Clinical validation of a novel automated cell-free DNA screening assay for trisomies 21, 13, and 18 in maternal plasma.

OBJECTIVE: To evaluate clinical performance of a new automated cell-free (cf)DNA assay in maternal plasma screening for trisomies 21, 18, and 13, and to determine fetal sex. METHOD: Maternal plasma samples from 1200 singleton pregnancies were analyzed with a new non-sequencing cfDNA method, which is based on imaging and counting specific chromosome targets. Reference outcomes were determined by either cytogenetic testing, of amniotic fluid or chorionic villi, or clinical examination of neonates. RESULTS: The samples examined included 158 fetal aneuploidies. Sensitivity was 100% (112/112) for trisomy 21, 89% (32/36) for trisomy 18, and 100% (10/10) for trisomy 13. The respective specificities were 100%, 99.5%, and 99.9%. There were five first pass failures (0.4%), all in unaffected pregnancies. Sex classification was performed on 979 of the samples and 99.6% (975/979) provided a concordant result. CONCLUSION: The new automated cfDNA assay has high sensitivity and specificity for trisomies 21, 18, and 13 and accurate classification of fetal sex, while maintaining a low failure rate. The study demonstrated that cfDNA testing can be simplified and automated to reduce cost and thereby enabling wider population-based screening.

Development of the glenohumeral joint after subscapular release and open relocation in children with brachial plexus birth palsy: long-term results in 61 patients.

BACKGROUND: We present the long-term results of remodeling of the glenohumeral joint after open subscapularis elongation and relocation of the humeral head in patients with an internal rotation contracture and joint incongruity due to brachial plexus birth palsy. METHODS: In this before-and-after study, 61 patients who underwent open subscapularis elongation and reduction of the glenohumeral joint were evaluated with respect to joint remodeling, with a mean follow-up period of 10.2 years (range, 7-16 years). The mean age at operation was 3.2 years (range, 8 months to 15 years). Measurements of the percentage of the humeral head anterior to the midscapular line (PHHA), glenoid version, and diameter of the humeral head were recorded using magnetic resonance imaging, comparing the affected joints preoperatively vs. postoperatively (n = 31) and comparing the operated vs. unaffected sides postoperatively (n = 61). RESULTS: The mean increase in PHHA was 27.6 percentage points (95% confidence interval, 22.4-32.7 percentage points; P < .01), from 13.2% to 40.8%. The glenoid retroversion changed by 14.8° (95% confidence interval, 11.1°-18.4°; P < .01), from 25.4° to 10.6°, approaching a normal value. All patients, even those older than 5 years, showed a clear benefit from surgery. CONCLUSIONS: Our study confirms that open subscapularis lengthening with joint repositioning, up to the age of 5 years, gives consistent remodeling of incongruent shoulders with surprisingly small differences between the operated and unaffected shoulders at long-term follow-up. The findings indicate that open reduction is useful also in adolescents and challenges the notion that older children should be treated with derotational humeral osteotomy.

Structures contributing to the shoulder contracture in brachial plexus birth palsy. An intraoperative biomechanical study.

Internal rotation contracture of the shoulder is a common sequel of the brachial plexus birth palsy. The purpose of this study is to describe the surgical method used in our centre and to measure the effect of sequentially releasing several anatomical structures that have been ascribed as the cause of the contracture. Twenty-four consecutive patients were operated on with an open release. We documented the increase in passive external rotation after each surgical step. We found small gains in passive external rotation when performing coracoidectomy and division of the upper part of the subscapularis tendon; 4° (95% confidence interval [CI] 2°-6° p < 0.01) and 6° (95% CI 4°-8° p < 0.01), respectively. A substantial gain in external rotation occurred when dividing the entire subscapularis tendon, 43° (95% CI 38°-48°, p < 0.01). Our findings indicate that a clinically relevant surgical release of the contracture requires lengthening of the entire subscapularis musculo-tendinous unit.Level of evidence: IV.

Clinical Validation of Fetal cfDNA Analysis Using Rolling-Circle-Replication and Imaging Technology in Osaka (CRITO Study).

BACKGROUND: Noninvasive prenatal genetic testing (NIPT) has been adopted as the first choice for aneuploidy screening. The purposes of this study were to investigate the accuracy of Vanadis® NIPT (hereafter CRITO-NIPT) in order to gain a deeper insight into the reasons for discrepancies, as well as to discuss the role of fetal ultrasound. METHODS: Between 2019 and 2020, CRITO-NIPT was performed in 1218 cases of patients who underwent CVS or amniocentesis after a detailed fetal ultrasound exam and genetic counseling. The CRITO-NIPT results were compared with the genetic results. In cases of test discrepancies, the placentae were collected for detailed genetic research, and the pre-procedure fetal ultrasound findings were referred to. RESULTS: The positive predictive value of T21, T18, and T13 was 93.55%, 88.46%, and 100%, respectively. In 90% of the of false positive (FP) cases, the placentae were examined. In 75% of the CRITO FP-T21 cases, placental mosaicism, or a demised twin's T21, were confirmed. There were complicated mosaic cases, including tetrasomy 21/trisomy7 and monosomy 21/trisomy21 cases. In one of three no-call cases, an intermediate deletion of chromosome 13 was detected. CONCLUSIONS: The CRITO study investigated the mechanism of false positives, and the detailed mechanisms in mosaic and no-call cases. There have hitherto been no reports that have provided insight by partitioning the placenta to compare the NIPT and invasive test results, nor that have provided detailed ultrasound findings in the cases of discordant results, revealing the demonstrated importance of, and necessity for, detailed ultrasonography. This article describes the potential of rolling-circle replication as a powerful biosensing platform, as well as the importance of examining the fetus in detail with ultrasound. However, we should remember that the potential applications raise ethical and social concerns that go beyond aneuploidy and its methodology.

Imaging single DNA molecules for high precision NIPT.

Cell-free DNA analysis is becoming adopted for first line aneuploidy screening, however for most healthcare programs, cost and workflow complexity is limiting adoption of the test. We report a novel cost effective method, the Vanadis NIPT assay, designed for high precision digitally-enabled measurement of chromosomal aneuploidies in maternal plasma. Reducing NIPT assay complexity is achieved by using novel molecular probe technology that specifically label target chromosomes combined with a new readout format using a nanofilter to enrich single molecules for imaging and counting without DNA amplification, microarrays or sequencing. The primary objective of this study was to assess the Vanadis NIPT assay with respect to analytical precision and clinical feasibility. Analysis of reference DNA samples indicate that samples which are challenging to analyze with low fetal-fraction can be readily detected with a limit of detection determined at <2% fetal-fraction. In total of 286 clinical samples were analysed and 30 out of 30 pregnancies affected by trisomy 21 were classified correctly. This method has the potential to make cost effective NIPT more widely available with more women benefiting from superior detection and false positive rates.

DNA repair genes are selectively mutated in diffuse large B cell lymphomas.

DNA repair mechanisms are fundamental for B cell development, which relies on the somatic diversification of the immunoglobulin genes by V(D)J recombination, somatic hypermutation, and class switch recombination. Their failure is postulated to promote genomic instability and malignant transformation in B cells. By performing targeted sequencing of 73 key DNA repair genes in 29 B cell lymphoma samples, somatic and germline mutations were identified in various DNA repair pathways, mainly in diffuse large B cell lymphomas (DLBCLs). Mutations in mismatch repair genes (EXO1, MSH2, and MSH6) were associated with microsatellite instability, increased number of somatic insertions/deletions, and altered mutation signatures in tumors. Somatic mutations in nonhomologous end-joining (NHEJ) genes (DCLRE1C/ARTEMIS, PRKDC/DNA-PKcs, XRCC5/KU80, and XRCC6/KU70) were identified in four DLBCL tumors and cytogenetic analyses revealed that translocations involving the immunoglobulin-heavy chain locus occurred exclusively in NHEJ-mutated samples. The novel mutation targets, CHEK2 and PARP1, were further screened in expanded DLBCL cohorts, and somatic as well as novel and rare germline mutations were identified in 8 and 5% of analyzed tumors, respectively. By correlating defects in a subset of DNA damage response and repair genes with genomic instability events in tumors, we propose that these genes play a role in DLBCL lymphomagenesis.