Utility of Next-Generation Sequencing-Based Chimerism Analysis for Early Relapse Prediction following Allogenic Hematopoietic Cell Transplantation.

Chimerism monitoring following allogeneic hematopoietic cell transplantation (HCT) plays a pivotal role in evaluating engraftment status and identifying early indicators of relapse. Recent advancements in next-generation sequencing (NGS) technology have introduced AlloSeq HCT as a more sensitive alternative to short tandem repeat (STR) analysis. This study aimed to compare AlloSeq HCT with STR, focusing on the prediction of early relapse post-allogeneic HCT. Chimerism levels in 29 HCT recipients were assessed using both STR and NGS, employing a total of 125 whole blood or bone marrow aspirate samples (68 post-HCT and 57 pre-HCT samples from recipients or donors). AlloSeq HCT exhibited high concordance with STR and demonstrated the potential for early detection of chimeric changes, particularly at extremely low levels. The combined advantages of high sensitivity and automated data analysis offered by AlloSeq HCT substantiate its clinical adoption for effective chimerism monitoring.

Prevalence and founder effect of DRC1 exon 1-4 deletion in Korean patients with primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder affecting ciliary structure and function. PCD exhibiting dynein regulatory complex subunit 1 (DRC1) exon 1-4 deletion has been reported in several Japanese PCD patients; however, no large scale studies have been performed. Here, we aimed to determine the prevalence and founder effect of this variant in the Korean population. Using an in-house copy number variation tool, we screened for DRC1 exon 1-4 deletion in 20 patients with PCD and exome data of 1435 patients in the Seoul National University Hospital repository. In cases of suspected DRC1 deletion, confirmatory gap-PCR was performed. In a PCD cohort, three of 20 (15%) patients were positive for DRC1 exon 1-4 deletion (NM_145038.5(DRC1): c.1-3952_540 + 1331del27748-bp) while pathogenic variants were found in CCDC39 (N = 1), DNAAF6 (N = 1), DNAH9 (N = 1). In the 1,435-sample exome data, seven patients (0.49%) were confirmed to have DRC1 exon 1-4 deletion. A chimeric sequence including the junction was searched from the 1000 Genomes Project data repository. One Japanese patient (0.96%) was found to have the same DRC1 exon 1-4 deletion, which was absent in other populations. This study demonstrated that the DRC1 exon 1-4 deletion is a founder mutation based on haplotype analysis. In summary, the prevalence of PCD based on DRC1 exon 1-4 deletion is particularly high in Korean and Japanese populations, which is attributed to the founder effect. Genetic testing for DRC1 exon 1-4 deletion should be considered as an initial screening tool for Korean and Japanese patients with PCD.

Relationship between left atrial reservoir strain, volumes, and geometry: Insights from simple theoretical model.

BACKGROUND: Left atrial (LA) volume is related to LA reservoir strain (LASR ), but the relationship is not fully resolved. We sought to model the relationship between LA end-diastolic and end-systolic volumes (LAEDV and LAESV) and LASR based on a geometrical approach to exploit the relationship between LASR and volume. METHODS: Modeling the LA as a hemisphere with radius r, LASR was recognized to vary linearly with r and LA volume with r3 . Expanding this cubic relation as a Taylor series resulted in a simple linear equation: LAESV/LAEDV = 1 + 3 × LASR . To validate this, 52 transthoracic echocardiograms were analyzed from 18 patients who underwent transcatheter edge-to-edge repair (TEER) with MitraClip with serial assessment pre-procedure, 1 month post-clip, and 12 months post-TEER. Linear regression was performed to compare the geometric equation to a statistical model created by a line of best fit to relate LAESV/LAEDV to LASR . RESULTS: The statistical and geometric model both resulted in a strong correlation (r = .8, p < .001, respectively). The slope of the line in the statistical model was 3.3, which was statistically indistinguishable from the expected slope of 3 based on the geometric model (Figure 2A). Using the geometric model to compare the measured versus calculated LAESV/LAEDV also resulted in a strong correlation (r = .8, p < .001)(Figure 2B). CONCLUSION: We describe the relationship between LA volume and strain mathematically by considering the geometry of the LA. This model enhances our understanding of the interaction between atrial strain and volume. Further research is necessary to validate this using 3D atrial volumes in a broader cohort of subjects.

Valvular regurgitation flow jet assessment using in vitro 4D flow MRI: Implication for mitral regurgitation.

PURPOSE: The purpose of this study was to evaluate the accuracy of four-dimensional (4D) flow MRI for direct assessment of peak velocity, flow volume, and momentum of a mitral regurgitation (MR) flow jets using an in vitro pulsatile jet flow phantom. We systematically investigated the impact of spatial resolution and quantification location along the jet on flow quantities with Doppler ultrasound as a reference for peak velocity. METHODS: Four-dimensional flow MRI data of a pulsatile jet through a circular, elliptical, and 3D-printed patient-specific MR orifice model was acquired with varying spatial resolution (1.5-5 mm isotropic voxel). Flow rate and momentum of the jet were quantified at various axial distances (x = 0-50 mm) and integrated over time to calculate Voljet and MTIjet . In vivo assessment of Voljet and MTIjet was performed on 3 MR patients. RESULTS: Peak velocities were comparable to Doppler ultrasound (3% error, 1.5 mm voxel), but underestimated with decreasing spatial resolution (-40% error, 5 mm voxel). Voljet was similar to regurgitant volume (RVol) within 5 mm, and then increased linearly with the axial distance (19%/cm) because of flow entrainment. MTIjet remained steady throughout the jet (2%/cm) as theoretically predicted. Four and 9 voxels across the jet were required to measure flow volume and momentum-time-integral within 10% error, respectively. CONCLUSION: Four-dimensional flow MRI detected accurate peak velocity, flow rate, and momentum for in vitro MR-mimicking flow jets. Spatial resolution significantly impacted flow quantitation, which otherwise followed predictions of flow entrainment and momentum conservation. This study provides important preliminary information for accurate in vivo MR assessment using 4D flow MRI.

Consistent count region-copy number variation (CCR-CNV): an expandable and robust tool for clinical diagnosis of copy number variation at the exon level using next-generation sequencing data.

PURPOSE: Despite the importance of exonic copy number variations (CNVs) in human genetic diseases, reliable next-generation sequencing-based methods for detecting them are unavailable. We developed an expandable and robust exonic CNV detection tool called consistent count region (CCR)-CNV. METHODS: In total, about 1000 samples of the truth set were used for validating CCR-CNV. We compared CCR-CNV performance with 2 well-known CNV tools. Finally, to overcome the limitations of CCR-CNV, we devised a combined approach. RESULTS: The mean sensitivity and specificity of CCR-CNV alone were above 95%, which was superior to that of other CNV tools, such as DECoN and Atlas-CNV. However, low covered region and positive predictive value and high false discovery rate act as obstacles to its use in clinical settings. The combined approach showed much improved performance than CCR-CNV alone. CONCLUSION: In this study, we present a novel diagnostic tool that allows the identification of exonic CNVs with high confidence using various reagents and clinical next-generation sequencing platforms. We validated this method using the largest multiple ligation-dependent probe amplification-confirmed data set, including sufficient copy normal control data. The approach, combined with existing CNV tools, allows the implementation of CCR-CNV in clinical settings.

Parallel Analysis of Pre- and Postoperative Circulating Tumor DNA and Matched Tumor Tissues in Resectable Pancreatic Ductal Adenocarcinoma: A Prospective Cohort Study.

BACKGROUND: Circulating tumor DNA (ctDNA) is a promising biomarker for early tumor detection and minimal residual disease (MRD) assessment in early-stage cancer, but quantifying minute amounts of ctDNA is challenging and well-designed studies on ctDNA in early-stage cancer are still lacking. Here, we adapted a sensitive next-generation sequencing (NGS) technology and performed parallel analysis of pre- and postoperative ctDNA and matched tumor tissues in a prospective cohort of patients with resectable pancreatic ductal adenocarcinoma (PDAC). METHODS: In total, 70 consecutive patients undergoing curative resection for resectable PDAC were enrolled. We performed integrated digital error suppression-enhanced cancer personalized profiling by deep sequencing NGS of triple-matched samples (pre/postoperative plasma cell-free DNA [cfDNA], tumor tissue, and genomic DNA) targeting 77 genes. RESULTS: Preoperative ctDNA was detected in 37.7% of the evaluable patients, with a median variant allele frequency of 0.09%. Twelve additional oncogenic mutations were detected exclusively in preoperative ctDNA but not in tissue. When quantitative concentrations of ctDNA were estimated in haploid genome equivalents per milliliter (hGE/mL), the risk of early recurrence was high in patients with postoperative ctDNA >1 hGE/mL. cfDNA variants from 24.5% of patients had features compatible with clonal hematopoiesis. CONCLUSIONS: An optimized NGS approach might add value beyond tissue analysis through the highly sensitive detection of minute amounts of ctDNA in resectable PDAC. Postoperative ctDNA concentration could be a tool for MRD assessment. Moreover, parallel analyses of matched tissues and leukocytes might be required to accurately detect clinically relevant ctDNA.

Intronic LINE-1 insertion in SLCO1B3 as a highly prevalent cause of rotor syndrome in East Asian population.

Rotor syndrome is caused by digenic loss-of-function variants in SLCO1B1 and SLCO1B3 but only a few studies have reported co-occurring inactivating variants from both genes. A rotor syndrome-causing long interspersed element-1 (LINE-1) insertion in SLCO1B3 had been reported to be highly prevalent in the Japanese population but there has been no additional report. In spite of its known association with various human diseases, LINE-1 is hard to detect with current sequencing technologies. In this study, we aimed to devise a method to screen the LINE-1 insertion variant and investigate the frequency of this variant in various populations. A chimeric sequence, that was generated by concatenating the reference sequence at the junction and a part of inserted LINE-1 sequence, was searched from 725 raw sequencing data files. In cases containing the chimeric sequence, confirmatory long-range PCR and gap-PCR were performed. In total, 95 (13.1%) of 725 patients were positive for the chimeric sequence, and all were confirmed to have the SLCO1B3 LINE-1 insertion by PCR-based tests. The same chimeric sequence was searched from the 1000 Genomes Project data repository and the carrier frequency was remarkably high in the East Asian populations (10.1%), especially in Southern Han Chinese (18.5%), but almost absent in other populations. This SLCO1B3 LINE-1 insertion should be screened in a population-specific manner under suspicion of Rotor syndrome and the methods proposed in this study would enable this in a simple way.

Longitudinal proteomic profiling provides insights into host response and proteome dynamics in COVID-19 progression.

In managing patients with coronavirus disease 2019 (COVID-19), early identification of those at high risk and real-time monitoring of disease progression to severe COVID-19 is a major challenge. We aimed to identify potential early prognostic protein markers and to expand understanding of proteome dynamics during clinical progression of the disease. We performed in-depth proteome profiling on 137 sera, longitudinally collected from 25 patients with COVID-19 (non-severe patients, n = 13; patients who progressed to severe COVID-19, n = 12). We identified 11 potential biomarkers, including the novel markers IGLV3-19 and BNC2, as early potential prognostic indicators of severe COVID-19. These potential biomarkers are mainly involved in biological processes associated with humoral immune response, interferon signalling, acute phase response, lipid metabolism, and platelet degranulation. We further revealed that the longitudinal changes of 40 proteins persistently increased or decreased as the disease progressed to severe COVID-19. These 40 potential biomarkers could effectively reflect the clinical progression of the disease. Our findings provide some new insights into host response to SARS-CoV-2 infection, which are valuable for understanding of COVID-19 disease progression. This study also identified potential biomarkers that could be further validated, which may support better predicting and monitoring progression to severe COVID-19.

Evidence of Severe Acute Respiratory Syndrome Coronavirus 2 Reinfection After Recovery from Mild Coronavirus Disease 2019.

BACKGROUND: Positive results from real-time reverse-transcription polymerase chain reaction (rRT-PCR) in recovered patients raise concern that patients who recover from coronavirus disease 2019 (COVID-19) may be at risk of reinfection. Currently, however, evidence that supports reinfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has not been reported. METHODS: We conducted whole-genome sequencing of the viral RNA from clinical specimens at the initial infection and at the positive retest from 6 patients who recovered from COVID-19 and retested positive for SARS-CoV-2 via rRT-PCR after recovery. A total of 13 viral RNAs from the patients' respiratory specimens were consecutively obtained, which enabled us to characterize the difference in viral genomes between initial infection and positive retest. RESULTS: At the time of the positive retest, we were able to acquire a complete genome sequence from patient 1, a 21-year-old previously healthy woman. In this patient, through the phylogenetic analysis, we confirmed that the viral RNA of positive retest was clustered into a subgroup distinct from that of the initial infection, suggesting that there was a reinfection of SARS-CoV-2 with a subtype that was different from that of the primary strain. The spike protein D614G substitution that defines the clade "G" emerged in reinfection, while mutations that characterize the clade "V" (ie, nsp6 L37F and ORF3a G251V) were present at initial infection. CONCLUSIONS: Reinfection with a genetically distinct SARS-CoV-2 strain may occur in an immunocompetent patient shortly after recovery from mild COVID-19. SARS-CoV-2 infection may not confer immunity against a different SARS-CoV-2 strain.

Using 5D flow MRI to decode the effects of rhythm on left atrial 3D flow dynamics in patients with atrial fibrillation.

PURPOSE: This study used a 5D flow framework to explore the influence of arrhythmia on thrombogenic hemodynamic parameters in patients with atrial fibrillation (AF). METHODS: A fully self-gated, 3D radial, highly accelerated free-running 5D flow sequence with interleaved four-point velocity-encoding was acquired using an in vitro arrhythmic flow phantom and in 25 patients with a history of AF (68 ± 8 y, 6 female). Self-gating signals were used to calculate AF burden, bin data, and tag each k-space line with its RRLength . Data were binned as an RR-resolved dataset with four RR-interval bins (RR1-RR4, short-to-long) for compressed sensing reconstruction. AF burden was calculated as interquartile range of all intrascan RR-intervals divided by median RR-interval, and left atrial (LA) stasis as the percent of the cardiac cycle where the velocity was <0.1 m/s. RESULTS: In vitro results demonstrated successful recovery of RR-binned flow curves using RR-resolved 5D flow compared to a real-time PC reference standard. In vivo, 5D flow was acquired in 8:48 minutes. AF burden was significantly correlated with 5D flow-derived peak (PV) and mean (MV) velocity and stasis (|ρ| = 0.54-0.75, P < .001). Sensitivity analyses determined a threshold for low versus high AF burden at 9.7%. High burden patients had increased LA mean stasis (up to +42%, P < .01), and lower MV and PV (-30%, -40.6%, respectively, P < .01). RR4 deviated furthest from respiratory-resolved reconstruction (end-expiration) with increased mean stasis (7.6% ± 14.0%, P = .10) and decreased PV (-12.7 ± 14.2%, P = .09). CONCLUSIONS: RR-resolved 5D flow can capture temporal and RR-resolved 3D hemodynamics in <10 minutes and offers a novel approach to investigate arrhythmias.

Plasma vs. serum in circulating tumor DNA measurement: characterization by DNA fragment sizing and digital droplet polymerase chain reaction.

Background Choosing the specimen type is the first step of the pre-analytical process. Previous reports suggested plasma as the optimal specimen for circulating tumor DNA (ctDNA) analysis. However, head-to-head comparisons between plasma and serum using platforms with high analytical sensitivity, such as droplet digital polymerase chain reaction (ddPCR), are limited, and several recent studies have supported the clinical utility of serum-derived ctDNA. This study aimed to compare the DNA profiles isolated from plasma and serum, characterize the effects of the differences between specimens on ctDNA measurement, and determine the major contributors to these differences. Methods We isolated cell-free DNA (cfDNA) from 119 matched plasma/serum samples from cancer patients and analyzed the cfDNA profiles by DNA fragment sizing. We then assessed KRAS mutations in ctDNA from matched plasma/serum using ddPCR. Results The amount of large DNA fragments was increased in serum, whereas that of cfDNA fragments (<800 bp) was similar in both specimens. ctDNA was less frequently detected in serum, and the KRAS-mutated fraction in serum was significantly lower than that in plasma. The differences in ctDNA fractions between the two specimen types correlated well with the amount of large DNA fragments and white blood cell and neutrophil counts. Conclusions Our results provided detailed insights into the differences between plasma and serum using DNA fragment sizing and ddPCR, potentially contributing to ctDNA analysis standardization. Our study also suggested that using plasma minimizes the dilution of tumor-derived DNA and optimizes the sensitivity of ctDNA analysis. So, plasma should be the preferred specimen type.

A comparison of complete blood count reference intervals in healthy elderly vs. younger Korean adults: a large population study.

Background The use of laboratory reference intervals based on younger populations is of questionable validity in older populations. We established reference intervals for 16 complete blood count (CBC) parameters in healthy elderly Koreans aged ≥60 years and compared them to those of individuals aged 20-59 years. Methods Among 64,532 individuals (39,609 men and 24,923 women) aged ≥20 years who underwent medical checkups, 8151 healthy subjects (12.6%, 5270 men and 2881 women, including 675 and 511, respectively, who were ≥60 years of age) were enrolled based on stringent criteria including laboratory, imaging and endoscopy results; previous medical history; and medication history. CBC parameters were measured using an Advia2120i instrument. Results Overall, healthy individuals aged ≥60 years did not require separate reference intervals from those aged <60 years except for red cell distribution width (RDW) and mean corpuscular hemoglobin (MCH) in women. However, subjects aged ≥60 years still required sex-specific reference intervals for red blood cell count, hemoglobin, hematocrit, MCH, monocytes and eosinophils. Separate reference intervals were required for MCH, eosinophils and basophils for certain age subgroups of men aged ≥60 years, and for MCH and RDW in certain age subgroups of women aged ≥60 years, compared to counterparts <60 years of age. Conclusions Healthy elderly Koreans can use the same reference intervals as younger populations. Thus, abnormal CBC results may not necessarily be attributable to physiologic changes but possible underlying diseases that should be investigated.

Mean Platelet Volume and Platelet Distribution Width Indicate that Platelets Remain Small for Most of Their Lifespans in Patients with Essential Thrombocythemia.

BACKGROUND: Platelets are large when young and immature and shrink as they age. The mean platelet volume (MPV) and platelet distribution width (PDW) reflect the volume and distribution of platelets, respectively. We compared the MPVs and PDWs of patients with immune thrombocytopenic purpura (ITP) or essential thrombocythemia (ET) to those of healthy individuals to test whether these values can reflect the pathomechanisms of these diseases. METHODS: Platelet counts, MPVs, and PDWs were measured in 153 healthy individuals and in 20 and 34 patients with ITP and ET, respectively, using an XN-3000 instrument. RESULTS: The MPVs and PDWs were significantly higher in ITP patients than in healthy individuals or ET patients. The MPVs were significantly lower in ET patients than in both ITP patients and healthy individuals; however, the PDWs in ET patients were similar to those in healthy individuals. The MPVs and PDWs did not correlate with platelet count in ITP or ET patients. CONCLUSIONS: Our data suggested an increased production of young, large platelets and nondiscriminatory destruction of circulating platelets irrespective of their ages in ITP. Decreased MPVs and unelevated PDWs in ET suggest that clonal process-produced platelets remain small throughout their lifespans.

Reclassification of BRCA1 and BRCA2 variants of uncertain significance: a multifactorial analysis of multicentre prospective cohort.

BACKGROUND: BRCA1 and BRCA2 (BRCA1/2) variants classified ambiguously as variants of uncertain significance (VUS) are a major challenge for clinical genetic testing in breast cancer; their relevance to the cancer risk is unclear and the association with the response to specific BRCA1/2-targeted agents is uncertain. To minimise the proportion of VUS in BRCA1/2, we performed the multifactorial likelihood analysis and validated this method using an independent cohort of patients with breast cancer. METHODS: We used a data set of 2115 patients with breast cancer from the nationwide multicentre prospective Korean Hereditary Breast Cancer study. In total, 83 BRCA1/2 VUSs (BRCA1, n=26; BRCA2, n=57) were analysed. The multifactorial probability was estimated by combining the prior probability with the overall likelihood ratio derived from co-occurrence of each VUS with pathogenic variants, personal and family history, and tumour characteristics. The classification was compared with the interpretation according to the American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG/AMP) guidelines. An external validation was conducted using independent data set of 810 patients. RESULTS: We were able to redefine 38 VUSs (BRCA1, n=10; BRCA2, n=28). The revised classification was highly correlated with the ACMG/AMP guideline-based interpretation (BRCA1, p for trend=0.015; BRCA2, p=0.001). Our approach reduced the proportion of VUS from 19% (154/810) to 8.9% (72/810) in the retrospective validation data set. CONCLUSION: The classification in this study would minimise the 'uncertainty' in clinical interpretation, and this validated multifactorial model can be used for the reliable annotation of BRCA1/2 VUSs.

Coexistence of anti-ß2-glycoprotein I domain I and anti-phosphatidylserine/prothrombin antibodies suggests strong thrombotic risk.

BACKGROUND: Highly specific assays for measuring antiphospholipid antibodies (aPLs) are required for accurate assessment of thrombotic risk. aPLs against ß2-glycoprotein I domain I (anti-ß2GPIdI) and against prothrombin complexed with phosphatidylserine (anti-PS/PT) have been recently identified as being associated with a hypercoagulable state. This study evaluated the synergism between anti-ß2GPIdI and anti-PS/PT for predicting thrombotic events. METHODS: A total of 180 patients with clinical suspicion of hypercoagulability were evaluated. The plasma levels of lupus anticoagulant (LA) and antibodies against anticardiolipin (anti-CL) (IgG and IgM), ß2GPI (IgG and IgM), PS/PT (IgG and IgM), and ß2GPI dI (IgG) were measured. RESULTS: IgG anti-ß2GPIdI and LA were highly associated with thrombosis. Mean values and positivity rates of IgG anti-ß2GPI dI and IgG anti-PS/PT were significantly higher in the triple-positive group (LA+, IgG anti-CL+, IgG anti-ß2GPI+) than in the other groups. Interestingly, the thrombotic risk [odds ratio (OR) 24.400, 95% confidence interval (CI) 1.976-63.273, p<0.001] of the newly defined triple positive group (LA+, IgG anti-CL+, IgG anti-ß2GPIdI+; OR 11.182, 95% CI 1.976-63.273, p=0.006) was more than twice that of the triple-positive group (LA+, IgG anti-CL+, IgG anti-ß2GPI+). Double positivity for IgG anti-PS/PT and IgG anti-ß2GPI also indicated significant thrombotic risk (OR 7.467, 95% CI 2.350-23.729, p=0.001). Furthermore, the thrombotic risk associated with double positivity for IgG anti-PS/PT and IgG anti-ß2GPIdI was markedly elevated (OR 33.654, 95% CI 6.322-179.141, p<0.001). CONCLUSIONS: Our data suggest that simultaneous measurement of IgG anti-ß2GPIdI and IgG anti-PS/PT may improve clinical decision-making for aPL-positive patients.

Viral RNA in Blood as Indicator of Severe Outcome in Middle East Respiratory Syndrome Coronavirus Infection.

We evaluated the diagnostic and clinical usefulness of blood specimens to detect Middle East respiratory syndrome coronavirus infection in 21 patients from the 2015 outbreak in South Korea. Viral RNA was detected in blood from 33% of patients at initial diagnosis, and the detection preceded a worse clinical course.

Microevolution of Outbreak-Associated Middle East Respiratory Syndrome Coronavirus, South Korea, 2015.

During the 2015 Middle East respiratory syndrome coronavirus outbreak in South Korea, we sequenced full viral genomes of strains isolated from 4 patients early and late during infection. Patients represented at least 4 generations of transmission. We found no evidence of changes in the evolutionary rate and no reason to suspect adaptive changes in viral proteins.

Genotype-phenotype analysis of von Hippel-Lindau syndrome in Korean families: HIF-α binding site missense mutations elevate age-specific risk for CNS hemangioblastoma.

BACKGROUND: von Hippel-Lindau (VHL) disease is a rare hereditary tumor syndrome caused by VHL gene mutations that is characterized by heterogeneous phenotypes such as benign/malignant tumors of the central nervous system, retina, kidney, adrenal gland, and pancreas. The genotype-phenotype correlation has not been well characterized in the Korean population so far. Therefore, this study aimed to evaluate the VHL mutation spectrum and genotype-phenotype correlations in Korean VHL patients. METHODS: Thirteen unrelated subjects with VHL mutations were included. Direct sequencing and multiplex ligation-dependent probe amplification were performed. Consequently, the clinical manifestations and family histories of the subjects were evaluated. RESULTS: We identified 10 different VHL mutations. The c.160_161delAT frameshift mutation was novel. Missense mutations clustered in 2 domains (α domain in exon 1; ß domain in exon 3). The most frequently observed mutation was c.208G > A (p.Glu70Lys). Milder phenotypes were observed in subjects with de novo mutations. Age-specific risk for CNS hemangioblastoma was significantly higher in subjects carrying missense mutations within the HIF-α binding site (P < 0.05). CONCLUSIONS: This study provides insight into the genotype-phenotype correlation in that amino acid substitutions in the HIF-α binding site may predispose patients to age-related risks of CNS hemangioblastoma.