Flat Pattern Peaks of Tacrolimus Absorption and Associated Pharmacogenomic Variants in Kidney Transplantation Recipients.

BACKGROUND: Tacrolimus is the most commonly used immunosuppressive drug in solid organ transplantation. After administering a conventional twice-daily dose of tacrolimus, peak levels were achieved within the first 1.5 to 2 hours. A group of patients showed different early absorption phase of tacrolimus after kidney transplantation. METHODS: Trough(C0) and 1.5-hour blood levels (C1.5) of tacrolimus were measured in 95 kidney transplantation recipients. Patients with a C1.5/C0 < 1.5 and > 1.5 were defined as those having flat pattern peaks and as controls, respectively. Transplantation outcomes were compared between the groups. Whole exome sequencing was performed to investigate the genetic susceptibility to flat pattern peaks. RESULTS: Twenty-eight patients showed flat pattern peaks. The mean C1.5/C0 values were 1.13 ± 0.22 and 3.78 ± 1.25 in the flat pattern peak and control groups, respectively. In multivariate analysis, flat pattern peak was an independent risk factor for biopsy-proven acute rejection (BPAR) and/or borderline change (P = 0.014). Patients having flat pattern peaks showed significantly lower post-transplant 36-month estimated glomerular filtration rate (P = 0.001). Two single nucleotide variants in ABCB1 genes, rs1922242 and rs2235035, were associated with flat pattern peaks (P = 0.019 and P = 0.027, respectively). CONCLUSION: Both of C1.5 and C0 should be measured to distinguish the patients showing unique initial absorption. A C1.5/C0 ratio lower than 1.5 was associated with an increased risk of BPAR and/or borderline change. Single nucleotide variants s in ABCB1 gene might influence the flat pattern peaks of tacrolimus absorption.

Homozygote CRIM1 variant is associated with thiopurine-induced neutropenia in leukemic patients with both wildtype NUDT15 and TPMT.

BACKGROUND: NUDT15 and TPMT variants are strong genetic determinants of thiopurine-induced hematological toxicity that results in therapeutic failure in pediatric acute lymphoblastic leukemia (ALL). However, many patients with both wild-type (WT) NUDT15 and TPMT still suffer from thiopurine toxicity and therapeutic failure. METHODS: Whole-exome sequencing was done for discovery (N = 244) and replication (N = 76) cohorts. Age- and sex-adjusted multiple regression analyses of both WT patients were performed to identify (p < 0.01, N = 188 for discovery) and validate (p < 0.05, N = 52 for replication) candidate variants for the tolerated last-cycle 6-mercaptopurine (6-MP) dose intensity percentage (DIP). Both independent and additive effects of the candidate variants on well-known NUDT15 and TPMT were evaluated by multigene prediction models. RESULTS: Among the 12 candidate variants from the discovery phase, the rs3821169 variant of the gene encoding Cysteine-Rich Transmembrane BMP Regulator 1 (CRIM1) was successfully replicated (p < 0.05). It showed high interethnic variability with an impressively high allele frequency in East Asians (T = 0.255) compared to Africans (0.001), Americans (0.02), Europeans (0.009), and South Asians (0.05). Homozygote carriers of the CRIM1 rs3821169 variant (N = 12, 5%) showed significantly lower last-cycle 6-MP DIPs in the discovery, replication, and combined cohorts (p = 0.025, 0.013, and 0.001, respectively). The traditional two-gene model (NUDT15 and TPMT) for predicting 6-MP DIP < 25% was outperformed by the three-gene model that included CRIM1, in terms of the area under the receiver operating characteristic curve (0.734 vs. 0.665), prediction accuracy (0.759 vs. 0.756), sensitivity (0.636 vs. 0.523), positive predictive value (0.315 vs. 0.288), and negative predictive value (0.931 vs. 0.913). CONCLUSIONS: The CRIM1 rs3821169 variant is suggested to be an independent and/or additive genetic determinant of thiopurine toxicity beyond NUDT15 and TPMT in pediatric ALL.

Germline Mutations for Novel Candidate Predisposition Genes in Sporadic Schwannomatosis.

BACKGROUND: Schwannomatosis is a late-onset tumor predisposition syndrome associated with the development of many different types of malignancies. A relevant genetic mechanism can be explained by three mutational events. The first-hit mutation is a germline mutation, and the SMARCB1 mutation on chromosome 22 is the most well-known genetic abnormality in patients with schwannomatosis. LZTR1 is another major predisposing gene in 22q-related schwannomatosis that lacks SMARCB1 variants. Although these two variants account for the occurrence of most familiar schwannomatoses, the genetic causes of sporadic schwannomatosis for the most part remain unknown. Therefore, current molecular diagnostic criteria cannot completely explain the basis of this disease. The common genetic background between schwannomatosis and other related malignant tumors is also unclear. Moreover, it is not easy to explain various clinical manifestations by only two known mutations. QUESTION/PURPOSES: (1) Are there important sequences outside the SMARCB1 or LZTR1 region on chromosome 22 that might carry a first-hit mutational predisposition to sporadic schwannomatosis? Or are there alternative evolutionarily conserved loci that might carry a first-hit mutational predisposition? (2) Is the age of disease onset associated to such genetic variants? METHODS: This study was a retrospective chart review and prospective genetic study on patients with schwannomatosis who were treated surgically. The clinical criteria to diagnose schwannomatosis were as follows: (1) histologically proven nonvestibular schwannomas; (2) no evidence of vestibular schwannomas on 3-mm brain MRI. A total of 21 patients were treated between March 2006 and June 2015. Since nine patients did not visit the outpatient clinic during the recruitment period, we obtained blood samples from 12 patients with schwannomatosis for a genetic analysis. After two patients were excluded because of their family history of schwannomatosis, genetic analyses were finally performed on 10 patients. Then, those with NF2, SMARCB1 or LZTR1 variants were screened by whole exome sequencing. All 10 patients passed our screening strategy. There were eight men and two women, with a median (range) age of 43 years (24 to 66) at the time of diagnosis. To select candidate genes, common ethnic variants and frequent mutations in in-house exome sequencing data were removed to exclude the population-specific polymorphisms not found in other population and to generalize the findings. Frameshift, nonsense, and splice-site variants were deemed pathogenic. Missense variants were classified as potentially pathogenic, variants of uncertain significance, or benign using in silico (via computer simulation) prediction algorithms, Sorting Intolerant From Tolerant (SIFT), Polymorphism Phenotyping v2 (PolyPhen-2), and Combined Annotation Dependent Depletion (CADD). A variant was considered potentially pathogenic if two or more algorithms predicted the variant to be damaging and benign if none considered it damaging. Then, potentially pathogenic variants only in the genes associated with cancer-predisposition or DNA damage repair were classified as the pathogenic candidate variants of sporadic schwannomatosis. The predictions for pathogenic candidate variants were checked again on Clinical Interpretation of Genetic Variants (InterVar) based on the American College of Medical Genetics guidelines and validated against Mendelian clinically applicable pathogenicity scores (M-CAP scores). RESULTS: We detected 26 variants; 13 variants across 10 genes were predicted to be pathogenic and found in seven patients, two each in ARID1A, PTCH2, and NOTCH2 and one each in MSH6, ALPK2, MGMT, NOTCH1, CIC, TSC2, and CDKN2A. One frameshift deletion in PTCH2 met the criteria for pathogenic or likely pathogenic classification, as recommended by the American College of Medical Genetics guidelines. Six missense mutations were classified as possibly pathogenic variants based on M-CAP scores. Four predicted pathogenic missense variants were detected in DNA damage repair (DDR) genes. Three DDR genes were affected: ARID1A, MGMT, and MSH6. Among the nine predicted pathogenic mutations detected in known cancer-predisposing genes, one was a frameshift deletion and the others were missense mutations. Seven tumor suppressor genes were involved: PTCH2, ALPK2, CIC, NOTCH1, NOTCH2, TSC2, and CDKN2A. One patient with multiple pathogenic variants in two DDR genes, ARID1A and MSH6, received a schwannomatosis diagnosis at 33 years old. Each of the other patients who had single variants in the DDR gene received their diagnoses at 41 years of age. The age at diagnosis was 40 years or older in patients with variants in cancer-predisposing genes, except for one patient who had multiple variants in TSC2 and CDKN2A. The carrier of those variants received the diagnosis at 24 years old. CONCLUSIONS: This study identified first-hit candidate mutations predisposing patients to schwannomatosis that were not related to SMARCB1 or LZTR1 variations in a cohort of patients with sporadic schwannomatosis. Patients with sporadic schwannomatosis without SMARCB1 or LZTR1 genetic variation may have developed the disease because of genomic variants related to cancer initiation in areas other than chromosome 22. Seven of 10 patients had predicted pathogenic germline mutations in DDR and cancer predisposition genes. We detected multiple cancer-related mutations in each patient. The age at the time schwannomatosis was diagnosed might be associated with a combination of variants and characteristics of the genes containing the variants; however, we did not have enough patients to confirm this association. CLINICAL RELEVANCE: The germline mutations identified in this study and the ideas related to the age of disease onset may provide potential candidate variants for future research on sporadic schwannomatosis and help to revise the current clinical and molecular diagnostic criteria. Further in vivo and in vitro studies are needed for these variants.

Identifying genetic variants underlying medication-induced osteonecrosis of the jaw in cancer and osteoporosis: a case control study.

BACKGROUND: Bisphosphonate-induced osteonecrosis of the jaw (BRONJ) presents with a typical pattern of jaw necrosis in patients who have been prescribed bisphosphonates (BPs) and other antiangiogenetic drugs to treat osteoporosis or bone-related complications of cancer. METHODS: This study divided 38 patients with BRONJ into two groups according to the prescribing causes: cancer (n = 13) and osteoporosis (n = 25), and underwent whole exome sequencing and compared them with normal controls (n = 90). To identify candidate genes and variants, we conducted three analyses: a traditional genetic model, gene-wise variant score burden, and rare-variant analysis methods. RESULTS: The stop-gain mutation (rs117889746) of the PZP gene in the BRONJ cancer group was significantly identified in the additive trend model analysis. In the cancer group, ARIDS, HEBP1, LTBP1, and PLVAP were identified as candidate genes. In the osteoporosis group, VEGFA, DFFA, and FAM193A genes showed a significant association. No significant genes were identified in the rare-variant analysis pipeline. Biologically accountable functions related to BRONJ occurrence-angiogenesis-related signaling (VEGFA and PLVAP genes), TGF-ß signaling (LTBP1 and PZP genes), heme toxicity (HEBP1) and osteoblast maturation (ARIDS)-were shown in candidate genes. CONCLUSION: This study showed that the candidate causative genes contributing to the development of BRONJ differ according to the BP dose and background disease.

Whole-exome sequencing identifies mutations of KIF22 in spondyloepimetaphyseal dysplasia with joint laxity, leptodactylic type.

Spondyloepimetaphyseal dysplasia with joint laxity (SEMDJL), leptodactylic (lepto-SEMDJL) or Hall type, is an autosomal-dominant skeletal dysplasia manifesting with short stature, joint laxity with dislocation(s), limb malalignment, and spinal deformity. Its causative gene mutation has not yet been discovered. We captured and sequenced the exomes of eight affected individuals in six unrelated kindreds (three individuals in a family and five simplex individuals). Five novel sequence variants in KIF22, which encodes a member of the kinesin-like protein family, were identified in seven individuals. Sanger sequencing of KIF22 confirmed that c.443C>T (p.Pro148Ser) cosegregated with the phenotype in the affected individuals in the family; c.442C>T (p.Pro148Leu) or c.446G>A (p.Arg149Gln) was present in four of five simplex individuals, but was absent in unaffected individuals in their family and 505 normal cohorts. KIF22 mRNA was detected in human bone, cartilage, joint capsule, ligament, skin, and primary cultured chondrocytes. In silico analysis of KIF22 protein structure indicates that Pro148 and Arg149 are important in maintaining hydrogen bonds in the ATP binding and motor domains of KIF22. We conclude that these mutations in KIF22 cause lepto-SEMDJL.

Genome-wide DNA methylation profiles according to Chlamydophila psittaci infection and the response to doxycycline treatment in ocular adnexal lymphoma.

PURPOSE: To compare genome-wide DNA methylation profiles according to Chlamydophila psittaci (Cp) infection status and the response to doxycycline treatment in Korean patients with ocular adnexal extranodal marginal zone B-cell lymphoma (EMZL). METHODS: Twelve ocular adnexal EMZL cases were classified into two groups (six Cp-positive cases and six Cp-negative cases). Among the 12 cases, eight were treated with doxycycline as first-line therapy, and they were divided into two groups according to their response to the treatment (four doxy-responders and four doxy-nonresponders). The differences in the DNA methylation states of 27,578 methylation sites in 14,000 genes were evaluated using Illumina bead assay technology. We also validated the top-ranking differentially methylated genes (DMGs) with bisulfite direct sequencing or pyrosequencing. RESULTS: The Infinium methylation chip assay revealed 180 DMGs in the Cp-positive group (74 hypermethylated genes and 106 hypomethylated genes) compared to the Cp-negative group. Among the 180 DMGs, DUSP22, which had two significantly hypomethylated loci, was validated, and the correlation was significant for one CpG site (Spearman coefficient=0.6478, p=0.0262). Regarding the response to doxycycline treatment, a total of 778 DMGs were revealed (389 hypermethylated genes and 336 hypomethylated genes in the doxy-responder group). In a subsequent replication study for representative hypomethylated (IRAK1) and hypermethylated (CXCL6) genes, the correlation between the bead chip analysis and pyrosequencing was significant (Spearman coefficient=0.8961 and 0.7619, respectively, p<0.05). CONCLUSIONS: Ocular adnexal EMZL showed distinct methylation patterns according to Cp infection and the response to doxycycline treatment in this genome-wide methylation study. Among the candidate genes, DUSP22 has a methylation status that was likely attributable to Cp infection. Our data also suggest that the methylation statuses of IRAK1 and CXCL6 may reflect the response to doxycycline treatment.

Clinical consequences in truncating mutations in exon 34 of NOTCH2: report of six patients with Hajdu-Cheney syndrome and a patient with serpentine fibula polycystic kidney syndrome.

It is debatable whether Hajdu-Cheney syndrome (HCS) and serpentine fibula-polycystic kidney syndrome (SFPKS) represent a single clinical entity with a variable degree of expression or two different entities, because both disorders share common clinical and radiological manifestations, including similar craniofacial characteristics, and defective bone mineralization. Since it was shown that heterozygous truncating mutations in NOTCH2 are responsible for both HCS and SFPKS, 37 patients with HCS and four patients with SFPKS are reported. To elucidate the clinical consequences of NOTCH2 mutations, we present detailed clinical information for seven patients with truncating mutations in exon 34 of NOTCH2, six with HCS and one with SFPKS. In addition, we review all the reported patients whose clinical manifestations are available. We found 13 manifestations including craniofacial features, acroosteolysis, Wormian bones, and osteoporosis in >75% of NOTCH2-positive patients. Acroosteolysis was observed in two patients with SFPKS and bowing fibulae were found in two patients with HCS. These clinical and molecular data would support the notion that HCS and SFPKS are a single disorder.

Comprehensive in vitro and in silico assessments of metabolic capabilities of 24 genomic variants of CYP2C19 using two different substrates.

Introduction: Most hepatically cleared drugs are metabolized by cytochromes P450 (CYPs), and Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines provide curated clinical references for CYPs to apply individual genome data for optimized drug therapy. However, incorporating novel pharmacogenetic variants into guidelines takes considerable time. Methods: We comprehensively assessed the drug metabolizing capabilities of CYP2C19 variants discovered through population sequencing of two substrates, S-mephenytoin and omeprazole. Results: Based on established functional assays, 75% (18/24) of the variants not yet described in Pharmacogene Variation (PharmVar) had significantly altered drug metabolizing capabilities. Of them, seven variants with inappreciable protein expression were evaluated as protein damaging by all three in silico prediction algorithms, Sorting intolerant from tolerant (SIFT), Polymorphism Phenotyping v2 (PolyPhen-2), and Combined annotation dependent depletion (CADD). The five variants with decreased metabolic capability (<50%) of wild type for either substrates were evaluated as protein damaging by all three in silico prediction algorithms, except CADD exact score of NM_000769.4:c.593T>C that was 19.68 (<20.0). In the crystal structure of the five polymorphic proteins, each altered residue of all those proteins was observed to affect the key structures of drug binding specificity. We also identified polymorphic proteins indicating different tendencies of metabolic capability between the two substrates (5/24). Discussion: Therefore, we propose a methodology that combines in silico prediction algorithms and functional assays on polymorphic CYPs with multiple substrates to evaluate the changes in the metabolism of all possible genomic variants in CYP genes. The approach would reinforce existing guidelines and provide information for prescribing appropriate medicines for individual patients.

Heterogeneous genetic landscape of congenital neutropenia in Korean patients revealed by whole exome sequencing: genetic, phenotypic and histologic correlations.

Congenital neutropenia (CN) is a hematological disease heterogeneous in its genetic, phenotypic and histologic aspects. We aimed to identify the genetic etiology of Korean CN patients in the context of bone marrow (BM) histology and clinical phenotype. Whole-exome sequencing (WES) or targeted sequencing was performed on the BM or peripheral blood specimens of 16 patients diagnosed with CN based on BM exam from 2009 to 2018. Absolute count of myeloperoxidase (MPO)-positive cells was calculated using ImageJ software. Semi-quantitation of MPO-positive cells in BM sections was performed by MPO grading (grades 0-3). Comprehensive retrospective review on real-world data of 345 pediatric patients with neutropenia including 16 patients in this study during the same period was performed. Seven disease-causing variants were identified in ELANE, G6PC3 and CXCR4 in 7 patients. A novel homozygous G6PC3 variant (K72fs) of which the mechanism was copy-neutral loss of heterozygosity was detected in two brothers. A low myeloid-to-erythroid ratio (0.5-1.5) was consistently observed in patients with ELANE mutations, while MPO-positive cells (40%-50%) with MPO grade 1 or 2 were detected in myelokathexis caused by G6PC3 and CXCR4 mutations. Meanwhile, disease-causing variants were detected in ELANE, TAZ and SLC37A4 in 5 patients by retrospective review of medical records. Our results suggest that following the immunological study and BM exam, WES or an expanded next generation sequencing panel that covers genes related to immunodeficiency and other inherited bone marrow failures as well as CN is recommended for neutropenia patient diagnosis.

Development and Validation of Targeted Gene Sequencing Panel Based Companion Diagnostic for Korean Patients with Solid Tumors.

Recently, several panels using two representative targeting methods have been developed but they do not reflect racial specificity, especially for Asians. We have developed and analytically validated the Korean Pan-cancer Companion Diagnostic (CDX) Panel to apply targeted anticancer drugs to Korean patients based on the molecular characteristics of tumors using tumor samples without matched patient normal samples. The panel included 31 genes with reported single nucleotide variants, 9 genes with reported copy number variations, and 15 genes with predictive responses to targeted drugs under clinical testing, enabling the panel to be analyzed for the targets of 30 targeted anticancer drugs. It is cost-effective and optimized for cancer type-specific therapy in Korean cancer patients across solid cancer types while minimizing the limitations of existing approaches. In addition, the optimized filtering protocol for somatic variants from tumor-only samples enables researchers to use this panel without matched normal samples. To verify the panel, 241 frozen tumor tissues and 71 formalin-fixed paraffin-embedded (FFPE) samples from several institutes were registered. This gene screening method is expected to reduce test turnaround time and cost, making it a balanced approach to investigate solid cancer-related gene regions.

Identifying genetic variants associated with ritodrine-induced pulmonary edema.

INTRODUCTION: Ritodrine is one of the most commonly used tocolytics in preterm labor, acting as a ß2-adrenergic agonist that reduces intracellular calcium levels and prevents myometrial activation. Ritodrine infusion can result in serious maternal complications, and pulmonary edema is a particular concern among these. The cause of pulmonary edema following ritodrine treatment is multifactorial; however, the contributing genetic factors remain poorly understood. This study investigates the genetic variants associated with ritodrine-induced pulmonary edema. METHODS: In this case-control study, 16 patients who developed pulmonary edema during ritodrine infusion [case], and 16 pregnant women who were treated with ritodrine and did not develop pulmonary edema [control] were included. The control pregnant women were selected after matching for plurality and gestational age at the time of tocolytic use. Maternal blood was collected during admission for tocolytic treatment, and whole exome sequencing was performed with the stored blood samples. RESULTS: Gene-wise variant burden (GVB) analysis resulted in a total of 71 candidate genes by comparing the cumulative effects of multiple coding variants for 19729 protein-coding genes between the patients with pulmonary edema and the matched controls. Subsequent data analysis selected only the statistically significant and deleterious variants compatible with ritodrine-induced pulmonary edema. Two final candidate variants in CPT2 and ADRA1A were confirmed by Sanger sequencing. CONCLUSIONS: We identified new potential variants in genes that play a role in cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) regulation, which supports their putative involvement in the predisposition to ritodrine-induced pulmonary edema in pregnant women.

Personal genome testing on physicians improves attitudes on pharmacogenomic approaches.

In this era of clinical genomics, the accumulation of knowledge of pharmacogenomics (PGx) is rising dramatically and attempts to utilize it in clinical practice are also increasing. However, this advanced knowledge and information have not yet been sufficiently utilized in the clinical field due to various barriers including physician factors. This study was conducted to evaluate the attitudes of physicians to PGx services by providing them their own genomic data analysis report focusing on PGx. We also tried to evaluate the clinical applicability of whole exome sequencing (WES)-based functional PGx test. In total 88 physicians participated in the study from September 2015 to August 2016. Physicians who agreed to participate in the study were asked to complete a pre-test survey evaluating their knowledge of and attitude toward clinical genomics including PGx. Only those who completed the pre-test survey proceeded to WES and were provided with a personal PGx analysis report in an offline group meeting. Physicians who received these PGx reports were asked to complete a follow-up survey within two weeks. We then analyzed changes in their knowledge and attitude after reviewing their own PGx analysis results through differences in their pre-test and post-test survey responses. In total, 70 physicians (79.5%) completed the pre-test and post-test surveys and attended an off-line seminar to review their personal PGx reports. After physicians reviewed the report, their perception of and attitude towards the PGx domain and genomics significantly changed. Physician' awareness of the likelihood of occurrence of adverse drug reactions and genetic contribution was also changed significantly. Overall, physicians were very positive about the value and potential of the PGx test but maintained a conservative stance on its actual clinical use. Results revealed that physicians' perception and attitude to the utility of PGx testing was significantly changed after reviewing their own WES results.

Star Allele-Based Haplotyping versus Gene-Wise Variant Burden Scoring for Predicting 6-Mercaptopurine Intolerance in Pediatric Acute Lymphoblastic Leukemia Patients.

Nudix Hydrolase 15 (NUDT15) and Thiopurine S-Methyltransferase (TPMT) are strong genetic determinants of thiopurine toxicity in pediatric acute lymphoblastic leukemia (ALL) patients. Since patients with NUDT15 or TPMT deficiency suffer severe adverse drug reactions, star (*) allele-based haplotypes have been used to predict an optimal 6-mercaptopurine (6-MP) dosing. However, star allele haplotyping suffers from insufficient, inconsistent, and even conflicting designations with uncertain and/or unknown functional alleles. Gene-wise variant burden (GVB) scoring enables us to utilize next-generation sequencing (NGS) data to predict 6-MP intolerance in children with ALL. Whole exome sequencing was performed for 244 pediatric ALL patients under 6-MP treatments. We assigned star alleles with PharmGKB haplotype set translational table. GVB for NUDT15 and TPMT was computed by aggregating in silico deleteriousness scores of multiple coding variants for each gene. Poor last-cycle dose intensity percent (DIP < 25%) was considered as 6-MP intolerance, resulting therapeutic failure of ALL. DIPs showed significant differences ( p < 0.05) among NUDT15 poor (PM, n = 1), intermediate (IM, n = 48), and normal (NM, n = 195) metabolizers. TPMT exhibited no PM and only seven IMs. GVB showed significant differences among the different haplotype groups of both NUDT15 and TPMT ( p < 0.05). Kruskal-Wallis test for DIP values showed statistical significances for the seven different GVB score bins of NUDT15. GVB NUDT15 outperformed the star allele-based haplotypes in predicting patients with reduced last-cycle DIPs at all DIP threshold levels (i.e., 5%, 10%, 15%, and 25%). In NUDT15-and-TPMT combined interaction analyses, GVB NUDT15 , TPMT outperformed star alleles [area under the receiver operating curve (AUROC) = 0.677 vs. 0.645] in specificity (0.813 vs. 0.796), sensitivity (0.526 vs. 0.474), and positive (0.192 vs. 0.164) and negative (0.953 vs. 0.947) predictive values. Overall, GVB correctly classified five more patients (i.e., one into below and four into above 25% DIP groups) than did star allele haplotypes. GVB analysis demonstrated that 6-MP intolerance in pediatric ALL can be reliably predicted by aggregating NGS-based common, rare, and novel variants together without hampering the predictive power of the conventional haplotype analysis.

Discovery of donor genotype associated with long-term survival of patients with hematopoietic stem cell transplantation in refractory acute myeloid leukemia.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been the only treatment option for acute myeloid leukemia (AML) refractory to induction chemotherapy, with only 10-20% of patients achieving long-term survival. Certain donor genotypes may confer leukemia-clearing effects after allo-HSCT. We performed whole-exome sequencing of five pairs of the germ lines in AML patients who achieved long-term remission after allo-HSCT and in their donors, and found two significant variants: EGFR c.2982C > T and CDH11 c.945G > A. To validate the protective effects of these leukemia-clearing genotypes (LCGs), AML patients who received allo-HSCT in a complete-remission status were also analyzed. Twenty-two of 96 donors (22.9%) had LCGs in their genomes, and overall survival was significantly longer in patients who received allo-HSCT from donors with germ-line LCGs (hazard ratio=0.47, 95% confidence interval=0.24-0.94, p = .033). These findings indicate that donor germ-line LCGs have phenotypically leukemia-clearing effects and are biomarkers for predicting clinical outcomes in allogeneic transplantation in AML patients.

APEX1 Polymorphism and Mercaptopurine-Related Early Onset Neutropenia in Pediatric Acute Lymphoblastic Leukemia.

PURPOSE: Mercaptopurine (MP) is one of the main chemotherapeutics for acute lymphoblastic leukemia (ALL). To improve treatment outcomes, constant MP dose titration is essential to maintain steady drug exposure, while minimizing myelosuppression. We performed two-stage analyses to identify genetic determinants of MP-related neutropenia in Korean pediatric ALL patients. MATERIALS AND METHODS: Targeted sequencing of 40 patients who exhibited definite MP intolerance was conducted using a novel panel of 211 pharmacogenetic-related genes, and subsequent analysis was performed with 185 patients. RESULTS: Using bioinformatics tools and genetic data, four functionally interesting variants were selected (ABCC4, APEX1, CYP1A1, and CYP4F2). Including four variants, 23 variants in 12 genes potentially linked to MP adverse reactions were selected as final candidates for subsequent analysis in 185 patients. Ultimately, a variant allele in APEX1 rs2307486was found to be strongly associated with MP-induced neutropenia that occurred within 28 days of initiating MP (odds ratio, 3.44; p=0.02). Moreover, the cumulative incidence of MP-related neutropenia was significantly higher in patients with APEX1 rs2307486 variants, as GG genotypes were associated with the highest cumulative incidence (p < 0.01). NUDT15 rs116855232 variants were strongly associated with a higher cumulative incidence of neutropenia (p < 0.01), and a lower median dose of tolerated MP throughout maintenance treatment (p < 0.01). CONCLUSION: We have identified that APEX1 rs2307486 variants conferred an increased risk of MP-related early onset neutropenia. APEX1 and NUDT15 both contribute to cell protection from DNA damage or misincorporation, so alleles that impair the function of either gene may affect MP sensitivities, thereby inducing MP-related neutropenia.

Deleterious genetic variants in ciliopathy genes increase risk of ritodrine-induced cardiac and pulmonary side effects.

BACKGROUND: Ritodrine is a commonly used tocolytic to prevent preterm labour. However, it can cause unexpected serious adverse reactions, such as pulmonary oedema, pulmonary congestion, and tachycardia. It is unknown whether such adverse reactions are associated with pharmacogenomic variants in patients. METHODS: Whole-exome sequencing of 13 subjects with serious ritodrine-induced cardiac and pulmonary side-effects was performed to identify causal genes and variants. The deleterious impact of nonsynonymous substitutions for all genes was computed and compared between cases (n = 13) and controls (n = 30). The significant genes were annotated with Gene Ontology (GO), and the associated disease terms were categorised into four functional classes for functional enrichment tests. To assess the impact of distributed rare variants in cases with side effects, we carried out rare variant association tests with a minor allele frequency ≤ 1% using the burden test, the sequence Kernel association test (SKAT), and optimised SKAT. RESULTS: We identified 28 genes that showed significantly lower gene-wise deleteriousness scores in cases than in controls. Three of the identified genes-CYP1A1, CYP8B1, and SERPINA7-are pharmacokinetic genes. The significantly identified genes were categorized into four functional classes: ion binding, ATP binding, Ca2+-related, and ciliopathies-related. These four classes were significantly enriched with ciliary genes according to SYSCILIA Gold Standard genes (P < 0.01), thus representing ciliary genes. Furthermore, SKAT showed a marginal trend toward significance after Bonferroni correction with Joubert Syndrome ciliopathy genes (P = 0.05). With respect to the pharmacokinetic genes, rs1048943 (CYP1A1) and rs1804495 (SERPINA7) showed a significantly higher frequency in cases than controls, as determined by Fisher's exact test (P < 0.05 and P < 0.01, respectively). CONCLUSIONS: Ritodrine-induced cardiac and pulmonary side effects may be associated with deleterious genetic variants in ciliary and pharmacokinetic genes.

Evaluation of exome variants using the Ion Proton Platform to sequence error-prone regions.

The Ion Proton sequencer from Thermo Fisher accurately determines sequence variants from target regions with a rapid turnaround time at a low cost. However, misleading variant-calling errors can occur. We performed a systematic evaluation and manual curation of read-level alignments for the 675 ultrarare variants reported by the Ion Proton sequencer from 27 whole-exome sequencing data but that are not present in either the 1000 Genomes Project and the Exome Aggregation Consortium. We classified positive variant calls into 393 highly likely false positives, 126 likely false positives, and 156 likely true positives, which comprised 58.2%, 18.7%, and 23.1% of the variants, respectively. We identified four distinct error patterns of variant calling that may be bioinformatically corrected when using different strategies: simplicity region, SNV cluster, peripheral sequence read, and base inversion. Local de novo assembly successfully corrected 201 (38.7%) of the 519 highly likely or likely false positives. We also demonstrate that the two sequencing kits from Thermo Fisher (the Ion PI Sequencing 200 kit V3 and the Ion PI Hi-Q kit) exhibit different error profiles across different error types. A refined calling algorithm with better polymerase may improve the performance of the Ion Proton sequencing platform.

A unique phenotype of 2q24.3-2q32.1 duplication: early infantile epileptic encephalopathy without mesomelic dysplasia.

The voltage-gated sodium channel genes and HOXD genes are clustered on chromosome 2q, and duplication of this region is associated with 2 clinical phenotypes: early-onset epilepsy and mesomelic dysplasia Kantaputra type, respectively. We report a case involving 2q24.3-2q32.1 duplication encompassing both the voltage-gated sodium channel and HOXD gene clusters, which were detected by a comparative genomic hybridization array. The associated clinical features were early-infantile-onset epilepsy, hypoplastic left heart syndrome, and global developmental delay. However, no features of mesomelic dysplasia were found. A fluorescent in situ hybridization study showed that the noncontiguous insertion of the duplicated chromosome 2q segment into chromosome 6q was inherited from the father, who has a balanced insertional translocation. The unique genotype-phenotype correlation in the present case suggests that dosage-sensitive effects might apply only to the voltage-gated sodium channel genes.

An interstitial, apparently-balanced chromosomal insertion in the etiology of Langer-Giedion syndrome in an Asian family.

Langer-Giedion syndrome (LGS; MIM 150230), also called trichorhinophalangeal syndrome type II (TRPS2), is a contiguous gene syndrome caused by a one-copy deletion in the chromosome 8q23-q24 region, spanning the genes TRPS1 and EXT1. We identified an LGS family with two affected and two unaffected siblings from unaffected parents. To investigate the etiology of recurrence of LGS in this family, array CGH was performed on all family members. We identified a 7.29 Mb interstitial deletion at chromosome region 8q23-q24 in the two affected siblings, but no such deletion in the unaffected family members. However, the mother and one of the two unaffected siblings carried a 1.29 Mb deletion at chromosome region 8q24.1, sharing the distal breakpoint with the larger deleted segment found in the affected siblings. Another unaffected sibling had a 6.0 Mb duplication, sharing the proximal breakpoint of the deletion in the affected siblings. Karyotypic and FISH analyses in the unaffected mother revealed an insertional translocation of 8q23-q24 genomic material into chromosome 13: 46,XX,ins(13;8)(q33;q23q24). This insertional translocation in the mother results in the recurrence of LGS in this family, highlighting the importance of submicroscopic rearrangements in the genetic counseling for LGS.