LOVD-DASH: A comprehensive LOVD database coupled with diagnosis and an at-risk assessment system for hemoglobinopathies.

Hemoglobinopathies are the most common monogenic disorders worldwide. Substantial effort has been made to establish databases to record complete mutation spectra causing or modifying this group of diseases. We present a variant database which couples an online auxiliary diagnosis and at-risk assessment system for hemoglobinopathies (DASH). The database was integrated into the Leiden Open Variation Database (LOVD), in which we included all reported variants focusing on a Chinese population by literature peer review-curation and existing databases, such as HbVar and IthaGenes. In addition, comprehensive mutation data generated by high-throughput sequencing of 2,087 hemoglobinopathy patients and 20,222 general individuals from southern China were also incorporated into the database. These sequencing data enabled us to observe disease-causing and modifier variants responsible for hemoglobinopathies in bulk. Currently, 371 unique variants have been recorded; 265 of 371 were described as disease-causing variants, whereas 106 were defined as modifier variants, including 34 functional variants identified by a quantitative trait association study of this high-throughput sequencing data. Due to the availability of a comprehensive phenotype-genotype data set, DASH has been established to automatically provide accurate suggestions on diagnosis and genetic counseling of hemoglobinopathies. LOVD-DASH will inspire us to deal with clinical genotyping and molecular screening for other Mendelian disorders.

Diagnosis of a Family with the Novel -α(21.9) Thalassemia Deletion.

The Qinzhou α-thalassemia (α-thal) or -α(21.9) deletion was first described at the Qinzhou Maternal and Child Health Care Hospital, Qinzhou, Guangxi, People's Republic of China (PRC) in 2013. The molecular biological mechanism by which this allele leads to α-thal involves the deletion of a 21.9 kb DNA fragment of the α-globin gene cluster (NG_000006.1), designated as -α(21.9). During routine screening, a new family with -α(21.9) was found by the research group. This is the first time that an adult patient with the -α(21.9)/αα genotype and a 6-month-old baby with the -α(21.9)/- -(SEA) (Southeast Asian) genotype were detected in one family. The discovery of this family demonstrates that there is a certain risk for the Qinzhou α-thal deletion in the southern regions of Guangxi Province, PRC. The detection of the adult patient with the -α(21.9)/αα genotype and the analysis of hematological data are important supplements for -α(21.9) research. Additionally, Hb Bart's (γ4) and Hb H (ß4) were detected in the 6-month-old, confirming that the baby with the -α(21.9)/- -(SEA) genotype also carries Hb H disease. The analysis of this family verifies that the -α(21.9) deletion is an α(+)-thal allele.

The diagnosis and molecular analysis of a novel 21.9kb deletion (Qinzhou type deletion) causing α+ thalassemia.

α-Thalassemia is a common single-gene genetic disease that can cause Hb Bart's hydrops fetalis and Hb H disease in tropical and subtropical regions. When examining conventional thalassemia genes, an only detected --(SEA) genotype sample needs further analysis. In doing so, we found a novel 21.9kb deletion (Qinzhou type deletion). The deletion position of the novel 21.9kb deletion is from 14373bp to 36299bp of the α-globin gene cluster (NG_000006.1); thus, there exists a 21927bp sequence deletion, into which a 29bp sequence is added. After sequence analysis, a group of Gap-PCR primers were synthesized to diagnose this novel thalassemia genotype. Through pedigree analysis, we deduced that the propositus obtained the novel alleles from her mother. The genotype of this propositus is --(SEA)/-α(21.9) and its phenotype conforms to the characteristics of Hb H disease, establishing that the combination between -α(21.9) genotype and α(0) genotype can lead to Hb H disease. By molecular analysis, we established that this case fits the characteristic of an α(+) thalassemia genotype.

NGS-based spinal muscular atrophy carrier screening of 10,585 diverse couples in China: a pan-ethnic study.

In this study, we performed a spinal muscular atrophy carrier screening investigation with NGS-based method. First, the validation for NGS-based method was implemented in 2255 samples using real-time PCR. The concordance between the NGS-based method and real-time PCR for the detection of SMA carrier and patient were up to 100%. Then, we applied this NGS-based method in 10,585 self-reported normal couples (34 Chinese ethnic groups from 5 provinces in South China) for SMA carrier screening. The overall carrier frequency was 1 in 73.8 (1.4%). It varied substantially between ethnic groups, highest in Dai ethnicity (4.3%), and no significant difference was found between five provinces. One couple was detected as carriers with an elevated risk of having an SMA affected baby. The distribution of SMN1:SMN2 genotype was also revealed in this study. Among the individuals with normal phenotype, the exon 7 copy-number ratio of SMN1 to SMN2 proved the gene conversion between them. With NGS-based method, we investigated SMA carrier status in Chinese population for the first time, and our results demonstrated that it is a promising alternative for SMA carrier screening and could provide data support and reference for future clinical application.

A Cell-free DNA Barcode-Enabled Single-Molecule Test for Noninvasive Prenatal Diagnosis of Monogenic Disorders: Application to ß-Thalassemia.

Noninvasive prenatal testing of common aneuploidies has become routine over the past decade, but testing of monogenic disorders remains a challenge in clinical implementation. Most recent studies have inherent limitations, such as complicated procedures, a lack of versatility, and the need for prior knowledge of parental genotypes or haplotypes. To overcome these limitations, a robust and versatile next-generation sequencing-based cell-free DNA (cfDNA) allelic molecule counting system termed cfDNA barcode-enabled single-molecule test (cfBEST) is developed for the noninvasive prenatal diagnosis (NIPD) of monogenic disorders. The accuracy of cfBEST is found to be comparable to that of droplet digital polymerase chain reaction (ddPCR) in detecting low-abundance mutations in cfDNA. The analytical validity of cfBEST is evidenced by a ß-thalassemia assay, in which a blind validation study of 143 at-risk pregnancies reveals a sensitivity of 99.19% and a specificity of 99.92% on allele detection. Because the validated cfBEST method can be used to detect maternal-fetal genotype combinations in cfDNA precisely and quantitatively, it holds the potential for the NIPD of human monogenic disorders.

Pilot study of expanded carrier screening for 11 recessive diseases in China: results from 10,476 ethnically diverse couples.

Expanded carrier screening (ECS) has been demonstrated to increase the detection rate of carriers compared with traditional tests. The aim of this study was to assess the potential value of ECS for clinical application in Southern China, a region with high prevalence of thalassemia and with diverse ethnic groups, and to provide a reference for future implementations in areas with similar population characteristics. A total of 10,476 prenatal/preconception couples from 34 self-reported ethnic groups were simultaneously tested and analyzed anonymously for 11 Mendelian disorders using targeted next-generation sequencing. Overall, 27.49% of individuals without self-reported family history of disorders were found to be carriers of at least 1 of the 11 conditions, and the carrier frequency varied greatly between ethnic groups, ranging from 4.15% to 81.35%. Furthermore, 255 couples (2.43%) were identified as carrier couples at an elevated risk having an affected baby, sixty-five of which would not have been identified through the existing screening strategy, which only detects thalassemia. The modeled risk of fetuses being affected by any of the selected disorders was 531 per 100,000 (95% CI, 497-567 per 100,000). Our data demonstrate the feasibility of ECS, and provide evidence that ECS is a promising alternative to traditional one-condition screening strategies. The lessons learned from this experience should be applicable for other countries or regions with diverse ethnic groups.

Rapid Targeted Next-Generation Sequencing Platform for Molecular Screening and Clinical Genotyping in Subjects with Hemoglobinopathies.

Hemoglobinopathies are among the most common autosomal-recessive disorders worldwide. A comprehensive next-generation sequencing (NGS) test would greatly facilitate screening and diagnosis of these disorders. An NGS panel targeting the coding regions of hemoglobin genes and four modifier genes was designed. We validated the assay by using 2522 subjects affected with hemoglobinopathies and applied it to carrier testing in a cohort of 10,111 couples who were also screened through traditional methods. In the clinical genotyping analysis of 1182 ß-thalassemia subjects, we identified a group of additional variants that can be used for accurate diagnosis. In the molecular screening analysis of the 10,111 couples, we detected 4180 individuals in total who carried 4840 mutant alleles, and identified 186 couples at risk of having affected offspring. 12.1% of the pathogenic or likely pathogenic variants identified by our NGS assay, which were undetectable by traditional methods. Compared with the traditional methods, our assay identified an additional at-risk 35 couples. We describe a comprehensive NGS-based test that offers advantages over the traditional screening/molecular testing methods. To our knowledge, this is among the first large-scale population study to systematically evaluate the application of an NGS technique in carrier screening and molecular diagnosis of hemoglobinopathies.

[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.

Rapid diagnosis of aneuploidy using segmental duplication quantitative fluorescent PCR.

The aim of this study was use a simple and rapid procedure, called segmental duplication quantitative fluorescent polymerase chain reaction (SD-QF-PCR), for the prenatal diagnosis of fetal chromosomal aneuploidies. This method is based on the co-amplification of segmental duplications located on two different chromosomes using a single pair of fluorescent primers. The PCR products of different sizes were subsequently analyzed through capillary electrophoresis, and the aneuploidies were determined based on the relative dosage between the two chromosomes. Each primer set, containing five pairs of primers, was designed to simultaneously detect aneuploidies located on chromosomes 21, 18, 13, X and Y in a single reaction. We applied these two primer sets to DNA samples isolated from individuals with trisomy 21 (n = 36); trisomy 18 (n = 6); trisomy 13 (n = 4); 45, X (n = 5); 47, XXX (n = 3); 48, XXYY (n = 2); and unaffected controls (n = 40). We evaluated the performance of this method using the karyotyping results. A correct and unambiguous diagnosis with 100% sensitivity and 100% specificity, was achieved for clinical samples examined. Thus, the present study demonstrates that SD-QF-PCR is a robust, rapid and sensitive method for the diagnosis of common aneuploidies, and these analyses can be performed in less than 4 hours for a single sample, providing a competitive alternative for routine use.

Rapid detection of Down's syndrome using quantitative real-time PCR (qPCR) targeting segmental duplications on chromosomes 21 and 11.

OBJECTIVE: Development of a qPCR test for the detection of trisomy 21 using segmental duplications. METHODS: Segmental duplications in the TTC3 gene on chromosome 21 and the KDM2A gene on chromosome 11 were selected as molecular markers for the diagnostic qPCR assay. A set of consensus primers selected from the conserved regions of these segmental duplications were used to amplify internal diverse sequences that were detected and quantified with different probes labeled with distinct fluorescence. The copy numbers of these two fragments were determined based on the ΔCq values of qPCR. The results of qPCR for prenatal and neonatal screening of Down's syndrome were compared with the conventional karyotype analysis by testing 82 normal individuals and 50 subjects with Down's syndrome. RESULTS: The ΔCq values of segmental duplications on chr21 and 11 ranged between 0.33 and 0.75 in normal individuals, and between 0.91 and 1.18 in subjects with Down's syndrome. The ΔCq values of these two segmental duplications clearly discriminated Down's syndrome from normal individuals (P<0.001). Furthermore, the qPCR results were consistent with karyotype analysis. CONCLUSION: Our qPCR can be used for rapid prenatal and neonatal screening of Down's syndrome.

Detection of three common α-thalassemia in non-deletion types and six common thalassemia in deletion types by QF-PCR.

OBJECTIVE: Thalassemia is one of the most common monogenic hereditary diseases in tropical and subtropical regions. An effective way to avoid the birth of severe thalassemia patients is to strengthen the thalassemia screening of couples before wives are pregnant. Thalassemia gene carriers can be diagnosed by molecular biology in order to conduct effective guidance for fertility. DESIGNS AND METHODS: For --(SEA) and --(THAI) of α-thalassemia and HPFH-SEA and DBT of ß-thalassemia, we design the fGap-PCR primer; for α(CS)α, α(QS)α and α(WS)α, we design the fAS-PCR primer; for -α(3.7)and -α(4.2), we design the QF-PCR primer; and lastly, we use universal primers and multiple-tailed primers to make a single-tube QF-PCR system. RESULTS: When the QF-PCR system is used to diagnose 123 screening samples of thalassemia genotyping, the typing result is consistent with conventional diagnosis of Gap-PCR and PCR-RDB. CONCLUSIONS: Compared with conventional Gap-PCR and PCR-RDB, this QF-PCR system is easy to operate, has high precision, and can diagnose genotypes in a large scale. Its automatic operation is more suitable for the large-scale screening of the thalassemia gene.