Characterization of a Novel 71.8 kb α0-Thalassemia Deletion and Subsequent Summary of a Practical Procedure for Thalassemia Molecular Diagnosis.

Thalassemia is the most common monogenic disorder around the world. Based on the principle of genotype-phenotype correlation, identification of thalassemia mutations is the essential prerequisite for clinical diagnosis and management. Because only common mutations are routinely detected, the identification of rare or undetermined mutations is a challenge for clinical laboratories. Herein, a proband presenting with inconsistent phenotype-genotype correlation after routine molecular screening was investigated by multiplex ligation-dependent probe amplification (MLPA), targeted-next generation sequencing (targeted-NGS), gap-polymerase chain reaction (gap-PCR) and Sanger sequencing. Eventually, a novel 71.8 kb deletion (- -71.8) was identified and characterized, which included HBZ (ζ), HBA2 (α2), and HBA1 (α1) genes and was causing α0-thalassemia (α0-thal). Furthermore, we summarized a practical procedure based on accumulated experience in studies and clinical practice, which can be a guide for molecular screening and clinical diagnosis of thalassemia, especially for identification of undetermined or novel mutations.

Mutations in C1orf194, encoding a calcium regulator, cause dominant Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease is a hereditary motor and sensory neuropathy exhibiting great clinical and genetic heterogeneity. Here, the identification of two heterozygous missense mutations in the C1orf194 gene at 1p21.2-p13.2 with Charcot-Marie-Tooth disease are reported. Specifically, the p.I122N mutation was the cause of an intermediate form of Charcot-Marie-Tooth disease, and the p.K28I missense mutation predominately led to the demyelinating form. Functional studies demonstrated that the p.K28I variant significantly reduced expression of the protein, but the p.I122N variant increased. In addition, the p.I122N mutant protein exhibited the aggregation in neuroblastoma cell lines and the patient's peroneal nerve. Either gain-of-function or partial loss-of-function mutations to C1ORF194 can specify different causal mechanisms responsible for Charcot-Marie-Tooth disease with a wide range of clinical severity. Moreover, a knock-in mouse model confirmed that the C1orf194 missense mutation p.I121N led to impairments in motor and neuromuscular functions, and aberrant myelination and axonal phenotypes. The loss of normal C1ORF194 protein altered intracellular Ca2+ homeostasis and upregulated Ca2+ handling regulatory proteins. These findings describe a novel protein with vital functions in peripheral nervous systems and broaden the causes of Charcot-Marie-Tooth disease, which open new avenues for the diagnosis and treatment of related neuropathies.

[Rapid detection of alpha-globin gene αααanti-3.7 triplets with droplet digital PCR].

OBJECTIVE: To establish a rapid method for detection of alpha-globin gene αααanti-3.7 based on droplet digital PCR (ddPCR) technique. METHODS: The differential sequence between the X1 and Y1 box of α1 gene was selected as the amplicon of the target gene with ß-actin as the reference gene. The specific primers and TaqMan probes were designed, and then a quantitative method for detecting the copy number was established based on ddPCR technique. The sensitivity and accuracy of the method were evaluated by detecting 28 samples of known genotypes and 60 clinical samples. RESULTS: The ddPCR-based method accurately identified the genotypes of all the 28 samples with known genotypes and detected 5 cases of αα/αααanti-3.7 from the 60 clinical samples, and the results were verified by MLPA. The sensitivity and accuracy of this method were both 100% for detecting alpha-globin gene αααanti-3.7. CONCLUSION: This ddPCR-based method for detecting αααanti-3.7 triplet can be applied for population screening and in routine clinical molecular diagnosis with simple operation, rapid analysis and accurate results.

Evaluation and comparison of three assays for molecular detection of spinal muscular atrophy.

BACKGROUND: Spinal muscular atrophy (SMA) is mainly caused by deletions in SMA-related genes. The objective of this study was to develop gene-dosage assays for diagnosing SMA. METHODS: A multiplex, quantitative PCR assay and a CNVplex assay were developed for determining the copy number of SMN1, SMN2, and NAIP. Reproducibility and specificity of the two assays were compared to a multiple ligation-dependent probe amplification (MLPA) assay. To evaluate reproducibility, 30 samples were analyzed three times using the three assays. A total of 317 samples were used to assess the specificity of the two assays. RESULTS: The multiplex quantitative PCR (qPCR) assay had higher reproducibility. Intra-assay CVs were 3.01%-8.52% and inter-assay CVs were 4.12%-6.24%. The CNVplex assay had ratios that were closer to expected (0.49-0.5 for one copy, 1.03-1.0 for two copies, and 1.50-1.50 for three copies). Diagnostic accuracy rates for the two assays were 100%. CONCLUSIONS: The multiplex qPCR assay was a simple, rapid, and cost-effective method for routine SMA diagnosis and carrier screening. The CNVplex assay could be used to detect SMAs with complicated gene structures. The assays were reliable and could be used as alternative methods for clinical diagnosis of SMA.

[Hemoglobin H disease with a rare α-thalassemia gene mutation (--SEA/α*92A>Gα): pedigree analysis and genetic diagnosis].

OBJECTIVE: To identify a rare α-thalassemia gene mutation in a family from south China and perform a pedigree analysis and genetic diagnosis of hemoglobin H (HbH) disease caused by this mutation. METHODS: Peripheral blood samples were collected from the family members for analysis of the hematological phenotype and routine test of thalassemia genes. DNA sequencing was carried out for samples that showed genotype and phenotype inconsistency. RESULTS: A rare α-thalassemia *92A>G gene mutation was detected within this family. The proband and his sister were confirmed to have non-deletional HbH disease with α--SEA/α*92A>Gα genotype. The proband's brother was confirmed to have an α-thalassemia trait with the genotype of -α3.7/α*92A>Gα. The proband's father was identified as an α-thalassemia silent carrier with the genotype of αα/α*92A>Gα. CONCLUSION: A rare α-thalassemia *92A>G gene mutation was identified for first time in south China. The description of the basic phenotypic characteristics of α-thalassemia trait and silent carrier caused by this mutation enriches the α-thalassemia gene mutation spectrum in Chinese population and helps in population screening, clinical molecular diagnosis and genetic counseling.

Development of a capillary zone electrophoresis method for rapid determination of human globin chains in α and ß-thalassemia subjects.

Thalassemia is an inherited autosomal recessive blood disorder characterized by the underproduction of globin chains as a consequence of globin gene defects, resulting in malfunctioning red blood cells and oxygen transport. Analysis of globin chains is an important aspect of thalassemia research. In this study we developed a capillary zone electrophoresis (CZE) method for human globin determination in the diagnosis of thalassemia and hemoglobin variants. To demonstrate the utility of this approach, α/ß area ratios were determined for samples from 310 thalassemia patients and healthy controls. The separation was performed on uncoated capillary with simple preparation. Distinct globin peaks were resolved in 17 min, and coefficients of variation (CV) for migration time and areas ranged from 0.37%-1.69% and 0.46%-6.71%, respectively. Receiver operating characteristic (ROC) curve analysis of the α/ß area ratios gave 100% sensitivity and specificity for indicating ß-TI/TM, and 100% sensitivity and 97.4% specificity for Hb H disease. Hemoglobin G-Honolulu (Hb G-Honolulu) and Hb Westmead (Hb WS) were successfully detected using this CZE method. This automated methodology is simple, rapid and cost-effective for the fast determination of human globin chains, which could be an important diagnostic tool in the field of hemoglobinopathies.

Molecular characterization and copy number of SMN1, SMN2 and NAIP in Chinese patients with spinal muscular atrophy and unrelated healthy controls.

BACKGROUND: Spinal muscular atrophy (SMA) is caused by SMN1 dysfunction, and the copy number of SMN2 and NAIP can modify the phenotype of SMA. The aim of this study was to analyze the copy numbers and gene structures of SMA-related genes in Chinese SMA patients and unrelated healthy controls. METHODS: Forty-two Chinese SMA patients and two hundred and twelve unrelated healthy Chinese individuals were enrolled in our study. The copy numbers and gene structures of SMA-related genes were measured by MLPA assay. RESULTS: We identified a homozygous deletion of SMN1 in exons 7 and 8 in 37 of 42 patients (88.1%); the other 5 SMA patients (11.9%) had a single copy of SMN1 exon 8. The proportions of the 212 unrelated healthy controls with different copy numbers for the normal SMN1 gene were 1 copy in 4 individuals (1.9%), 2 copies in 203 (95.7%) and 3 copies in 5 (2.4%). Three hybrid SMN genes and five genes that lack partial sequences were found in SMA patients and healthy controls. Distributions of copy numbers for normal SMN2 and NAIP were significantly different (P < 0.001) in people with and without SMA. CONCLUSION: The copy numbers and gene structures of SMA-related genes were different in Chinese SMA patients and healthy controls.

Rapid determination of human globin chains using reversed-phase high-performance liquid chromatography.

Reversed-phase high-performance liquid chromatography (RP-HPLC) of human globin chains is an important tool for detecting thalassemias and hemoglobin variants. The challenges of this method that limit its clinical application are a long analytical time and complex sample preparation. The aim of this study was to establish a simple, rapid and high-resolution RP-HPLC method for the separation of globin chains in human blood. Red blood cells from newborns and adults were diluted in deionized water and injected directly onto a micro-jupiter C18 reversed-phase column (250 mm × 4.6 mm) with UV detection at 280 nm. Under the conditions of varying pH or the HPLC gradient, the globin chains (pre-ß, ß, δ, α, (G)γ and (A)γ) were denatured and separated from the heme groups in 12 min with a retention time coefficient of variation (CV) ranging from 0.11 to 1.29% and a peak area CV between 0.32% and 4.86%. Significant differences (P<0.05) among three groups (normal, Hb H and ß thalassemia) were found in the area ratio of α/pre-ß+ß applying the rapid elution procedure, while P≥0.05 was obtained between the normal and α thalassemia silent/trait group. Based on the ANOVA results, receiver operating characteristic (ROC) curve analysis of the δ/ß and α/pre-ß+ß area ratios showed a sensitivity of 100.0%, and a specificity of 100.0% for indicating ß thalassemia carriers, and a sensitivity of 96.6% and a specificity of 89.6% for the prediction of hemoglobin H (Hb H) disease. The proposed cut-off was 0.026 of δ/ß for ß thalassemia carriers and 0.626 of α/pre-ß+ß for Hb H disease. In addition, abnormal hemoglobin hemoglobin E (Hb E) and Hb Westmead (Hb WS) were successfully identified using this RP-HPLC method. Our experience in developing this RP-HPLC method for the rapid separation of human globin chains could be of use for similar work.

A quantitative assay to detect α-thalassemia deletions and triplications using multiplex nested real-time quantitative polymerase chain reaction.

Increasing evidence indicates that copy number variants (CNVs) have great relevance to common human diseases. In α-thalassemia, clinical phenotypes are related to genotypes, specifically copy number changes in the human α-globin gene cluster. Assays are available for high-throughput screening of unknown CNVs genome-wide and also for targeted CNV genotyping at loci associated with genetic disorders. Here we describe a universal quantitative approach based on nested real-time quantitative polymerase chain reaction for accurate determination of copy numbers at multiple particular gene loci. We used the α-globin gene as a model system, obtaining the reproducibility and sensitivity to analyze different gene copies and testing 95 DNA samples with 16 different known genotypes. Our results showed that this approach has high sensitivity and low standard deviations for correctly genotyping DNA samples containing different copy numbers of the α1 and α2 globin genes. Our method is rapid, simple, and reliable, and it could be used to simultaneously screen for α-thalassemia deletions or triplications. Moreover, it has potential as a versatile technology for the rapid genotyping of known CNVs in a targeted region.

[Large-scale population-based genetic screening and prenatal diagnosis for thalassemias in Zhuhai City of Guangdong Province].

OBJECTIVE: To report the results of preventive control program of severe thalassemias in Zhuhai City of Guangdong Province from 1998 to 2010. METHODS: As the guide centre of marriage and childbearing and the greatest maternity hospital in Zhuhai City of Guangdong Province, Zhuhai Municipal Maternity and Child Healthcare Hospital constructed the genetic screening network for thalassemias testing and referred for follow-up and for genetic counseling. The couples for premarital medical examination or regular healthcare examination in pregnancy were enrolled to this preventive control program. A conventional strategy of screening for heterozygote was used to identify the α- and ß-thalassemia traits in women and their spouses according to the standard procedures of hematological phenotype analysis which was recommended by Thalassemia International Federation (TIF). Then those suspected couples at risk were diagnosed for α- and ß-thalassemia by PCR-based DNA assays. The couples at risk for severe thalassemias were counseled and offered prenatal diagnosis and termination of pregnancy in case of an affected fetus in the rights of consent and of option voluntarily. RESULTS: From January 1998 to December 2010, 85 522 brides and grooms-to-be for premarital screening and 41 503 pregnant women in addition to 14 141 partners for prenatal screening were recorded, the covering rates of premarital screening and prenatal screening in the city were 92.698% (from 1998 to 2003) and 27.667% (from 2004 to 2010), respectively. Totally 10 726 cases were found to be the carriers of thalassemias, with 7393 for α-thalassemia (5.237%, 7 393/141 166) and 3333 for ß-thalassemia (2.361%, 3 333/141 166). A total of 257 couples at-risk for severe thalassemias were detected including 190 for α-thalassemia and 67 for ß-thalassemia. Among them, 251 (97.7%, 251/257) couples were performed prenatal diagnosis. During the preventive control program, a total of 72 fetuses with severe thalassemias including hemoglobin H disease were voluntarily terminated. In Zhuhai City, the average annual birth rate of fetuses with severe thalassemia was declined by 32.9% (49/149). CONCLUSIONS: This study has reduced effectively birth rate of perinatal infants with severe thalassemias in Zhuhai City by genetic screening and prenatal diagnosis of thalassemia in the large population of 13 years. Our summary comes out of technical proposals for prenatal screening and diagnosis, which could be take example by preventative control of thalassemia in other regions of China where are prevalent.

Reliable detection of paternal SNPs within deletion breakpoints for non-invasive prenatal exclusion of homozygous α-thalassemia in maternal plasma.

Reliable detection of large deletions from cell-free fetal DNA (cffDNA) in maternal plasma is challenging, especially when both parents have the same deletion owing to a lack of specific markers for fetal genotyping. In order to evaluate the efficacy of a non-invasive prenatal diagnosis (NIPD) test to exclude α-thalassemia major that uses SNPs linked to the normal paternal α-globin allele, we established a novel protocol to reliably detect paternal SNPs within the (--(SEA)) breakpoints and performed evaluation of the diagnostic potential of the protocol in a total of 67 pregnancies, in whom plasma samples were collected prior to invasive obstetrics procedures in southern China. A group of nine SNPs identified within the deletion breakpoints were scanned to select the informative SNPs in each of the 67 couples DNA by multiplex PCR based mini-sequencing technique. The paternally inherited SNP allele from cffDNA was detected by allele specific real-time PCR. A protocol for reliable detection of paternal SNPs within the (--(SEA)) breakpoints was established and evaluation of the diagnostic potential of the protocol was performed in a total of 67 pregnancies. In 97% of the couples one or more different SNPs within the deletion breakpoint occurred between paternal and maternal alleles. Homozygosity for the (--(SEA)) deletion was accurately excluded in 33 out of 67 (49.3%, 95% CI, 25.4-78.6%) pregnancies through the implementation of the protocol. Protocol was completely concordant with the traditional reference methods, except for two cases that exhibited uncertain results due to sample hemolysis. This method could be used as a routine NIPD test to exclude gross fetal deletions in α-thalassemia major, and could further be employed to test for other diseases due to gene deletion.

Rapid, accurate detection of TMPRSS6 gene causative mutations with a high-resolution melting assay.

Mutations of the TMPRSS6 gene are considered the major genetic factors for iron-refractory iron deficiency anemia (IRIDA). Artificial clone libraries containing 17 known mutations of the TMPRSS6 gene were used to develop a high-resolution melting (HRM) assay for the detection of 17 TMPRSS6 gene mutations. The melting temperatures and melting curves were able to distinguish the different genotypes of the 17 TMPRSS6 gene mutations. We used replicate experiments to evaluate the reproducibility of the assay, and the coefficients of variation were in the range 0.0091% to 0.0873%. A total of 145 Chinese patients with IDA were screened with this assay and no TMPRSS6 gene causative mutation was found in any patient. The HRM assay was proved to be rapid, accurate and cost-effective method to identify the TMPRSS6 gene mutations and can be used in the clinical diagnosis of IRIDA.

[A community-based genetic screening of large-scale population and prenatal diagnosis for alpha and beta thalassemia in Zhuhai city of Guangdong province].

OBJECTIVE: To describe a community-based model for prevention and control of severe alpha and beta thalassemias in Zhuhai city of Guangdong province. METHODS: Couples for premarital medical examination or regular healthcare examination in pregnancy were enrolled in this prospective screening program, which was supported by the two-level network composed of 6 local hospitals for testing thalassemias and follow-up for genetic counseling. A conventional heterozygote screening strategy was used to determine alpha and beta thalassemia traits in women and their partners according to the standard procedures of hematological phenotype analysis. Then confirmative diagnosis of alpha and beta thalassemia was performed on those couples suspected at-risk for severe thalassemia by using the PCR-based molecular diagnostic assays. The couples at-risk for severe thalassemia were counseled and offered prenatal diagnosis and termination of pregnancy in case of an affected fetus. RESULTS: During the period between January 1998 and December 2005, the screened records included 85522 young females and their partners for premarital screening and 10439 pregnant women for prenatal screening, with 71.38% coverage of total population recorded in this city for premarital screening. Six thousands five hundreds and sixty-three individuals in total were found to be the carriers of thalassemias, with 4312 for alpha thalassemia (4.5%) and 2251 for beta thalassemia (2.3%), respectively. One hundred and forty-eight couples were diagnosed to be at-risk for thalassemias, including 103 for alpha thalassemia and 45 for beta thalassemia, respectively. Successful prenatal diagnosis was made for 142 (98 for alpha thalassemia and 44 for beta thalassemia) out of 148 (95.9%) pregnancies at-risk for severe thalassemias. Twenty-three cases of hydrops fetalis, 4 of Hb H diseases and 14 of beta thalassemia were identified. All 41 pregnancies with affected fetuses were voluntarily terminated. Thus, this has led to a marked decrease of severe thalassemia syndrome since the program started. CONCLUSION: We presented the first community-based prospective screening program in China for control of alpha and beta thalassemia in Zhuhai city with a population of 1.29 million through premarital or prenatal screening. This model could be used for control of thalassemias and other hemoglobinopathies in other regions of China and also in other developing countries.

Rapid genotyping of known mutations and polymorphisms in beta-globin gene based on the DHPLC profile patterns of homoduplexes and heteroduplexes.

BACKGROUND: Beta-thalassemia represents a great heterogeneity as over 200 mutations have been identified for the beta-globin gene responsible for this disease. A rapid genotyping test with high accuracy, selectivity, and reproducibility suitable for the determination of known mutations is needed for prenatal screening and post-natal diagnosis of this disease in clinical setting. DESIGN AND METHODS: We have performed the validation of a DHPLC assay for direct genotyping of known causative mutations in beta-globin gene using the chromatographic pattern-based strategy under partially-denaturing conditions. RESULTS: DHPLC assay was established based on the analysis of 795 DNA samples from a group of various genotypes for the 20 mutations and 8 polymorphisms in beta-globin gene then validated on 319 tests in a blind study. The results obtained with this assay were in concordance with the results obtained by DNA sequence analysis. CONCLUSION: This simple method can meet the requirements of direct genotyping of known beta-thalassemia mutations and/or polymorphisms in the clinical setting for Chinese and in general as a model for other populations.

[Identification of a missense mutation in SEDL gene from a Chinese family with X-linked spondyloepiphyseal dysplasia tarda].

OBJECTIVE: To identify the SEDL gene mutation in a Chinese family with X-linked spondyloepiphyseal dysplasia tarda (SEDL) and to establish a genotyping assay for rapid diagnosis of this X-linked recessive disorder. METHODS: Clinical diagnoses were made based on physical examination, radiological examination and pedigree analysis for this family. Four primer pairs flanking the SEDL exons 3-6 including their exon/intron boundaries were designed. A rapid genotyping assay based on denaturing high performance liquid chromatography (DHPLC) was established to screen the point mutations of the SEDL gene. Genomic DNA was extracted by standard methods from 18 members in the three generations of the pedigree and subjected to PCR-denaturing high performance liquid chromatography (PCR-DHPLC) assay followed by direct DNA sequencing. RESULTS: A c.218C>T mutation in exon 4 of the SEDL gene, which resulted in a substitution of serine 218 with leucine, was identified in this family. Among the 18 members, 3 patients, 5 obligate female carriers and 2 unmarried young females were found to have the missense mutation, and other 8 healthy individuals were not detected to carry the mutation, in which genotype-phenotype correlations were well established in each member investigated in this family. CONCLUSION: A c.218C>T missense mutation in the SEDL gene was firstly reported in Chinese population and the results of this study expand the spectrum of SEDL mutations. The PCR-DHPLC assay is a useful tool to rapidly detect the SEDL mutation in clinical and prenatal diagnosis.