The value of single-molecule real-time technology in the diagnosis of rare thalassemia variants and analysis of phenotype–genotype correlation

To compare single-molecule real-time technology (SMRT) and conventional genetic diagnostic technology of rare types of thalassemia mutations, and to analyze the molecular characteristics and phenotypes of rare thalassemia gene variants, we used 434 cases with positive hematology screening as the cohort, then used SMRT technology and conventional gene diagnosis technology [(Gap-PCR, multiple ligation probe amplification technology (MLPA), PCR-reverse dot blot (RDB)] for thalassemia gene screening. Among the 434 enrolled cases, conventional technology identified 318 patients with variants (73.27%) and 116 patients without variants (26.73%), SMRT identified 361 patients with variants (83.18%), and 73 patients without variants (16.82%). The positive detection rate of SMRT was 9.91% higher than conventional technology. Combination of the two methods identified 485 positive alleles among 49 types of variant. The genotypes of 354 cases were concordant between the two methods, while 80 cases were discordant. Among the 80 cases, 76 cases had variants only identified in SMRT method, 3 cases had variants only identified in conventional method, and 1 false positive result by the traditional PCR detection technology. Except the three variants in HS40 and HBG1-HBG2 loci, which was beyond the design of SMRT method in this study, all the other discordant variants identified by SMRT were validated by further Sanger sequencing or MLPA. The hematological phenotypic parameters of 80 discordant cases were also analyzed. SMRT technology increased the positive detection rate of thalassemia genes, and detected rare thalassemia cases with variable phenotypes, which had great significance for clinical thalassemia gene screening.

Deterioration of Avellino corneal dystrophy in a Chinese family after LASIK

AIM: To reveal the importance of TGFBI gene screening for candidates with a family history of corneal disease or granular opacities in corneal stroma before refractive surgery. METHODS: A 37-year-old male (proband) underwent bilateral laser-assisted in situ keratomileusis (LASIK) in 2002, with right vision decreased significantly in 2006. The proband and other 32 members of the family underwent a detailed ophthalmic examination, including vision acuity, intraocular pressure, slit-lamp photograph, fundus examination, optical coherence tomography (OCT) of cornea, and in vivo confocal microscope (IVCM) and peripheral blood was used for genomic DNA extraction. Seventeen TGFBI gene exons were analyzed via polymerase chain reaction amplification and direct sequencing. RESULTS: Slit-lamp, IVCM, and OCT images showed that a large amount of dense and confluent granular opaque were seen at the interfaces of the flap and remnant stromal bed in right and light degree in left eye. Sanger sequencing showed that there was a 371G>A mutation (CGC>CAC) in exon 4, which indicated that he harbored a heterozygote R124H mutation, identifying the diagnosis of Avellino corneal dystrophy (ACD). Among the other 32 family members, 6 of them harbored the identical mutation to that in the proband. CONCLUSION: ACD will worsen and recur after LASIK. Preoperative gene-screening for TGFBI mutations is important in diagnosing ACD.

mcr-1 Identified in Fecal Escherichia coli and Avian Pathogenic E. coli (APEC) From Brazil

Colisitin-associated resistance in bacteria of food producing animals has gained significant attention with the mcr gene being linked with resistance. Recently, newer variants of mcr have emerged with more than nine variants currently recognized. Reports of mcr associated resistance in Escherichia coli of poultry appear to be relatively limited, but its prevalence requires assessment since poultry is one of the most important and cheapest sources of the world’s protein and the emergence of resistance could limit our ability to treat disease outbreaks. Here, 107 E. coli isolates from production poultry were screened for the presence of mcr 1–9. The isolates were collected between April 2015 and June 2016 from broiler chickens and free-range layer hens in Rio de Janeiro, Brazil. All isolates were recovered from the trachea and cloaca of healthy birds and an additional two isolates were recovered from sick birds diagnosed with colibacillosis. All isolates were screened for the presence of mcr-1 to 9 using PCR and Sanger sequencing for confirmation of positive genes. Additionally, pulse field gel electrophoresis (PFGE) analysis, avian fecal E. coli (APEC) virulence associated gene screening, plasmid replicon typing and antimicrobial resistance phenotype and resistance gene screening, were also carried out to further characterize these isolates. The mcr-1 gene was detected in 62 (57.9%) isolates (61 healthy and 1 APEC) and the mcr-5 gene was detected in 3 (2.8%) isolates; mcr-2, mcr-3, mcr-4, mcr-6, mcr-7, mcr-8, and mcr-9 were not detected in any isolate. In addition, mcr 1 and 5 positive isolates were phenotypically resistant to colistin using the agar dilution assay (> 8ug/ml). PFGE analysis found that most of the isolates screened had unique fingerprints suggesting that the emergence of colistin resistance was not the result of clonal dissemination. Plasmid replicon types IncI2, FIB, and B/O were found in 38, 36, and 34% of the mcr positive isolates and were the most prevalent replicon types detected; tetA and tetB (32 and 26%, respectively) were the most prevalent antimicrobial resistance genes detected and iutA, was the most prevalent APEC virulence associated gene, detected in 50% of the isolates. Approximately 32% of the isolates examined could be classified as APEC-like, based on the presence of 3 or more genes of APEC virulence associated path panel (iroN, ompT, hlyF, iss, iutA). This study has identified a high prevalence of mcr-1 in poultry isolates in Brazil, suggesting that animal husbandry practices could result in a potential source of resistance to the human food chain in countries where application of colistin in animal health is practiced. Emergence of the mcr gene and associated colisitin resistance in production poultry warrants continued monitoring from the animal health and human health perspective.

Pathogenic Germline Mutations of DNA Repair Pathway Components in Early-Onset Sporadic Colorectal Polyp and Cancer Patients

Given recent increases in the proportion of early-onset colorectal cancer (CRC), researchers are urgently working to establish a multi-gene screening test for both inherited and sporadic cancer-susceptible individuals. However, the incidence and spectrum of germline mutations in young sporadic CRC patients in East Asian countries and, especially, in sporadic polyp carriers and normal individuals are unknown. Peripheral blood samples were collected from 43 colonoscopy-proved normal controls and from 50 polyp patients and 49 CRC patients with no self-reported family history of cancer. All participants were under 50 years old. Next-generation sequencing with a panel of 30 CRC-associated susceptibility genes was employed to detect pathogenic germline mutations. The germline mutation carrier rates were 2.3%, 4.0%, and 12.2% in the normal, polyp, and cancer groups, respectively. A total of seven different mutations in six DNA repair pathway-related genes (MLH1, BRCA1, BRCA2, CHEK2, BLM, and NTHL1) were detected in nine participants. One frameshift mutation in BRCA2 and one frameshift mutation in the CHEK2 gene were found in a normal control and two colorectal polyp patients, respectively. One young sporadic CRC patient carried two heterozygous mutations, one in MLH1 and one in BRCA1. Three mutations (MLH1 p.Arg265Cys, MLH1 p.Tyr343Ter and CHEK2 p.Ile158TyrfsTer10) were each found in two independent patients and were considered “founder” mutations. This is the first report to demonstrate high percentage of germline mutations in young sporadic colorectal polyp, CRC, and general populations. A multi-gene screening test is warranted for the proactive identification of cancer-predisposed individuals.