Siping Li

Large Cohort Screening of G6PD Deficiency and the Mutational Spectrum in the Dongguan District in Southern China

Background Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common enzymatic disorder of the erythrocytes that affects 400 million people worldwide. We developed a PCR-reverse dot blot (RDB) assay to screen twenty genotypes of seventeen Chinese G6PD mutations and investigate the spectrum of G6PD deficiency mutations in Dongguan District, Guangdong Province, in southern China. Method The PCR-RDB assay consists of multiplex PCR amplification of seven fragments in the G6PD target sequence of wild-type and mutant genomic DNA samples followed by hybridization to a test strip containing allele-specific oligonucleotide probes. A total of 16,464 individuals were analyzed by a combination of phenotypic screening and genotypic detection using the PCR-RDB assay and DNA sequence analysis. Results The PCR-RDB assay had a detection rate of 98.1%, which was validated by direct sequencing in a blind study with 100% concordance. The G6PD deficiency incidence rate in Dongguan District is 4.08%. Thirty-two genotypes from 469 individuals were found. The two most common variants were c.1376G>T and c.1388G>A, followed by c.95A>G, c.871G>A, c.392G>T, and c.1024 C>T. In addition, two rare mutations (c.703C>A and c.406C>T) were detected by DNA sequencing analysis. In our study, 65 cases harbored the C1311T/IVS polymorphism and 67 cases were homozygote. Conclusion The PCR-RDB assay we established is a reliable and effective method for screening G6PD mutations in the Chinese population. Data on the spectrum of mutations in the Dongguan District is beneficial to the clinical diagnosis and prevention of G6PD deficiency.

A reverse dot blot assay for the expanded screening of eleven Chinese G6PD mutations.

BACKGROUND Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a multiethnic inherited disease with a particularly high prevalence in tropical and subtropical regions including southern China. A convenient and reliable method is required to detect common G6PD mutations in the Chinese population. METHODS We developed a reverse dot blot (RDB) assay for the expanded screening of eleven mutations (c.95A>G, c.392G>T, c.871G>A, c.1004C>T, c.1004C>A, c.1024C>T, c.1360C>T, c.1376G>T, c.1381G>A, c.1387C>T, c.1388G>A). The method consists of a single-tube multiplex PCR amplification of four fragments in the G6PD target sequence of wild-type and mutant genomic DNA samples followed by hybridization to a test strip containing allele-specific oligonucleotide probes. We applied our method to a group of 213 unrelated Chinese patients. RESULTS The test had a detection rate of 95.8%, validated by direct sequencing in a blind study with 100% concordance. CONCLUSIONS The results demonstrate that our reverse dot blot assay is an easy, reliable, high-yield and cost-effective method for genetic screening to identify G6PD patients and carriers among the Chinese population.

A reverse dot blot assay for the screening of twenty mutations in four genes associated with NSHL in a Chinese population

Background Congenital deafness is one of the most distressing disorders affecting humanity and exhibits a high incidence worldwide. Most cases of congenital deafness in the Chinese population are caused by defects in a limited number of genes. A convenient and reliable method for detecting common deafness-related gene mutations in the Chinese population is required. Methods We developed a PCR-reverse dot blot (RDB) assay for screening 20 hotspot mutations of GJB2, GJB3, SLC26A4, and MT-RNR1, which are common non-syndromic hearing loss (NSHL)–associated genes in the Chinese population. The PCR-RDB assay consists of multiplex PCR amplifications of 10 fragments in the target sequence of the four above-mentioned genes in wild-type and mutant genomic DNA samples followed by hybridization to a test strip containing allele-specific oligonucleotide probes. We applied our method to a set of 225 neonates with deafness gene mutations and 30 normal neonates. Results The test was validated through direct sequencing in a blinded study with 100% concordance. Conclusions The results demonstrated that our reverse dot blot assay is a reliable and effective genetic screening method for identifying carriers and individuals with NSHL among the Chinese population.

Molecular epidemiology of the enteroviruses associated with hand, foot and mouth disease/herpangina in Dongguan, China, 2015

Enteroviruses (EVs) are the etiological agents involved in most cases of hand, foot and mouth disease (HFMD) and herpangina (HA). Information on the epidemiology profiles of EVs in China is very limited, as the present surveillance system of China focuses on CAV16 and EV71, and no published data are available in Dongguan. The aim of this study is to determine the prevalence of EVs among patients with HFMD and HA in Dongguan, China, during 2015. A total of 271 clinical stool specimens that were clinically determined to be positive for enteroviruses were genotyped by semi-nested polymerase chain reaction (PCR) for the VP1 genes of EVs. The results showed that a total of 14 enterovirus genotypes were identified among HFMD and HA patients in this study. CVA6 was the most common genotype for HFMD, and CVA2 accounted for the majority of HA cases in this study. Sequence and phylogenetic analysis showed that all of the CVA6 and CVA2 strains identified in our study displayed a close genetic relationship to strains identified in other cities in China. This study also demonstrates that there are associations between particular causative enterovirus genotypes and some clinical symptoms, which may provide useful information for improving case prevention, diagnosis and treatment of HFMD and HA.

A novel mutation in the MYO7A gene is associated with Usher syndrome type 1 in a Chinese family

OBJECTIVES We aimed to investigate the genetic causes of hearing loss in a Chinese proband with autosomal recessive congenital deafness. METHODS The targeted capture of 159 known deafness genes and next-generation sequencing were performed to study the genetic causes of hearing loss in the Chinese family. Sanger sequencing was employed to verify the variant mutations in members of this family. RESULTS The proband harbored two mutations in the MYO7A gene in the form of compound heterozygosity. She was found to be heterozygous for a novel insertion mutation c.3847_3848 ins TCTG (p.N1285LfsX24) in exon 30 and for the known mutation c.2239_2240delAG (p.R747S fsX16)in exon 19. The novel mutation was absent in the 1000 Genomes Project. These variants were carried in the heterozygous state by the parents and were therefore co-segregated with the genetic disease. Clinical re-assessment, including detailed audiologic and ocular examinations, revealed congenital deafness and retinitis pigmentosa in the proband. Collectively, the combination of audiometric, ophthalmologic and genetic examinations successfully confirmed the phenotype of Usher syndrome type 1 (USH1). CONCLUSION This study demonstrates that the novel mutation c.3847_3848insTCTG (p. N1285LfsX24) in compound heterozygosity with c.2239_2240delAG in the MYO7A gene is the main cause of USH1 in the proband. Our study expands the mutational spectrum of MYO7A and provides a foundation for further investigations elucidating the MYO7A-related mechanisms of USH1.

Prevalence of S gene mutations within the major hydrophilic region of hepatitis B virus in patients in Dongguan, southern China

HBsAg point mutations within the major hydrophilic region (MHR) have frequently been reported to be associated with diagnostic failure, vaccine escape and immunotherapy escape. However, the prevalence of escape mutations in chronic hepatitis B (CHB) patients has not been systematically studied in patients from southern China within the past decade. This study aimed to determine the prevalence of escape mutations within the MHR of hepatitis B virus in patients in Dongguan, southern China. Between June 2015 and May 2016, 391 patients who were chronically infected with HBV were enrolled in the study, including 240 patients with the genotype B strain and 151 with the genotype C strain. The most frequent mutated position was s126 (4.3%), followed by s100 (3.3%), s101 (2.8%), s133 (2.8%), s145 (2.3%), s120 (2.0%) and s129 (1.8%). Furthermore, the mutations sY100C, sQ101R/K, sS114A, sP120T, sT/I126A/N/S, sQ129R, sM133L/T/S and sG145R/A were prevalent in at least one genotype, with a frequency higher than 1%, which indicated that these mutations were relatively common. In addition, sQ101K/R was found only in genotype C isolates (P < 0.05), and sT126A was only discovered in genotype B isolates (P = 0.047), indicating that such mutations were genotype-associated mutations. Notably, combinations of escape mutations within the MHR were also frequently discovered in genotypes B (5.0%) and C (6.6%), with no significant difference (P = 0.498). These results indicated that we should increase the surveillance HBsAg mutations among HBV-infected patients in China.

A novel mutation in the SLC26A4 gene in a Chinese family with non-syndromic hearing loss and enlarged vestibular aqueduct

OBJECTIVES To identity the genetic causes of hearing loss in a Han Chinese family with enlarged vestibular aqueduct syndrome. METHODS Multiplex PCR technology combined with Ion Torrent™ next-generation sequencing technology was used to search for pathogenic mutations. A group of 1500 ethnically-matched normal hearing subjects screened for mutations in deafness-related genes using the same method in previously studied were included as a control. RESULTS The proband and his little sister suffered from typical features of sensorineural hearing loss with enlarged vestibular aqueduct (EVA). Both subjects harbored two compound heterozygous mutations in the SLC26A4 gene. A novel mutation named c.2110 G > C (p.Glu704Gln) in exon 19 and another previously reported mutation c.1673 A > T (p.Asn558Ile) were identified. These mutations were carried in the heterozygous state by the parents and therefore co-segregated with the genetic disease. The c.2110 G > C (p.Glu704Gln) mutation was absent in 1500 healthy newborns. Protein alignment indicated high evolutionary conservation of the p.E704 residue, and this mutation was predicted by online tools to be damaging and deleterious. CONCLUSION This study demonstrates that the novel mutation c.2110 G > C (p.Glu704Gln) in compound heterozygosity with c.1673 A > T (p.Asn558Ile) in the SLC26A4 gene corresponds to the EVA in this family. Our study will provide a foundation for elucidating the SLC26A4-related mechanisms of hearing loss.

A Novel α-Thalassemia Nonsense Mutation on the α2-Globin Gene: HBA2: c.184A>T

Abstract We report a novel mutation on the α2-globin gene, HBA2: c.184A>T, detected in a Chinese proband. This mutation resulted in a Lys→Term substitution at position 62 of the α2-globin gene, causing a premature termination of translation. This mutation did not cause severe hematological abnormalities in the carriers. From the properties of substituted residues on the α2-globin gene, it is generally expected that this mutation causes unstable and truncated protein, thus this mutation should be detected in couples at-risk for α-thalassemia (α-thal).

Coinheritance of α- and β-Thalassemia with a Novel Mutation (HBB: c.268_281delAGTGAGCTGCACTG) in a Chinese Family

Abstract We report a novel mutation (HBB: c.268_281delAGTGAGCTGCACTG) in a Chinese proband, who was also an α-thalassemia (α-thal) Southeast Asian (αα/– –SEA) deletion carrier and displayed characteristic hematological features of β-thalassemia (β-thal) traits. The proband and carriers in her family presented hematological abnormalities. This novel mutation results in a frameshift and consequently creates a premature stop codon at codon 90 of the HBB gene. Thus, couples at-risk for β-thal should also be tested for this mutation. Double heterozygotes for α- and β-thal are easily misdiagnosed as pure β-thal carriers, which should be noted in the process of risk assessment and counseling.