Hyunwoong Park

The Phantom Menace for Patients with Hepatobiliary Diseases: Shewanella haliotis, Often Misidentified as Shewanella algae in Biochemical Tests and MALDI-TOF Analysis

Although Shewanella algae has been known to have weak pathogenicity, case reports on infections with this species have been steadily increasing. S. algae and S. haliotis are difficult to distinguish from each other with conventional phenotypic methods. We reviewed the microbiological and clinical features of S. algae and S. haliotis infections at our institute. Bacterial culture and identification reports from patient samples from 2010 to 2014 were reviewed to screen the cases of Shewanella infections. In addition to conventional biochemical tests, 16S rRNA gene sequence analysis and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were performed for 19 stored bacterial isolates. Medical records were reviewed for clinical characteristics and laboratory findings. All isolates were identified as S. algae by using VITEK 2. MALDI-TOF also identified all isolates as S. algae with a 99.9 confidence value. In contrast, 16S rRNA analysis identified 10 isolates as S. algae and 9 isolates as S. haliotis. Both S. algae (60%) and S. haliotis (77%) infections were strongly associated with diseases of the hepatobiliary tract and pancreas. To distinguish between S. algae and S. haliotis, 16S rRNA gene sequence analysis seems more accurate than biochemical tests or MALDI-TOF. Patients with underlying diseases in the hepatobiliary tract and pancreas seem to be susceptible to these marine pathogens.

Pitfalls of Multiple Ligation-Dependent Probe Amplifications in Detecting DMD Exon Deletions or Duplications.

Multiple ligation-dependent probe amplifications (MLPAs) are a key technology for the molecular diagnosis of Duchenne/Becker muscular dystrophy, which is mainly caused by large gene arrangements. However, little is known about the false-positive rates of MLPA for this disease. Here, we review MLPA analysis results from 398 patients suspected to have Duchenne/Becker muscular dystrophy. MLPA assay was used for screening the entire coding region. If these amplifications produced normal results, direct sequencing was performed to search for sequence variations and to determine single-exon deletions, duplications, or indeterminate results. Using MLPA, 290 cases (72.9%) showed exon deletion or duplication results. Among those, 75 cases (25.9%) resulted in a deletion or duplication of a single exon. Direct sequencing revealed that 11 single-exon deletion cases resulted in false-positives due to sequence variations within the patient population interfering with probe binding at the probe-hybridization sites. Abnormal MLPA results were closely related to the type of sequence change and the position within the probe-hybridization locus. The most common type was C-T transition (n = 19, 55.9%). Abnormal MLPA results correlated with CA mismatch and low melting temperature (≤75°C). False-positive events for large gene rearrangements involving a single exon in DMD accounted for approximately 15% (11/75). Therefore, careful design of MLPA probes is required to avoid false-positive results.

Mutational spectrum of the SPAST and ATL1 genes in Korean patients with hereditary spastic paraplegia

Hereditary spastic paraplegia (HSP) is a genetically heterogeneous group of diseases characterized by insidiously progressive lower-extremity weakness and spasticity. Spastic paraplegia 4 (SPAST) is the most common type of uncomplicated autosomal dominant HSP (40% of such cases), and spastic paraplegia 3A (ATL1) is the second most common. Here, we conducted mutational analysis of the SPAST and/or ATL1 genes in 206 unrelated patients with HSP. DNA sequencing and multiplex ligation-dependent probe amplification was used to analyze SPAST or ATL1 pathogenic variants. To confirm splice-site pathogenic variants, mRNA transcripts were analyzed by reverse transcription-polymerase chain reactions and sequencing. Among the 52 patients with medical records and SPAST or ATL1 gene pathogenic variants or novel unclassified variants, 50 showed spasticity or weakness in their lower extremities. We identified 16 known and 18 novel SPAST pathogenic variants and 2 known and a novel splicing pathogenic variants in ATL1. We also identified 4 unclassified SPAST variants in 5 patients and an unclassified ATL1 variant in 1 patient. Further, a novel leaky-splicing variant (c.1537-11A>G) was found in SPAST, which caused skipping of exon 13 or exons 13-14. Among the 206 unrelated patients with HSP, SPAST or ATL1 pathogenic variants and potentially pathogenic variants were identified in 52 patients, a low pathogenic variant rate compared to previous results. Results from our study suggest that other genes may be involved in HSP in the Korean population.

Consecutive analysis of mutation spectrum in the dystrophin gene of 507 Korean boys with Duchenne/Becker muscular dystrophy in a single center.

Duchenne and Becker muscular dystrophies (DMD and BMD) are allelic X‐linked recessive muscle diseases caused by mutations in the large and complex dystrophin gene.

Application of Multigene Panel Sequencing in Patients with Prolonged Rate-corrected QT Interval and No Pathogenic Variants Detected in KCNQ1, KCNH2, and SCN5A

Long QT syndrome (LQTS) is an inherited cardiac disease characterized by a prolonged heart rate-corrected QT (QTc) interval. We investigated the genetic causes in patients with prolonged QTc intervals who were negative for pathogenic variants in three major LQTS-related genes (KCNQ1, KCNH2, and SCN5A). Molecular genetic testing was performed using a panel including 13 LQTS-related genes and 67 additional genes implicated in other cardiac diseases. Overall, putative genetic causes of prolonged QTc interval were identified in three of the 30 patients (10%). Among the LQTS-related genes, we detected a previously reported pathogenic variant, CACNA1C c.1552C>T, responsible for cardiac-only Timothy syndrome. Among the genes related to other cardiac diseases, a likely pathogenic variant, RYR2 c.11995A>G, was identified in a patient with catecholaminergic polymorphic ventricular tachycardia. Another patient who developed dilated cardiomyopathy with prolonged QTc interval was found to carry a likely pathogenic variant, TAZ c.718G>A, associated with infantile dilated cardiomyopathy. Comprehensive screening of genetic variants using multigene panel sequencing enables detection of genetic variants with a possible involvement in QTc interval prolongation, thus uncovering unknown molecular mechanisms underlying LQTS.

Molecular Epidemiological Features and Antibiotic Susceptibility Patterns of Streptococcus dysgalactiae subsp. equisimilis Isolates from Korea and Japan

Background The molecular characterization of Streptococcus dysgalactiae subsp. equisimilis (SDSE) has not yet been performed in Korea. This study aimed to find the differences or similarities in the clinical features, molecular epidemiological findings, and antimicrobial resistance patterns of SDSE from two countries (Korea and Japan). Methods SDSE isolates were collected from Korea (N=69) from 2012–2016 and Japan (N=71) from 2014–2016. Clinical characteristics, emm genotypes, and sequence types (STs) were compared. Microdilution tests were performed using different antimicrobials, and their resistance determinants were screened. Results Median ages were 69 years in Korea and 76 years in Japan. The most common underlying diseases were diabetes and malignancy. Blood-derived isolates comprised 36.2% and 50.7% of Korean and Japanese isolates, respectively; mortality was not different between the two groups (5.8% vs 9.9%, P=0.53). Among Korean isolates with 20 different combined ST-emm types, ST127-stG245 (N=16), ST128-stG485 (N=10), and ST138-stG652 (N=8) were prevalent. Among Japanese isolates with 29 different combined types, ST17-stG6792 (N=11), ST29-stG485 (N=7), and ST205-stG6792 (N=6) were prevalent. Resistance rates to erythromycin, clindamycin, and minocycline were 34.8%, 17.4%, and 30.4% in Korea and 28.2%, 14.1%, and 21.4% in Japan, respectively. Conclusions SDSE infections commonly occurred in elderly persons with underlying diseases. There was a significant difference in the distribution of ST-emm types between the two countries. Antimicrobial resistance rates were comparable with different frequencies of resistance determinants in each country.

Effects of interleukin-4 and interleukin-12B gene polymorphisms on hepatitis B virus vaccination.

Approximately 10% of individuals do not respond to hepatitis B virus (HBV) vaccination, i.e. non-responders (NRs). We aimed to investigate the association of interleukin (IL)-4 and IL-12B gene polymorphisms with responsiveness to the HBV vaccine in Korean infants. Among 300 healthy infants (9-12 month), SNPs for the IL-4 gene (rs2243250, rs2070874, and rs2227284) and for the IL-12B gene (rs3213094 and rs17860508) were compared between subgroups in terms of the response to HBV vaccination. The percentages of NRs (< 10 mIU/mL), low-titer responders (LRs, 10-100 mIU/mL), and high-titer responders (HRs, ≥ 100 mIU/mL) were 20.3%, 37.7% and 42.0%, respectively. No SNPs differed in frequency between NRs and responders or between LRs and HRs. We divided the subjects into two groups according to the time interval from the 3rd dose of HBV vaccination to Ab quantification: > 6 months from the 3rd dose (n = 87) and ≤ 6 months from the 3rd dose (n = 213). In the ≤ 6 month subjects, rs2243250C and rs2227284G were significantly frequent in the lower-titer individuals (NRs + LR) than HRs (40.1 vs. 25.9%, p = 0.014 and 45.1 vs. 33.0%, p = 0.018, respectively), and the rs2243250C and rs2227284G frequencies were significantly different among the three subgroups (13.2 vs. 26.9 vs. 25.9%, p = 0.040 and 15.5 vs. 29.6 vs. 33.0%, p = 0.038, respectively). In conclusion, those results suggest that IL-4 gene polymorphisms may play a role in the response to the HBV vaccine in Korean infants.

Spectrum of MNX1 Pathogenic Variants and Associated Clinical Features in Korean Patients with Currarino Syndrome

Background The major genetic cause of Currarino syndrome (CS), a congenital malformation syndrome typically characterized by sacral agenesis, anorectal malformation, and presence of a pre-sacral mass, is known to be pathogenic variants in motor neuron and pancreas homeobox 1 (MNX1), which exist in almost all familial cases and 30% of sporadic cases. Less commonly, a large deletion or a complex rearrangement involving the 7q36 region is associated with CS. We investigated the spectrum of MNX1 pathogenic variants and associated clinical features in the Korean patients with CS. Methods We enrolled 25 patients with CS, including 24 sporadic cases and one familial case. Direct sequencing of MNX1 and multiplex ligation-dependent probe amplification were performed. We also analyzed clinical phenotypes and evaluated genotype-phenotype correlations. Results We identified six novel variants amongst a total of six null variants, one missense variant, and one large deletion. The null variants included four frameshift variants (p.Gly98Alafs*124, p.Gly145Alafs*77, p.Gly151Leufs*67, and p.Ala216Profs*5) and two nonsense variants (p.Tyr186* and p.Gln212*). The missense variant, p.Lys295Gln, was located in the highly-conserved homeobox domain and was predicted to be deleterious. A large deletion involving the 7q36 region was detected in one patient. Pathogenic variants in MNX1 were detected in 28% of all CS cases and 25% of sporadic cases. The clinical phenotype was variable in patients with and without pathogenic variants; no significant genotype-phenotype correlation was observed. Conclusions This study revealed the spectrum and phenotypic variability of MNX1 pathogenic variants in the Korean population.

Persistence of Group B Streptococcus in the Urogenital Area

Dear Editor, Early onset neonatal infections might be due to transmission from the maternal vaginal carriage of group B streptococcus (GBS) [1]. Urinary GBS suggests a heavy load of GBS [2]. Persistence of GBS in the urogenital area is not clearly understood and may cause recurrent infections. The aim of this study was to investigate how long GBS remains in the urogenital tract. The study involved 14 patients whose urine (n=9) and vaginal swab (n=5) repeatedly tested positive for GBS throughout 2011–2015. The interval between the first and second isolation ranged from two to 40 months for urine and from two to six months for vaginal swabs. Intervals of less than two months were exclud ed. The vaginal swabs were taken from pregnant women for preterm screening of GBS. Bacterial identification and antimicrobial susceptibility test were performed by using the Vitek-2 system (bioMerieux Inc., Marcy l’Etoile, France). Multilocus sequence typing (MLST) was performed according to previous reports for the collected isolates [3]. Briefly, bacterial DNA was extracted by using the GENEDIA Mycobacteria DNA Prep Kit (Green Cross Medical Science, Eumseng, Korea), and PCR was conducted for seven housekeeping genes with the AccuPower Taq PCR PreMix (Bioneer, Daejeon, Korea). DNA sequencing was performed at Macrogen Inc. (Seoul, Korea) using a dye-terminator method. Allocation of each allele and sequence type (ST) was assigned by using https://pubmlst.org/ [4]. This study was exempted from the approval by the institutional review board of Changwon Gyeongsang National University Hospital, Korea. None of the isolates were resistant to ampicillin, penicillin-G, ceftriaxone, co-trimoxazole, linezolid, or vancomycin. Therefore, susceptibility to erythromycin, clindamycin, tetracycline, and levofloxacin was compared between the first and second isolations (Table 1). Of the isolates from the 14 patients, those of patients 2 and 3 showed a change in susceptibility to tetracycline, while those of patient 12 showed a change in susceptibility to erythromycin, clindamycin, and tetracycline. Seven different STs were identified among the first isolates of the 14 patients. ST1 and ST19 were the most common (n=3), followed by ST2, ST335, ST654 (n=2), and ST10 and ST28 (n=1) (Table 1). Interestingly, only patients with ST19 exhibited different STs in their second specimen (patient 2, ST2 in the urine; patient 12, ST877 in the vaginal swab), whereas the other patients demonstrated the same ST. The allele combination of the seven housekeeping genes (adhP, pheS, atr, glnA, sdhA, glcK, and tkt) was 1-1-3-2-2-2-2 for ST19, 1-1-3-1-1-2-2 for ST2, and 166-1-2-1-1-2-2 for ST877. The interval of patient 2 was 40 months, and that of patient 12 was two months. The longest interval with the same ST was 14 months (patient 6) in

In-stent restenosis-prone coronary plaque composition: A retrospective virtual histology-intravascular ultrasound study

BACKGROUND The mechanism of in-stent restenosis (ISR) is multifactorial, which includes biological, mechanical and technical factors. This study hypothesized that increased inflammatory reaction, which is known to be an important atherosclerotic process, at a culprit lesion may lead to higher restenosis rates. METHODS The study population consisted of 241 patients who had undergone percutaneous coronary intervention with virtual histology-intravascular ultrasound (VH-IVUS) and a 9-month follow-up coronary angiography. Compared herein is the coronary plaque composition between patients with ISR and those without ISR. RESULTS Patients with ISR (n = 27) were likely to be older (66.2 ± 9.5 years vs. 58.7 ± 11.7 years, p = 0.002) and have higher levels of high-sensitivity C-reactive protein (hs-CRP, 1.60 ± 3.59 mg/dL vs. 0.31 ± 0.76 mg/dL, p < 0.001) than those without ISR (n = 214). VH-IVUS examination showed that percent necrotic core volume (14.3 ± 8.7% vs. 19.5 ± 9.1%, p = 0.005) was higher in those without ISR than those with ISR. Multivariate analysis revealed that hs-CRP (odds ratio [OR] 3.334, 95% con-fidence interval [CI] 1.158-9.596, p = 0.026) and age (OR 3.557, 95% CI 1.242-10.192, p = 0.018) were associated with ISR. CONCLUSIONS This study suggests that ISR is not associated with baseline coronary plaque composition but is associated with old age and increased expression of the inflammatory marker of hs-CRP. (Cardiol J 2018; 25, 1: 7-13).