Data sharing to improve concordance in variant interpretation across laboratories: results from the Canadian Open Genetics Repository.

BACKGROUND: This study aimed to identify and resolve discordant variant interpretations across clinical molecular genetic laboratories through the Canadian Open Genetics Repository (COGR), an online collaborative effort for variant sharing and interpretation. METHODS: Laboratories uploaded variant data to the Franklin Genoox platform. Reports were issued to each laboratory, summarising variants where conflicting classifications with another laboratory were noted. Laboratories could then reassess variants to resolve discordances. Discordance was calculated using a five-tier model (pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), likely benign (LB), benign (B)), a three-tier model (LP/P are positive, VUS are inconclusive, LB/B are negative) and a two-tier model (LP/P are clinically actionable, VUS/LB/B are not). We compared the COGR classifications to automated classifications generated by Franklin. RESULTS: Twelve laboratories submitted classifications for 44 510 unique variants. 2419 variants (5.4%) were classified by two or more laboratories. From baseline to after reassessment, the number of discordant variants decreased from 833 (34.4% of variants reported by two or more laboratories) to 723 (29.9%) based on the five-tier model, 403 (16.7%) to 279 (11.5%) based on the three-tier model and 77 (3.2%) to 37 (1.5%) based on the two-tier model. Compared with the COGR classification, the automated Franklin classifications had 94.5% sensitivity and 96.6% specificity for identifying actionable (P or LP) variants. CONCLUSIONS: The COGR provides a standardised mechanism for laboratories to identify discordant variant interpretations and reduce discordance in genetic test result delivery. Such quality assurance programmes are important as genetic testing is implemented more widely in clinical care.

Alpha-1-antitrypsin molecular testing in Canada: A seven year, multi-centre comparison.

BACKGROUND: Laboratory confirmation of alpha-1-antitrypsin (A1AT) deficiency may be achieved by multiple methods. Here, we compare the relative comprehensiveness and efficiency of pathogenic variant (PV) detection of four different protocols utilized at different diagnostic centres in Canada. METHODS: Diagnostic results from 2011 to 2018 at clinical laboratories in British Columbia (BC), Alberta (AB), Ontario (ON), and Québec (QC) were reviewed. The four labs utilize the following protocols: BC-CGID (serum A1AT Concentration/Genotyping/Isoelectric focussing (IEF)/SERPINA1 DNA sequencing), AB-CID (serum A1AT Concentration/IEF/DNA sequencing), ON-CD (serum A1AT Concentration/DNA sequencing), and QC-G (Genotyping). As the respective catchment areas varied in size and ethnic composition, the comprehensiveness of PV detection was assessed by comparing the frequency of individual genotypes to the ZZ genotype, which is clearly identified by all protocols. RESULTS: Collectively 5399 index patients were tested identifying 396 ZZ genotypes. Serum A1AT concentration as a determinant of further testing efficiently identified PV. ON-CD had the highest detection rate for PV; genotypes with at least one PV, other than S, Z or F, were identified at 0.67/ZZ as compared to <0.2/ZZ (all others). However, ON-CD had the highest rates of undefined molecular variants (UMV) (0.16/ZZ) or likely benign variants (LBV) (0.08/ZZ), compared to all others (<0.12/ZZ and < 0.06/ZZ, respectively). The F variant was identified at 0.10/ZZ, only in the ON-CD and the AB-CID protocols. Collectively, MMalton was the next most common variant, identified as a compound heterozygous genotype at 0.04/ZZ, only in the ON-CD and BC-CGID protocols. CONCLUSION: Strategies which readily detect variants across the full coding sequence of SERPINA1 detect more PV as well as more UMV and LBV.

Data sharing as a national quality improvement program: reporting on BRCA1 and BRCA2 variant-interpretation comparisons through the Canadian Open Genetics Repository (COGR).

PurposeThe purpose of this study was to develop a national program for Canadian diagnostic laboratories to compare DNA-variant interpretations and resolve discordant-variant classifications using the BRCA1 and BRCA2 genes as a case study.MethodsBRCA1 and BRCA2 variant data were uploaded and shared through the Canadian Open Genetics Repository (COGR; http://www.opengenetics.ca). A total of 5,554 variant observations were submitted; classification differences were identified and comparison reports were sent to participating laboratories. Each site had the opportunity to reclassify variants. The data were analyzed before and after the comparison report process to track concordant- or discordant-variant classifications by three different models.ResultsVariant-discordance rates varied by classification model: 38.9% of variants were discordant when using a five-tier model, 26.7% with a three-tier model, and 5.0% with a two-tier model. After the comparison report process, the proportion of discordant variants dropped to 30.7% with the five-tier model, to 14.2% with the three-tier model, and to 0.9% using the two-tier model.ConclusionWe present a Canadian interinstitutional quality improvement program for DNA-variant interpretations. Sharing of variant knowledge by clinical diagnostic laboratories will allow clinicians and patients to make more informed decisions and lead to better patient outcomes.

Charcot-Marie-Tooth 1B caused by expansion of a familial myelin protein zero (MPZ) gene duplication.

Charcot-Marie-Tooth (CMT) disease is a group of hereditary disorders affecting the motor and sensory nerves of the peripheral nervous system. CMT patterns of inheritance include dominant, recessive, and X-linked disorders. Charcot-Marie-Tooth disease, type 1B (CMT1B, OMIM 118200) is an autosomal dominant neuropathy caused by mutations in myelin protein zero (MPZ, OMIM 159440), a structural protein of peripheral myelin. Most causative MPZ mutations are missense sequence variants; however, recent clinical reports have described cases of CMT1B caused by increased dosage of the MPZ gene, with over-expression of the MPZ protein suspected to be causative of the disorder. We report an unusual case of early onset de novo CMT1B, caused by amplification of a familial, apparently benign, MPZ duplication.

Non-syndromic language delay in a child with disruption in the Protocadherin11X/Y gene pair.

Protocadherin11 is located on both the X and Y chromosomes in Homo sapiens but only on the X chromosome in other hominid species. The pairing of PCDH11Y with PCDH11X arose following a duplicative 3.5 Mb translocation from the ancestral X chromosome to the Y chromosome several million years ago. The genes are highly expressed in fetal brain and spinal cord. The evolutionary consequence of this duplication has been proposed to include the sexual dimorphism of cerebral asymmetry and the hominid specific transition to the capacity for language. We report a case of a male child referred for genetic investigation of severe language delay. Microarray analysis indicated the presence of a 220 Kb intragenic deletion at Xq21.31 involving the PCDH11X gene. Fluorescence in situ hybridization using a BAC probe mapping to intron 2 of the Protocadherin11X/Y gene pair confirmed loss of the locus on both the X and Y chromosomes. The X chromosome deletion was maternally inherited, but the Y chromosome deletion was found to be a de novo occurrence in this child. This finding lends support to the hypothesis that the Protocadherin11X/Y gene plays a role in language development in humans and that rare copy number variation is a possible mechanism for communication disorders.

The detection of chromosome anomalies by QF-PCR and residual risks as compared to G-banded analysis.

OBJECTIVE: To determine the detection rate of clinically significant chromosome abnormalities using quantitative fluorescent polymerase chain reaction (QF-PCR) of fetal DNA in comparison with G-banded analysis of cultured amniotic fluid cells and determine the residual risk if QF-PCR were performed alone for low-risk cases. METHODS: Amniotic fluid samples were prospectively categorized based on the likelihood of the fetus having a chromosome anomaly. QF-PCR results were compared with the G-banded findings. The distribution of patients and the rates of clinically significant anomalies in each risk category were determined. RESULTS: A total of 4176 amniotic fluid samples were studied. Among these, 331 cases with abnormalities were detected by both methods and an additional 19 abnormal cases were detected by G-banding only. Five of those undetected by QF-PCR were considered clinically significant, four of which were referred due to an elevated a priori risk (>4%). If QF-PCR is performed in all cases and G-banding performed only in higher risk cases, the residual risk for a clinically significant chromosome abnormality will be as low as 0.083%. CONCLUSIONS: This study suggests that QF-PCR alone is appropriate for patients with uncomplicated pregnancies, who are referred solely for an increased risk of a common trisomy.

A novel complex mutation in MSH2 contributes to both Muir-Torre and Lynch Syndrome.

Mutations in mismatch repair genes lead to Lynch Syndrome, the most common form of inherited colorectal cancer. In this report, we describe a novel complex germline mutation c.[1601_1661+92dup; 1591_1611del] of the mismatch repair gene, MSH2. This mutation, which segregates with the disease phenotype, was discovered in a Lynch syndrome kindred that also shows a history of the Muir-Torre syndrome. Interestingly, several tumors from this family displayed microsatellite instability, a hallmark of Lynch syndrome tumors but no consistent, concomitant loss of MSH2 protein expression. In addition, a subset of tumors showed neither prototypical feature of microsatellite instability nor immunohistochemistry deficiency, highlighting the importance of a detailed molecular analysis of rare genetic alterations. This mutation and the atypical clinical manifestations observed underscore the genetic complexity underlying Lynch syndrome, and the importance of comprehensive molecular screening in the diagnosis and early detection of colorectal and other associated cancers.

Alternatively spliced, truncated human BRCA2 isoforms contain a novel coding exon.

The protein truncation test (PTT) employs in vitro transcription and translation of amplified cDNA and exonic gDNA to reveal truncating germ-line mutations. In a series of PTT analyses, abnormal splicing in the region encompassing exons 20-23 of BRCA2 was discovered in leucocytes from high-risk breast cancer patients. Although sequencing of the genomic DNA in this region failed to reveal a detectable mutation in these patients, cDNA obtained from this region of BRCA2 uncovered numerous alternative splice isoforms. PTT analysis and nested RT-PCR using RNA from leucocytes from healthy individuals, normal tissue and breast and ovarian cancer tumours demonstrated the presence of these alternatively spliced transcripts in all cases. The splice forms appeared to be more prominent in RNA from aged blood, suggesting that isoform expression was conditional. It is therefore important to distinguish naturally occurring alternative splicing from true splice defects due to mutations when interpreting PTT results.

Molecular characterization of an inherited ring (19) demonstrating ring opening.

Ring chromosomes arise following breakage in both chromosome arms and rejoining of the centric segment at the broken ends or by end-to-end fusion of the telomeres. The phenotype of ring carriers is unpredictable, and developmental abnormalities may occur even when the ring appears to be structurally balanced. This is believed to be due to mitotic instability from abnormal segregation and sister chromatid exchange in somatic cells. Although ring chromosomes usually arise as de novo events, transmittal from mosaic carriers to offspring sometimes occurs. In such cases, offspring with ring mosaicism in combination with a normal cell line remain unexplained. In this report, we used detailed molecular and cytogenetic analyses of a prenatally detected, inherited ring (19) to observe the behavior of the ring chromosome in culture, and to investigate the mechanism of inherited ring chromosome mosaicism.