Molecular characterization of a novel 83.9-kb deletion of the α-globin upstream regulatory elements by long-read sequencing.

Inherited deletions of upstream regulatory elements of α-globin genes give rise to α-thalassemia, which is an autosomal recessive monogenic disease. However, conventional thalassemia target diagnosis often fails to identify these rare deletions. Here we reported a family with two previous pregnancies of Hb Bart's hydrops fetalis and was seeking for prenatal diagnosis during the third pregnancy. Both parents had low level of Hemoglobin A2 indicating α-thalassemia. Conventional Gap-PCR and PCR-reverse dot blot showed the father carried -SEA deletion but did not identify any variants in the mother. Multiplex ligation-dependent probe amplification identified a deletion containing two HS-40 probes but could not determine the exact region. Finally, a long-read sequencing (LRS)-based approach directly identified that the exact deletion region was chr16: 48,642-132,584, which was located in the α-globin upstream regulatory elements and named (αα)JM after the Jiangmen city. Gap-PCR and Sanger sequencing confirmed the breakpoint. Both the mother and fetus from the third pregnancy carried heterozygous (αα)JM, and the fetus was normally delivered at gestational age of 39 weeks. This study demonstrated that LRS technology had great advantages over conventional target diagnosis methods for identifying rare thalassemia variants and assisted better carrier screening and prenatal diagnosis of thalassemia.

Comprehensive Analysis of Fragile X Syndrome: Full Characterization of the FMR1 Locus by Long-Read Sequencing.

BACKGROUND: Fragile X syndrome (FXS) is the most frequent cause of inherited X-linked intellectual disability. Conventional FXS genetic testing methods mainly focus on FMR1 CGG expansions and fail to identify AGG interruptions, rare intragenic variants, and large gene deletions. METHODS: A long-range PCR and long-read sequencing-based assay termed comprehensive analysis of FXS (CAFXS) was developed and evaluated in Coriell and clinical samples by comparing to Southern blot analysis and triplet repeat-primed PCR (TP-PCR). RESULTS: CAFXS accurately detected the number of CGG repeats in the range of 93 to at least 940 with mass fraction of 0.5% to 1% in the background of normal alleles, which was 2-4-fold analytically more sensitive than TP-PCR. All categories of mutations detected by control methods, including full mutations in 30 samples, were identified by CAFXS for all 62 clinical samples. CAFXS accurately determined AGG interruptions in all 133 alleles identified, even in mosaic alleles. CAFXS successfully identified 2 rare intragenic variants including the c.879A > C variant in exon 9 and a 697-bp microdeletion flanking upstream of CGG repeats, which disrupted primer annealing in TP-PCR assay. In addition, CAFXS directly determined the breakpoints of a 237.1-kb deletion and a 774.0-kb deletion encompassing the entire FMR1 gene in 2 samples. CONCLUSIONS: Long-read sequencing-based CAFXS represents a comprehensive assay for identifying FMR1 CGG expansions, AGG interruptions, rare intragenic variants, and large gene deletions, which greatly improves the genetic screening and diagnosis for FXS.

Design, synthesis and antibacterial activity evaluation of ebselen derivatives in NDM-1 producing bacteria.

New Delhi-ß-lactamase-1 (NDM-1) is a type of metal-ß-lactamase. NDM-1-expressing bacteria can spread rapidly across the globe via plasmid transfer, which greatly undermines the clinical efficacy of the carbapenem. Research on NDM-1 inhibitors has attracted extensive attention. However, there are currently no clinically available NDM-1 inhibitors. Our research group has reported that 1,2-benzisoselenazol-3(2H)-one derivatives as covalent NDM-1 inhibitors can restore the efficacy of meropenem (Mem) against NDM-1 producing strains. In this study, 22 compounds were designed and synthesized, which restored the Mem susceptibility of NDM-1-expressing Escherichia coli. and its minimum inhibitory concentration (MIC) was reduced by 2-16 times. Representative compound A4 showed significant synergistic antibacterial activity against NDM-1-producing carbapenem-resistant Enterobacteriaceae (CRE) isolates. The in vitro NDM-1 enzyme inhibitory activity test showed that the IC50 was 1.26 ± 0.37 µM, which had low cytotoxicity. When combined with meropenem, it showed good combined antibacterial activity. Electrospray ionization mass spectrometry (ESI-MS) analysis demonstrates that compound A4 covalently binds to NDM-1 enzyme. In summary, compound A4 is a potent NDM-1 covalent inhibitor and provides a potential lead compound for drug development in resistant bacteria.

A high-fidelity long-read sequencing-based approach enables accurate and effective genetic diagnosis of spinal muscular atrophy.

BACKGROUND: We aimed to develop a high-fidelity long-read sequencing (LRS)-based approach to detect SMN gene variants in one step. It is challenging for conventional step-wise methods to simultaneously detect all kinds of variations between homologous SMN1 and SMN2. METHODS: In this study, LRS was developed to analyze copy numbers (CNs), full sequences, and structure of SMN1 and SMN2. The results were compared with those from the step-wise methods in 202 samples from 67 families. RESULTS: LRS achieved 100% (202/202) and 99.5% (201/202) accuracy for SMN1 and SMN2 CNs, respectively. It corrected SMN1 CNs from MLPA, which was caused by SNVs/indels that located in probe-binding region. LRS identified 23 SNVs/indels distributing throughout SMN1, including c.22dup and c.884A > T in trans-configuration, and a de novo variant c.41_42delinsC for the first time. LRS also identified a SMN2 variant c.346A > G. Moreover, it successfully determined Alu-mediated 8978-bp deletion encompassing exon 2a-5 and 1415-bp deletion disrupting exon 1, and the exact breakpoints of large deletions. Through haplotype-based pedigree trio analysis, LRS identified SMN1 2 + 0 carriers, and determined the distribution of SMN1 and SMN2 on two chromosomes. CONCLUSIONS: LRS represents a more comprehensive and accurate diagnosis approach that is beneficial to early treatment and effective management of SMA.

Evaluating the clinical utility of a long-read sequencing-based approach in genetic testing of fragile-X syndrome.

BACKGROUND: Fragile X syndrome (FXS) arises from the FMR1 CGG expansion. Comprehensive genetic testing for FMR1 CGG expansions, AGG interruptions, and microdeletions is essential to provide genetic counseling for females carrying premutation alleles. However, conventional PCR-based FMR1 assays mainly focus on CGG repeats, and could detect AGG interruption only in males. METHODS: The clinical utility of a long-read sequencing-based assay termed comprehensive analysis of FXS (CAFXS) was evaluated in 238 high-risk samples by comparing to conventional PCR assays. RESULTS: PCR assays identified five premuation and three full mutation categories alleles in all the samples, and CAFXS successfully called all the FMR1 CGG expansion. CAFXS identified 24-bp microdeletions upstream to the trinucleotide region with 30 CGG repeats, which was miscalled by the length-based PCR methods. CAFXS also identified a 187-bp deletion in about 1/7 of the sequencing reads in a male patient with mosaic full mutation alleles. CAFXS allowed for precise constructing the FMR1 CGG repeat and AGG interruption pattern in all the samples, and identified a novel and alternative CGA interruption in one normal female sample. CONCLUSIONS: CAFXS represents a more comprehensive and accurate approach for FXS genetic testing that potentially enables more informed genetic counseling compared to PCR-based methods.

Case report: Long-read sequencing identified a novel 14.9-kb deletion of the α-globin gene locus in a family with α-thalassemia in China.

Background: Thalassemia is a hereditary blood disease resulting from globin chain synthesis impairment because of α- and/or ß-globin gene variants. α-thalassemia is characterized by non-deletional and deletional variants in the HBA gene locus, of which rare deletional variants are difficult to detect by conventional polymerase chain reaction (PCR)-based methods. Case report: We report the case of a one-month-old boy, who and his mother had abnormal hematological parameters, while his father had normal hematology. Conventional PCR-reverse dot blot (RDB) was performed for all family members to analyze the 23 most common thalassemia variants in China, but did not identify any pathologic variants. Single-molecule real-time (SMRT) long-read sequencing (LRS) technology was then performed and identified an unreported 14.9-kb large deletion (hg38 chr16:168,803-183,737) of the α-globin gene locus, which disrupted both HBA1 and HBA2 genes in the proband and his mother. The exact breakpoints of the deletion were confirmed by gap-PCR and Sanger sequencing. Conclusion: We have detected a novel large deletion in α-globin gene locus in China, which not only enriches the variant spectrum of thalassemia, but also demonstrates the accuracy and efficiency of LRS in detecting rare and novel deletions.

The comprehensive analysis of thalassemia alleles (CATSA) based on single-molecule real-time technology (SMRT) is a more powerful strategy in the diagnosis of thalassemia caused by rare variants.

Thalassemia is one of the most widely distributed monogenic disorders in the world and affects the largest number of people. It can manifest a wide spectrum of phenotypes from asymptomatic to fatal, which is associated with the degree of imbalance between α- and ß-globin chains. Therefore, individuals with different genotypes could present with a similar phenotype. Genetic analysis is always needed to make a correct diagnosis. However, routine genetic analysis of thalassemia used in the Chinese population identifies only 23 common variants, resulting in many cases undiagnosed or being misdiagnosed. In this study, we applied a long-read sequencing-based approach termed comprehensive analysis of thalassemia alleles (CATSA) to 30 subjects whose hematologic screening results could not be explained by the routine genetic test results. The identification of additional variants and the correction of genotypes allowed the interpretation of the clinical phenotype in 24 subjects, which have been confirmed to be correct by independent experiments. Moreover, we identified a novel 8.4-kb deletion containing the entire HBB and HBD genes as well as part of the HBBP1 gene, expanding the genotype spectrum of ß-thalassemia. CATSA showed a great advantage over other genetic tests in the diagnosis of thalassemia caused by rare variants.

Identification of a novel 10.3 kb deletion causing α0-thalassemia by third-generation sequencing: Pedigree analysis and genetic diagnosis.

BACKGROUND: α-thalassemia is an inherited blood disorder caused by variants in the α-globin gene cluster. Identification of the pathogenic α-globin gene variants is important for the diagnosis and management of thalassemia. METHODS: Two suspected families from Xiantao, Hubei Province were recruited in this study. The family members underwent hemoglobin testing. Polymerase Chain Reaction based reverse dot blot (PCR-RDB) was employed to identify the known variants. Next-generation sequencing (NGS) and third-generation sequencing (TGS) were performed to screen the potential disease-causing variants, which were validated by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). RESULTS: Hematological analysis suggested that proband A had α-thalassemia traits, and proband B had HbH disease traits. However, only a -α3.7 mutation had been detected by PCR-RDB and NGS in the proband of family B. Subsequent TGS identified a novel 10.3 kb deletion (NC_000016.10:g.172342-182690del) covering the HBA1, HBQ1 and HBA2 genes in the α-globin gene cluster in both family A and B, which was confirmed by Sanger sequencing and MLPA. These results indicated that the novel deletion is likely responsible for α-thalassemia. CONCLUSION: A novel α-thalassemia deletion was identified for the two families by TGS. Our work broadened the molecular spectrum of α-thalassemia, and was beneficial for the diagnosis, genetic counseling and management of α-thalassemia.

Knocking out Analysis of the CpxP gene using Crispr/Cas9 in Escherichia coli MG1655.

Based on the analysis of cpxP genes among Escherichia coli strains, cpxP gene-targeting short guide RNA (sgRNA) was designed and inserted into the pGL3-MGP-RNA. The donor sequences (MG-HR) for homologous repair were designed and cloned by PCR. MG-HR and pGL3-MGP-RNA were transformed into E. coli MG1655 (pCas9). The cpxP gene expression cassette was amplified by PCR and subcloned into pBBR1MCS-2. Then the pBBR-cpxP was independently transformed into E. coli MG1655. The results of motility experiment suggest that cpxP gene had a significant effect on the movement ability of E. coli strain. The CpxP protein had a significant inhibition of bacterial activity. The lastest 81 CpxP proteins sequences were selected and analyzed by multi-sequence alignment and molecular cluster. The CpxP proteins were roughly divided into three categories. Our results suggest that the CpxP protein was involved in bacterial motility, infection and pathogenicity.