine thalassemia screening.