Characterization of novel HLA-A*24:608N allele discovered in Koreans.

A novel null HLA-A*24 allele, HLA-A*24:608N, was identified in five Korean subjects including three from a family and two separate individuals. This study was performed to discern its immunological function in transplantation settings. Because this null variant had deletions of approximately 12 k base pairs from intron 3 to 3' end of the HLA-A gene, low resolution HLA typing and amplicon-based next generation sequencing (NGS) typing methods had failed to assign it. Hybrid capture-based NGS method confirmed that this novel variant had a large deletion. T-lymphocyte crossmatching by complement-dependent lymphocytotoxicity and flow cytometry with a serum consisting anti-HLA-A24 antibody revealed negative results, implying that an individual with this allele would not carry a functioning A24 antigen. These findings highlight the importance of identifying a null HLA allele by employing appropriate molecular method and providing expected crossmatching outcomes in a real-world transplantation setting.

Discovery of BRCA1/BRCA2 founder variants by haplotype analysis.

A significant number of hereditary breast or ovarian cancers are caused by germline variants, mostly BRCA1/BRCA2 genes. Because genetic predispositions vary by ethnicity, several studies have reported founder variants of BRCA1/BRCA2 genes. Such founder variants were reported primarily based on their relevant population frequencies. We reviewed the variant data relating to BRCA1 and BRCA2 genes from January 2012 to March 2019 at Samsung Medical Center, Seoul, Korea. Among the cases with pathogenic variants (PVs) or likely pathogenic variants (LPVs), we defined recurrent variants as those found in more than five unrelated patients. Using single nucleotide polymorphisms, we analyzed patient haplotypes. There were 14 recurrent variants in the BRCA1 gene and seven variants in the BRCA2 gene. Of note, three variants in each gene were primarily detected in Korean populations. Among them, the c.5339T>C (p.Leu1780Pro) BRCA1 variant had a long block sized 74.5 kb. In BRCA2, the c.1399A>T (p.Lys467*) variant had a long block sized 35.5 kb. We suggest that BRCA1 c.5339T>C (p.Leu1780Pro) and BRCA2 c.1399A>T (p.Lys467*) are founder variants of the Korean population. These two recurrent variants were ethnicity-prevalent, primarily found in Korean populations, and the sizes of the linkage disequilibrium blocks are longer than others.

Variation spectrum of MECP2 in Korean patients with Rett and Rett-like syndrome: a literature review and reevaluation of variants based on the ClinGen guideline.

Rett syndrome (RTT) is a progressive neurodevelopmental disorder caused by variants in MECP2. Emerging evidence of ethnic specificity of genetic variations has allowed precise diagnostic approaches with tailored therapies. In this study, we reviewed the variation spectrum of MECP2 in Korean RTT(-like) patients and compared it with previous reports in multiple ethnic groups. We reevaluated variants found in Korean RTT patients according to the new Clinical Genome Resource guideline to reinterpret and reclassify variants of uncertain significance in MECP2. Among 377 cases, 56 (14.9%) showed pathogenic variants, and three novel variants, p.(Ala277Argfs*7), p.(Ala378Glyfs*8), and p.(Arg270_Ser332del), were identified. Comprehensive data from Korea revealed an overall consistent variation spectrum with those from other ethnicities. Through the reevaluation of variants, nine that previously had insufficient evidence for pathogenicity were reclassified into pathogenic variants. Our study provided insight on the genetic contribution of MECP2 in RTT and a useful background for genetic counseling in the Korean population.

Case Report: Novel Splicing Variant in SH2D1A in a Patient With X-Linked Lymphoproliferative Syndrome Type 1.

X-linked lymphoproliferative disease type 1 (XLP1), an X-linked recessive genetic disorder, is associated with primary immunodeficiency. Patients with XLP1 are susceptible to Epstein-Barr virus (EBV) infection. SH2D1A gene is known as the causative gene. We found a novel hemizygous variant of SH2D1A, c.162_201+31delinsTACAAGGACATATACA, from a 5-year-old male patient who had been diagnosed with EBV infection and Hodgkin's lymphoma. In targeted next-generation sequencing (NGS), complex variants at exon 2 were not consistently identified with two software programs. They showed a soft-clipped read pattern. The variant had a 71-bp deletion and a 16-bp insertion across exon 2 as confirmed by direct sequencing. As the variant was located within the exon-intron boundary, two aberrant transcripts were shown by RNA study. Although NGS method has a limitation in detecting large deletion/duplication variants, proper bioinformatics pipeline and careful review of data might enable the detection of complex variants.

Necessity of multiplex ligation probe amplification in genetic tests: Germline variant analysis of the APC gene in familial adenomatous polyposis patients.

BACKGROUND: Familial adenomatous polyposis (FAP) is an autosomal dominant hereditary cancer syndrome characterized by hundreds to thousands of colorectal adenomatous polyps. Without treatment, progression to colorectal cancer is inevitable. Most pathogenic mutations in the APC gene were nonsense or frameshift mutations; however, some previous studies reported large deletions or duplications. METHOD: We reviewed the results of APC gene analyses from January 2010 to December 2020. We analyzed the entire coding sequence of the APC gene by Sanger sequencing to detect genetic abnormalities. Moreover, multiplex ligation-dependent probe amplification (MLPA) testing was performed starting in September 2017, and a multigene panel study that includes the APC gene was begun in July 2019. RESULTS: In the 266 collected cases, pathogenic variants/likely pathogenic variants (PV/LPVs) in the APC gene were detected in 73 patients, and variants of uncertain significance were found in 13 patients. Among those variants, 14 variants were novel. We performed MLPA for 29 patients, and 7 of them (24.1%) were positive for a large duplication/deletion. Among the 73 cases in the multigene panel study, 17 cases (23.3%) of APC gene variation were detected. CONCLUSION: We retrospectively analyzed the APC gene in Korean patients suspected to have FAP. Variants truncated by nonsense and frame shift mutations were common PV/LPVs. However, the detection rate in the MLPA study was higher than in previous studies of Caucasian populations. We suggest that MLPA should be performed for patients likely to have FAP but in whom no variant is detected by sequencing methods.

Comprehensive clinical characterization of patients with BRCA1: c.5017_5019del germline variant.

Purpose: We provide evidence for the reclassification of the BRCA1:c.5017_5019del variant by presenting the clinicopathological characteristics, clinical outcomes, and family history of breast or ovarian cancer in 17 patients with this variant. Methods: This study included breast or ovarian cancer patients tested for BRCA1/2 genes between January 2008 and June 2020 at 10 medical centers in Korea. We retrospectively reviewed 17 probands from 15 families who had the BRCA1:c.5017_5019del variant according to the electronic medical records. Results: We present 10 breast cancer patients and 7 ovarian cancer patients from 15 families identified as having BRCA1:c.5017_5019del and a total of 19 cases of breast cancer and 14 cases of ovarian cancer in these families. The ratio of breast-to-ovarian cancer was 1.3:1. Breast cancer patients with this variant showed a rich family history of breast or ovarian cancer, 8 patients (80.0%). The mean age at diagnosis was 45.4 years and 6 patients (60.0%) were categorized into hormone-receptor-negative breast cancer. Also, the ovarian cancer patients with this variant showed strong family histories of breast and/or ovarian cancer in 4 patients (57.1%). Conclusion: We presented clinical evidence for the reclassification of BRCA1:c.5017_5019del as a likely pathogenic variant (LPV). Reclassification as LPV could result in the prophylactic treatment and medical surveillance of probands, family testing recommendations, and appropriate genetic counseling of their families.

Clinicopathological Characterization of Double Heterozygosity for BRCA1 and BRCA2 Variants in Korean Breast Cancer Patients.

PURPOSE: Double heterozygosity (DH) for BRCA1 and BRCA2 variant is very rare with only a few cases reported, and most those in Caucasians. In this article, we present seven unrelated cases of DH for BRCA1/2 identified from a single institution in Korea, and describe the characteristics and phenotype of DH individuals compared to those with a single BRCA variant. MATERIALS AND METHODS: This study included 27,678 patients diagnosed with breast cancer and surgically treated at Samsung Medical Center (SMC) between January 2008 and June 2020. In total, 4,215 high-risk breast cancer patients were tested for the BRCA1/2 genes, and electronic medical records from 456 cases with pathogenic/likely pathogenic variants (PVs/LPVs) were reviewed. RESULTS: A younger mean age at diagnosis was associated with DH than a single variant of BRCA1/2. More triple-negative breast cancer (TNBC) and higher nuclear and histologic grade cancer occurred with DH than BRCA2 variant. All 7 cases of DH were unrelated, and their mutation combinations were different. There were no Ashkenazi founder variants detected. CONCLUSION: We suggest that patients with DH for BRCA1/2 variants develop breast cancer at a younger age, but the histopathologic features are similar to those of BRCA1.

Genetic Counseling and Long-Term Surveillance Using a Multidisciplinary Approach in von Hippel-Lindau Disease.

BACKGROUND: von Hippel-Lindau (VHL) disease is an autosomal dominant disorder caused by variants of the VHL tumor suppressor gene (VHL). Early detection and treatment are essential to prevent morbidity and mortality. We evaluated the effectiveness of surveillance strategies and the utility of a VHL clinic with a multidisciplinary team for the first time in Korea. METHODS: The VHL clinic was organized at the Samsung Medical Center in 2011 and consisted of a multidisciplinary team, including an endocrinologist, urologist, general surgeon, neurosurgeon, ophthalmologist, otolaryngologist, and radiologist. Biochemical and imaging surveillance and personalized genetic counseling were conducted at the VHL clinic and patients were referred to the necessary departments upon detection of disease manifestation. We divided the patients in three groups (I-III) based on their compliance to VHL clinic attendance. RESULTS: Between 2011 and 2018, 50 VHL patients were identified by VHL molecular analysis and referred to the VHL clinic. Most patients regularly participated in imaging of the central nervous system (43/50, 86.0%) and of the abdomen (46/50, 92.0%). However, there were differences in compliance to determination of the catecholamine level, audiometry, and ophthalmic examination among the three groups. CONCLUSIONS: We present the results of using a multidisciplinary team approach and showed that the VHL clinic strategy is useful for the comprehensive surveillance and management of VHL disease. We hope that VHL clinics will be widely set up in hospitals to improve prognosis in patients with VHL.

Flow Cytometry for the Diagnosis of Primary Immunodeficiency Diseases: A Single Center Experience.

PURPOSE: While there is an urgent need for diagnosis and therapeutic intervention in patients with primary immunodeficiency diseases (PIDs), current genetic tests have drawbacks. We retrospectively reviewed the usefulness of flow cytometry (FCM) as a quick tool for immunophenotyping and functional assays in patients suspected to have PIDs at a single tertiary care institute. METHODS: Between January 2001 and June 2018, patients suspected of having PIDs were subjected to FCM tests, including lymphocyte subset analysis, detection of surface- or intracellular-target proteins, and functional analysis of immune cells, at Samsung Medical Center, Seoul, Korea. The genetic diagnosis was performed using Sanger or diagnostic exome sequencing. RESULTS: Of 60 patients diagnosed with definite or probable PID according to the European Society of Immune Deficiencies criteria, 24 patients were provided with useful information about immunological dysfunction after initial FCM testing. In 10 patients, the PID diagnosis was based on abnormal findings in FCM testing without genetic tests. The FCM findings provided strong evidence for the diagnosis of severe combined immunodeficiency (n = 6), X-linked chronic granulomatous diseases (CGD) (n = 6), leukocyte adhesion deficiency type 1 (n = 3), X-linked agammaglobulinemia (n = 11), autoimmune lymphoproliferative syndrome-FASLG (n = 1), and familial hemophagocytic lymphohistiocytosis type 2 (n = 1), and probable evidence for autosomal recessive-CGD (n = 2), autosomal dominant-hyper-immunoglobulin E (IgE)-syndrome (n = 1), and STAT1 gain-of-function mutation (n = 1). In PIDs derived from PIK3CD (n = 2), LRBA (n = 2), and CTLA4 mutations (n = 3), the FCM test provided useful evidence of immune abnormalities and a tool for treatment monitoring. CONCLUSIONS: The initial application of FCM, particularly with known protein targets on immune cells, would facilitate the timely diagnosis of PIDs and thus would support clinical decisions and improve the clinical outcome.

Comparative evaluation of QuantiFERON-TB Gold Plus for diagnosis of latent tuberculosis infection during solid organ transplantation.

Background: The diagnosis of latent tuberculosis infection (LTBI) in solid organ transplantation (SOT) patients under lifelong immunosuppression has profound effects on preoperative and postoperative management. Interferon-gamma release assay (IGRA) is widely used to screen LTBI before or after transplantation. Methods: We evaluated the effect of posttransplantation immunosuppression on IGRA and influencing factors by measuring interval change of QuantiFERON-TB Gold Plus (QFT-Plus) between pretransplantation (pre-QFT-Plus) and posttransplantation (post-QFT-Plus) state in 20 patients who previously had reactive IGRA but not taken LTBI treatment. Results: Eleven (55%) out of 20 pre-QFT-Plus reactive patients became nonreactive state in repeated QFT-Plus (post-QFT-Plus) at 194-413 days (median, 257 days) after transplantation (discordant group). Even in persistently reactive group (concordant group), interferon-gamma (IFN-γ) levels after transplantation were decreased about 34% and 36% of their pretransplantation levels for TB1 and TB2, respectively. The only significant factors that affect interval change of QFT-Plus between pre- and post-SOT status were the concentrations of IFN-γ in pre-QFT-Plus (6.93 vs. 0.44 IU/mL in TB1 and 7.33 vs. 0.71 IU/mL in TB2). Conclusions: The reactivity of QFT-Plus is significantly compromised by immunosuppressive therapy, which increase the risk of false negative, particularly in patients with low level of IGRA reactivity. Therefore, interpretation of IGRA under immunosuppressive treatment require a caution and eventually, more sensitive tuberculosis-specific cytokine markers might be needed.

Performance evaluation of the QMAC-dRAST for staphylococci and enterococci isolated from blood culture: a comparative study of performance with the VITEK-2 system.

Objectives: To evaluate the performance of a rapid antimicrobial susceptibility testing (AST) platform based on microfluidic chip technology, the QMAC-dRAST, which enables AST from colony isolates or positive blood culture broth (PBCB), and to compare the performance of the QMAC-dRAST for staphylococci and enterococci with that of the VITEK-2 system based on reference broth microdilution (BMD). Methods: A total of 110 staphylococcal and enterococcal isolates from blood cultures were included. AST was performed directly using the QMAC-dRAST with PBCB. Thereafter, colony isolates derived from subculture of PBCB were used for the QMAC-dRAST, the VITEK-2 system and BMD. Results: The overall agreement between the QMAC-dRAST with PBCB and BMD was 91.5%. There were 1.2% very major errors (VMEs), 4.3% major errors (MEs) and 5.4% minor errors (mEs). The QMAC-dRAST with colony isolates yielded 94.6% agreement and error rates of 1.0% VMEs, 1.8% MEs and 4.0% mEs. The VITEK-2 system showed 96.2% agreement and error rates of 2.3% VMEs, 0.5% MEs and 2.6% mEs. The incubation time in the QMAC-dRAST was significantly shorter than in the VITEK-2 system (median of 6 versus 10 h; P < 0.0001). Conclusions: The QMAC-dRAST system provided rapid results and represents an alternative to conventional AST methods. The QMAC-dRAST with colony isolates produced more reliable results for staphylococci and enterococci than the QMAC-dRAST with PBCB. The QMAC-dRAST system also performed comparably to BMD and the VITEK-2 system.

Clinical utility of FISH analysis in addition to G-banded karyotype in hematologic malignancies and proposal of a practical approach.

BACKGROUND: Fluorescence in situ hybridization (FISH) analysis can provide important information in the management of patients with hematologic malignancies. However, FISH performed in addition to G-banded karyotype can be labor-intensive and expensive. The aim of this study was to evaluate whether FISH gives additional information in the setting of adequate conventional cytogenetics in cases of hematologic malignancies. METHODS: Bone marrow aspirates were obtained from 135 patients at diagnosis (56 AML, 32 MDS, 20 ALL, and 27 MM) between 2005 and 2010. Interphase FISH was performed using the following probes: BCR/ABL1, AML1/ETO, PML/RARA, CBFB, MLL, EGR1, CEP8, and D7S486 for AML; CEP8, D20S108, EGR1, and D7S486 for MDS; BCR/ABL1, MLL, CDKN2A (p16), ETV6, and 6q21/c-myc for ALL; IgH, TP53, D13S25, IgH/CCND1, IgH/MAF, IgH/FGFR3, and 1q21/8p21 for MM. We compared the results of FISH with the corresponding aberrations identified by G-banded karyotype. RESULTS: Additional genetic aberrations detected by FISH (which were not identified by G-banded karyotype) were 4%, 9%, 50%, and 67% in AML, MDS, ALL, and MM, respectively. In ALL, CDKN2A and ETV6 FISH revealed additional genetic aberrations in 33% and 28% of cases, respectively. In MM, FISH was of benefit in detecting IgH, D13S25, TP53, and 1q21 rearrangements, not detected by G-banded karyotype (31%, 36%, 20%, and 40%, respectively). CONCLUSION: These results suggest that performing FISH in addition to G-banded karyotype may contribute little additional genetic information in AML and MDS, whereas routine FISH analysis appears to be an efficient screening method in ALL and MM.