A modified Patte classification system for rotator cuff tendon retraction to predict reparability and tendon healing in arthroscopic rotator cuff repair.

PURPOSE: The purpose of this study was to propose a modified Patte classification system for tendon retraction, including the cut-off points for predicting reparability and rotator cuff healing after arthroscopic rotator cuff repair (ARCR) and assess its prediction accuracy and measurement reliability. METHODS: This retrospective study included 463 consecutive patients scheduled to undergo ARCR for full-thickness supraspinatus tears. Receiver operating characteristic (ROC) curve analysis was used to determine the cut-off points for predicting reparability and tendon healing. The modified Patte classification system, in which these cut-off points were combined with the original Patte classification, classified the tendon retraction as stages I-V. The prediction accuracy of reparability and tendon healing was assessed using the area under the curve (AUC). Measurement reliability was determined using Cohen's κ statistics. RESULTS: Of the 402 included patients, 32 rotator cuff tears were irreparable and 71 of the remaining 370 were diagnosed with healing failure. ROC analysis determined the cut-off point of reparability at the medial one-fifth and that of tendon healing at the medial one-third of the humeral head. The AUC of the modified Patte classification for predicting reparability and tendon healing was 0.897 (excellent) and 0.768 (acceptable), respectively. Intra-rater reliability was almost perfect (mean κ value: 0.875), and inter-rater reliability was substantial (0.797). CONCLUSION: Diagnostic performance of the modified Patte classification system was excellent for reparability and acceptable for rotator cuff healing, with high measurement reliability. The modified Patte classification system can be easily implemented in clinical practice for planning surgical procedures and counselling patients in the day-by-day clinical work. LEVEL OF EVIDENCE: Level III.

Repair Tension During Arthroscopic Rotator Cuff Repair is Correlated With Preoperative Tendon Retraction and Postoperative Rotator Cuff Integrity.

PURPOSE: This study aimed to examine the correlation of repair tension during arthroscopic rotator cuff repair (ARCR) with preoperative factors and to evaluate whether measuring tension during ARCR is effective for predicting rotator cuff integrity after ARCR. METHODS: Patients who underwent ARCR from May 2014 to June 2017 were enrolled in this study. Inclusion criteria were patients with medium or larger-sized tears and with a minimum of 6 months' follow-up. Patients with a partial repair were excluded. Intraoperative repair tension was measured according to Davidson's method. Correlation of repair tension with preoperative factors was evaluated with Pearson and Spearman correlation coefficient tests. Logistic regression analysis was performed on intraoperative factors, including repair tension, to identify independent predictors of retear after ARCR. Receiver operating characteristic (ROC) curve was used to determine the cutoff value of repair tension for retear. RESULTS: One-hundred twenty patients met the inclusion criteria. Mean repair tension was 26.6 ± 12.6 N, and retear was found in 29 shoulders (24.2%). Among the preoperative factors, tear size in the mediolateral (P < .001) and anteroposterior (P < .001) directions, DeOrio and Cofield's classification (P <0.001), geometric classification (P <.001), and fatty infiltration of supraspinatus (P = .006) and infraspinatus (P = .003) were significantly correlated with repair tension. However, multivariable logistic regression analysis identified only tear size in the mediolateral direction as an independent predictor of repair tension (P = .036). Logistic regression analysis showed that repair tension (P = .02) and geometric classification (P < .001) are significant factors affecting rotator cuff integrity after ARCR. ROC curve analysis showed the cutoff value of repair tension of large to massive tears for retear to be 35.6 N. CONCLUSION: This study demonstrated that intraoperative repair tension is strongly correlated with tear size in the mediolateral direction based on preoperative magnetic resonance imaging and that measuring tension during ARCR is effective for predicting rotator cuff integrity after ARCR. LEVEL OF EVIDENCE: Level IV, prognostic study.

Apalutamide for metastatic, castration-sensitive prostate cancer in the Japanese population: A subgroup analysis of the randomized, double-blind, placebo-controlled phase 3 TITAN study.

OBJECTIVE: To evaluate the efficacy and safety of apalutamide + androgen deprivation therapy versus androgen deprivation therapy alone in Japanese patients with metastatic castration-sensitive prostate cancer from the phase 3, randomized, global TITAN study. METHODS: Men with metastatic castration-sensitive prostate cancer randomly (1:1) received 240 mg apalutamide + androgen deprivation therapy or matching placebo + androgen deprivation therapy. The primary efficacy endpoints were radiographic progression-free survival and overall survival. Secondary efficacy endpoints were time to cytotoxic chemotherapy, pain progression, chronic opioid use, and skeletal-related events. Safety was also assessed. RESULTS: Of the 1052 patients included in the TITAN study, 51 (4.85%) were Japanese (apalutamide group, n = 28; placebo group, n = 23). In all, 81.8% of patients in the apalutamide and 71.8% in the placebo group did not experience radiographic progression or death, and the hazard ratio for radiographic progression-free survival favored treatment with apalutamide (hazard ratio 0.712, 95% confidence interval 0.205-2.466; P = 0.59). At 24 months, 85.7% of patients in the apalutamide group and 81.5% in the placebo group were alive, and the hazard ratio for overall survival favored apalutamide (hazard ratio 0.840, 95% confidence interval 0.210-3.361; P = 0.805). In the interim analysis, the median radiographic progression-free survival and overall survival were not reached in the apalutamide group and time to cytotoxic chemotherapy was delayed following apalutamide treatment. The safety profile of apalutamide in the Japanese subpopulation was comparable with that of the global population, except for skin rash. CONCLUSIONS: The results of the present analyses suggest that apalutamide + androgen deprivation therapy in Japanese patients had favorable efficacy compared with androgen deprivation therapy alone, and these findings are comparable to those in the overall population. Apalutamide + androgen deprivation therapy can be considered as one of the therapeutic options for a broad spectrum of metastatic castration-sensitive prostate cancer regardless of prior treatment and disease extent in Japanese patients.

Estimation of the number of contributors to mixed samples of DNA by mitochondrial DNA analyses using massively parallel sequencing.

We evaluated whether the number of contributors to mixed DNA samples can be estimated by analyzing the D-loop of mitochondrial DNA using massively parallel sequencing. The A- (positions 16,209-16,400) and B- (positions 30-284) amplicons in hypervariable regions 1 and 2, respectively, were sequenced using MiSeq with 2 × 251 cycles. Sequence extraction and trimming were performed using CLC Genomics Workbench 11 and the number of observed haplotypes was counted for each amplicon type using Microsoft Excel. The haplotype ratios were calculated by dividing the number of counted reads of the corresponding haplotype by the total number of sequence reads. Haplotypes that were over the threshold (5%) were defined as positive haplotypes. The number of larger positive haplotypes in either of the two amplicon types was defined as the number of contributors. Samples were collected from seven individuals. Seventeen mixed samples were prepared by mixing DNA from two to five contributors at various ratios. The number of contributors was correctly estimated from almost all of the mixed samples containing equal amounts of DNA from two to five people. In mixed samples of two or three people, the minor components were detected down to a ratio of 20:1 or 8:2:1. However, heteroplasmy, base deletions, and sharing of the same haplotypes caused incorrect estimations of the number of contributors. Although this method still has room for improvement, it may be useful for estimating the number of contributors in a mixed sample, as it does not rely on forensic mathematics.

Thrombosis of the draining vein causes intracranial haemorrhage in the natural history of brain arteriovenous malformation: case report.

Rupture of arteriovenous malformations (AVM) may be caused by venous outflow restriction, but there is no radiographic evidence of this. We report a case showing only a venous thrombus preceding intracranial haemorrhage from AVM. This is the first evidence that venous thrombus results in AVM rupture in its natural history.

Posterior cerebral artery giant aneurysm associated with bilateral internal carotid artery occlusion in a Klippel-Trenaunay syndrome patient: a case report.

We experienced an extremely rare case of a giant P1-P2 partially thrombosed aneurysm associated with bilateral ICA occlusion in a Klippel-Trenaunay syndrome patient. In our experience, direct surgical clipping via a pterional approach is generally favored for aneurysms located in the junction of the P1-P2 segments, even if they are giant.

Comparable clinical and structural outcomes after arthroscopic rotator cuff repair in diabetic and non-diabetic patients.

PURPOSE: To compare clinical outcome and rotator cuff integrity after arthroscopic rotator cuff repair (ARCR) in patients with and without diabetes mellitus. METHODS: This retrospective study involved 264 consecutive patients who underwent ARCR from 2012 to 2015. Inclusion criteria were a medium or large-sized tear and a minimum of 1-year follow-up. Clinical outcome measures included range of motion (ROM) and the Japanese Orthopaedic Association (JOA) and University of California, Los Angeles (UCLA) scores preoperatively and at final follow-up. Rotator cuff retear was evaluated with magnetic resonance imaging at 3 months post-surgery and final follow-up. Diabetic patients with poor control were pre-operatively hospitalized for intensive diabetic control. RESULTS: Our inclusion criteria were met by 30 diabetic patients and 126 non-diabetic patients. Demographic data were not significantly different between the groups, except body mass index (p = 0.021). Preoperative JOA and UCLA scores of the diabetic patients were significantly lower than those of the non-diabetic patients (p < 0.001, and p = 0.006, respectively); however, the scores at final follow-up were not different. ROM was significantly restricted in the diabetic patients before surgery (forward flexion, abduction, internal rotation: p < 0.001, external rotation: p = 0.035), but at the final follow-up, there was no significant difference except for internal rotation (p = 0.005). The retear rate in diabetic patients (23.3%) was not significantly different from that in non-diabetic patients (15.1%). CONCLUSIONS: Diabetic patients who had good perioperative glycemic control showed clinical and structural outcomes comparable to non-diabetic patients after ARCR. Intensive perioperative glycemic control and patient education are recommended for preoperative uncontrolled diabetic patients. LEVEL OF EVIDENCE: III.

Diagnostic Value of the Supine Napoleon Test for Subscapularis Tendon Lesions.

PURPOSE: The purpose of this study was to compare the diagnostic value of the supine Napoleon test (a variation of the belly-press test that reduces compensatory motion) with other clinical tests for subscapularis tendon (SSC) tears. METHODS: One hundred thirty consecutive patients who were scheduled for arthroscopic rotator cuff repair were evaluated preoperatively with the lift-off test, Napoleon test, bear-hug test, and supine Napoleon test. The supine Napoleon test was performed by placing the patient's hand on their belly while they lay supine with an examiner holding their hand and shoulder to prevent compensatory motion. The patient was asked to move their elbow upward and the test was considered positive if they were unable to do so. During surgery, SSC lesions were classified with the Lafosse classification system. Sensitivity, specificity, accuracy, and positive and negative likelihood ratios (LR+, LR-) were calculated for each test. The ability to detect partial SSC tears was compared among the clinical tests. RESULTS: Fifty-two of 130 patients (40%) had SSC tears confirmed arthroscopically. For diagnosis of these tears, the supine Napoleon test was the most sensitive (84%), followed by the bear-hug test (74%), and the bear-hug test was the most specific (97%), followed by the supine Napoleon test (96%). The LR+ was greatest for the bear-hug test (28.4) and next greatest for the supine Napoleon test (21.9). The LR- was lowest for the supine Napoleon test (0.16) and the bear-hug test was second (0.27). The sensitivity of the supine Napoleon test (65%) was highest for detecting partial tears. CONCLUSIONS: In comparison with other clinical tests for SSC tears, the supine Napoleon test had the greatest diagnostic value for full-thickness SSC tears and was most able to detect partial tears. LEVEL OF EVIDENCE: Level III, diagnostic nonrandomized study.

Effect of losartan on serum uric acid in hypertension treated with a diuretic: the COMFORT study.

It has been shown that losartan, an angiotensin II receptor blocker (ARB), reduces serum uric acid levels. However, the effects of losartan on serum uric acid levels in the patients treated with a thiazide diuretic have not been fully elucidated. We have investigated the effects of losartan compared with other ARBs on blood variables and blood pressure control in hypertensive patients treated with a thiazide diuretic using data from the COMFORT study. The present analysis included a total of 118 hypertensive subjects on combination treatment with ARBs except for losartan and a diuretic who were randomly assigned to a daily regimen of a combination pill (losartan 50 mg/hydrochlorothiazide 12.5 mg) or to continuation of two pills, an ARB except for losartan and a diuretic. Blood pressures were evaluated at 1, 3, and 6 months after randomization and changes in blood variables including serum uric acid were evaluated during 6 months treatment period. Mean follow-up blood pressure levels were not different between the combination pill (losartan treatment) group and the control (ARBs except for losartan) group. On the other hand, serum uric acid significantly decreased in the combination pill group compared with the control group (-0.44 versus + 0.10 mg/dl; p = 0.01), although hematocrit, serum creatinine, sodium and potassium were not different between the groups. These results suggest that the treatment regimen switched from a combination therapy of ARBs except for losartan and a diuretic to a combination pill (losartan/ hydrochlorothiazide) decreases serum uric acid without affecting blood pressure control.

Safety screw fixation technique in a case of coracoid base fracture with acromioclavicular dislocation and coracoid base cross-sectional size data from a computed axial tomography study.

Coracoid base fracture accompanied by acromioclavicular joint dislocation with intact coracoclavicular ligaments is a rare injury. Generally, an open reduction with screw fixation is the first treatment choice, as it protects the important structures around the coracoid process. This report presents a new technique of screw fixation for coracoid base fracture and provides anatomic information on cross-sectional size of the coracoid base obtained by computed tomography (CT). An axial image of the coracoid base was visualized over the neck of the scapula, and a guidewire was inserted into this circle under fluoroscopic guidance. The wire was inserted easily into the neck of scapula across the coracoid base fracture with imaging in only 1 plane. In addition, 25 measurements of the coracoid base were made in 25 subjects on axial CT images. Average length of the long and short axes at the thinnest part of the coracoid base was 13.9 ± 2.0 mm (range 10.6-17.0) and 10.5 ± 2.2 mm (6.6-15.1), respectively. This new screw fixation technique and measurement data on the coracoid base may be beneficial for safety screw fixation of coracoid base fracture.

Y chromosome haplogroup N in a Japanese population is classified into three subclades, and two DYS385 loci, a duplicated Y-STR, are duplicated again in subclade N-M1819.

DYS385 is one of the major Y chromosome short tandem repeats (Y-STRs) in forensic genetics and exists as 2 copies in the human Y chromosome palindrome P4 region. In this study, we found that some samples were estimated to have ≥ 4 copies of DYS385 in Y chromosome haplogroup N in a Japanese population. Y chromosome haplogroup N is distributed widely in eastern/central Asia, Siberia, and eastern/northern Europe, and is also observed in Japan; however, little is known about haplogroup N subclades in the Japanese population. To reveal the link between increased DYS385 copy number and haplogroup N subclades, we sequenced single nucleotide polymorphisms to classify the subclades. As a result, the Japanese Y chromosomes of haplogroup N were classified into three subclades, and an increased DYS385 copy number was specific to subclade N-M1819* (N1b2*). These results are of use in forensic DNA analysis because Y-STR copy number is important for the interpretation of Y-STR typing results of male DNA mixtures and kinship analysis. We also found that DYS458.1 microvariants (DYS458 intermediate alleles with single-nucleotide insertion) were observed only in subclade N-CTS962 (N1b1b∼) samples. Given that previous studies reported that DYS458.1 microvariants are observed in Y chromosomes of haplogroup N in other populations, DYS458.1 might be used to infer haplogroup N subclades without limitation to the Japanese population. The results of this study will be beneficial not only to forensic genetics but also to anthropological studies.

Apalutamide and Goserelin for Androgen Receptor-Positive Salivary Gland Carcinoma: A Phase 2 Nonrandomized Clinical Trial, YATAGARASU.

PURPOSE: To assess efficacy and safety of apalutamide plus goserelin for androgen receptor (AR)-positive, unresectable or recurrent/metastatic salivary gland carcinoma (URM-SGC). PATIENTS AND METHODS: This was an open-label, single-arm, multicenter phase II study for patients with AR-positive URM-SGC. The primary endpoint was the overall response rate (ORR) by an independent central radiology review (ICRR) in the first 24 response evaluable patients who had been observed at least 24 weeks from study initiation (primary RE patients). The efficacy was to be declared when at least 8 of the 24 primary RE patients responded. RESULTS: 31 patients were enrolled. In the first 24 primary RE patients with a median follow-up of 7.4 months, confirmed ORR by ICRR was 25.0% (6/24 patients; 95%CI: 9.8%-46.7%; P =0.11 (one-sided)), which did not meet the predefined criteria of efficacy. Clinical benefit rate (ORR + rate of stable disease for at least 24 weeks) and median progression-free survival were 50.0% and 7.4 months, respectively. Both median duration of response and overall survival were not reached. Exploratory analyses showed a better ORR of 54.5% (6/11) in patients with AR-positivity ≥ 70% and no history of prior systemic therapy. Grade 3 or higher treatment-emergent adverse events were reported in 35.5% (11/31), which included skin rash, anemia, leukopenia, and cancer pain. CONCLUSIONS: Although this study did not meet the predefined efficacy criteria, apalutamide plus goserelin showed clinically meaningful efficacy in a subset of patients with AR-positive SGC and safety consistent with prior experience in prostate cancer.

Effects of the superoxide dismutase mimetic tempol on impaired endothelium-dependent and endothelium-independent relaxations in type II diabetic rats.

Endothelium-derived hyperpolarizing factor (EDHF)-mediated hyperpolarization and relaxation, and endothelium-independent relaxations to the nitric oxide donor sodium nitroprusside and the adenosine 5'-triphosphate (ATP)-sensitive K(+)-channel opener levcromakalim were both impaired in mesenteric arteries of type II diabetic Goto-Kakizaki rats. The treatment with the superoxide dismutase mimetic tempol or its combination with the angiotensin II type 1 receptor blocker candesartan failed to improve EDHF-mediated responses, although both treatments partially improved endothelium-independent relaxations. These findings suggest that increased oxidative stress may in part account for the impaired endothelium-independent relaxations in diabetes, while it does not play a major role in the impaired EDHF-mediated responses.

Comparison of tunnel orientation between transtibial and anteromedial portal techniques for anatomic double-bundle anterior cruciate ligament reconstruction using 3-dimensional computed tomography.

PURPOSE: The aim of this study was to compare femoral and tibial tunnel placement, angle, and length between transtibial and anteromedial portal techniques for anatomic double-bundle anterior cruciate ligament (ACL) reconstruction. METHODS: Fifty patients were randomized to the 2 groups, and a femoral tunnel was created through the tibial tunnel (transtibial) and the far anteromedial portal (AMP) in 25 patients each. Both groups underwent anatomic double-bundle ACL reconstruction with hamstring tendons. Volume-rendering computed tomography (CT) was used to evaluate femoral and tibial tunnel placement, and transparent 3-dimensional CT image reconstruction, to evaluate tunnel angles, on the seventh postoperative day. Femoral tunnel length was measured intraoperatively. RESULTS: Anteromedial bundle (AMB) and posterolateral bundle (PLB) femoral tunnels were placed significantly lower and deeper with the AMP technique (shallow/deep direction: 21% and 30%, high/low direction: 18% and 48%) than with the transtibial technique (25% and 34%, 12% and 43%). Except for the tibial tunnel angle in the axial plane, AMB and PLB femoral and tibial tunnel angles differed significantly in 3 dimensions. AMB and PLB femoral tunnel lengths were significantly shorter with the AMP technique (AMB: 33 mm, PLB: 32 mm) than with the transtibial technique (AMB: 49 mm, PLB: 37 mm) (P < .001 and P = .001). Both femoral tunnel lengths in the AMP group correlated significantly with the tunnel angle in the sagittal (AMB: r = 0.69, PLB: r = 0.51) and axial (AMB: r = 0.58, PLB: r = 0.75) planes. CONCLUSIONS: AMB and PLB femoral tunnels were placed significantly deeper, lower, and closer to the femoral footprint reported in previous cadaveric studies in the anteromedial portal technique than in the transtibial technique. Femoral tunnel length was significantly shorter in the anteromedial portal group than in the transtibial group. LEVEL OF EVIDENCE: Level II, prospective comparative study.

Safety, reactogenicity, and immunogenicity of Ad26.COV2.S: Results of a phase 1, randomized, double-blind, placebo-controlled COVID-19 vaccine trial in Japan.

BACKGROUND: This study evaluated safety, reactogenicity, and immunogenicity of a 2-month homologous booster regimen of Ad26.COV2.S in Japanese adults. METHODS: In this multicenter, placebo-controlled, Phase 1 trial, adults (Cohort 1, aged 20-55 years, N = 125; Cohort 2, aged ≥ 65 years, N = 125) were randomized 2:2:1 to receive Ad26.COV2.S 5 × 1010 viral particles (vp), Ad26.COV2.S 1 × 1011 vp, or placebo, followed by a homologous booster 56 days later. Safety, reactogenicity, and immunogenicity were assessed. RESULTS: Two hundred participants received Ad26.COV2.S and 50 received placebo. The most frequent solicited local adverse event (AE) was vaccination-site pain, and the most frequent solicited systemic AEs were fatigue, myalgia, and headache. After primary vaccination, neutralizing and binding antibody levels increased through Day 57 (post-prime) in both cohorts. Fourteen days after boosting (Day 71), neutralizing antibody geometric mean titers (GMTs) had almost reached their peak value in Cohort 1 (5 × 1010 vp: GMT = 1049; 1 × 1011 vp: GMT = 1470) and peaked in Cohort 2 (504; 651); at Day 85, GMTs had declined minimally in Cohort 2. For both cohorts, binding antibody levels peaked at Day 71 with minimal decline at Day 85. CONCLUSION: A single dose and homologous Ad26.COV2.S booster increased antibody responses with an acceptable safety profile in Japanese adults (ClinicalTrials.gov Identifier: NCT04509947).

Does a combination pill of antihypertensive drugs improve medication adherence in Japanese? A randomized controlled trial.

BACKGROUND: In order to achieve target blood pressure levels to prevent cardiovascular disease, combination therapy of antihypertensive drugs is often required, although it is thought that requiring a patient to take many different pills would reduce adherence to the medication regimen. Whether antihypertensive treatment with a single pill combining antihypertensive drugs would improve medication adherence and blood pressure control was investigated. METHODS AND RESULTS: A total of 207 hypertensive subjects were randomly assigned to a combination pill group (losartan 50mg/hydrochlorothiazide 12.5mg; n=103) or a control group (an angiotensin receptor blocker and a thiazide diuretic; n=104). Medication adherence was evaluated by pill counts at 1, 3, and 6 months after randomization. The mean adherence rates over 6 months were not different between the 2 groups: 98% in the combination pill group and 98% in the control group. Moreover, the 2 groups included similar numbers of subjects with relatively poor adherence rates (<90%) in each treatment period. The mean blood pressures over the 6-month treatment period were not different between the groups: 131/75 mmHg in the combination pill group and 130/75 mmHg in the control group (P=0.84/0.96). CONCLUSIONS: There were no appreciable effects of the combination pill of antihypertensive drugs on medication adherence or blood pressure control in Japanese patients over a 6-month period.

Middle meningeal artery embolization before craniotomy for infected organizing chronic subdural hematoma: A case report and review of the literature.

Background: Organizing chronic subdural hematoma (OSDH) is intractable and its radical treatment remains controversial. Middle meningeal artery embolization has emerged as an adjunctive treatment to craniotomy for OSDH. Case Description: The patient is an 86-year-old man. He had been taking warfarin for atrial fibrillation and was referred to the department for the treatment of bilateral chronic subdural hematoma (CSDH), which was found on head computed tomography after a fall. Bilateral burr hole drainages were performed, but his hematomas were organized, so the hematomas could not be drained sufficiently. The patient was discharged from the hospital without any neurological symptoms. Two months later, the patient presented with persistent fever and headache and had recurrent bilateral CSDHs. The hematoma on the right side was larger. Based on the initial intraoperative findings, OSDH was suspected, and craniotomy was performed on the right hematoma. Propionibacterium acnes were detected in the hematoma culture, and antimicrobial therapy was started postoperatively. Since the right hematoma recurred on the 7th postoperative day, bilateral middle meningeal artery (MMA) embolization with 20% n-butyl-2-cyanoacrylate was performed, followed by craniotomy for the left hematoma and drainage for the right recurrent hematoma. Antimicrobials were administered for 2 weeks after the last operations. Six months after the operations, both bilateral hematomas had almost disappeared. Conclusion: Craniotomy is effective for the treatment of infected OSDH, and MMA embolization is useful to reduce the risk of bleeding complications in the perioperative period, and may also reduce the recurrence of CSDH.

Poly_NumtS_430 or HSA_NumtS_587 observed in massively parallel sequencing of the mitochondrial HV1 and HV2 regions.

An increasing number of studies on massively parallel sequencing of mitochondrial DNA (mtDNA) have been reporting identification of various types of noise or off-target sequences. Herein, we report that an off-target haplotype (sequence length 192 bp) observed in MiSeq data of mtDNA at nucleotide position 16,209-16,400 was likely caused by polymorphic nuclear mitochondrial DNA sequences (NumtS). Buccal DNA samples from Volunteers #001-004 and Control DNA 007 were amplified with our multiplex system of the B (15,998-16,172), C (16,209-16,400), and E (30-289) regions using 2000 copies of mtDNA. A sample index was added using a Nextera XT index kit, and MiSeq Reagent Nano Kit v2 was used in 2 × 251 cycles on a MiSeq FGx. FASTQ files were analyzed by CLC Genomics Workbench 21.0.3. The extracted SAM files were analyzed using our original Excel macro to sum up the read counts as the phased variant calls for each region. An off-target haplotype differing at 19 sites against the revised Cambridge reference sequence was observed in Volunteer #001 (4 in 10 MiSeq data), Volunteer #002 (2 in 9), and Control DNA 007 (6 in 9). In a BLAST search, the sequence of the off-target haplotype matched perfectly to three polymorphic NumtS (Poly_NumtS_430 [KM281528.1], HSA_NumtS_587 [HE613849.1], and nuclear fossil [S80333.1]) and BAC clone of chromosome 11 (AC107937.2). The sequence also matched perfectly to a Filipino mtDNA (KC993973.1) which was inferred as a chimeric sequence of mtDNA and the HSA_NumtS_587. The sequence of the off-target haplotype was not contained in the latest human reference genome sequence (GRCh38.p13). In a phylogenetic tree, the off-target haplotype was genetically distant from modern human mtDNA and not directly connected to them. In conclusion, observed off-target haplotype amplified by our multiplex system was likely caused by Poly_NumtS_430 or HSA_NumtS_587.

Development and validation of a SYBR green-based mitochondrial DNA quantification method by following the MIQE and other guidelines.

In forensic mitochondrial DNA (mtDNA) analysis, quantitative PCR (qPCR) is usually performed to obtain high-quality sequence data for subsequent Sanger or massively parallel sequencing. Unlike methods for nuclear DNA quantification using qPCR, a calibrator is necessary to obtain mtDNA concentrations (i.e., copies/µL). Herein, we developed and validated a mtDNA quantification method based on a SYBR Green assay by following MIQE [Bustin et al., Clin. Chem. 55 (2009) 611-22] and other guidelines. Primers were designed to amplify nucleotide positions 16,190-16,420 in hypervariable region 1 for qPCR using PowerUp SYBR Green and QuantStudio 5. The optimized conditions were 0.3 µM each primer and an annealing temperature of 60 °C under a 2-step cycling protocol. K562 DNA at 100 pg/µL was converted into a mtDNA concentration of 16,400 copies/µL using linearized plasmid DNA. This mtDNA calibrator was obtained by cloning the synthesized DNA fragments of mtDNA (positions 16,140-16,470) containing a 100-bp inversion. The linear dynamic range of the K562 standard curve was 10,000-0.1 pg/µL (r2 ≥ 0.999). The accuracy was examined using NIST SRM 2372a, and its components A, B, and C were quantified with differences of -29.4%, -35.0%, and -22.0%, respectively, against the mtDNA concentrations calculated from published NIST data. We also examined the specificity of the primers, stability of the reaction mix, precision, tolerance against PCR inhibitors, and cross-reactivity against DNA from various animal taxa. Our newly developed mtDNA quantification method is expected to be useful for forensic mtDNA analysis.

Frequencies of D19S433 silent alleles in a Japanese population of 1501 individuals and their effect on likelihood ratios calculated in kinship tests.

Although silent alleles in D19S433 typing using the GlobalFiler PCR Amplification Kit have been reported, the exact frequency of the D19S433 silent alleles in population data of 1501 Japanese individuals, which are widely used for the assessment of Japanese STR typing results, is unclear. In this study, we examined the exact D19S433 silent allele frequency in this population data. We newly observed the G32A variant causing silent alleles at D19S433 in five samples. Combining them with data including 30 samples with the variant reported previously, we determined that the total frequency of the silent alleles (i.e. the frequency of the G32A variant) in the 1501 Japanese samples was 0.0117 (35/3002). Using the D19S433 allele frequency data, we evaluated the effect of presence/absence information for the D19S433 silent allele on kinship tests. Likelihood ratios (LRs) were calculated for both simulated parent-child and full sibling cases, revealing that the LR may change by approximately 10-2 to 103 fold when the presence/absence of the D19S433 silent allele is revealed in a kinship test. Therefore, if a sufficiently large or small LR is obtained, there is little need to determine the presence/absence of the D19S433 silent allele in Japanese kinship tests using GlobalFiler. This study will be beneficial for the assessment of Japanese human identification and kinship test results using GlobalFiler.