February 17, 2016

Our scientists will be available during the following times to discuss their posters. If you miss these times, visit us at booth 419.

THURSDAY, MARCH 10, 10:30a-12:00p

291- John Thompson, PhD: Two exomes are better than one; Orthogonal NGS for Clinical Diagnostics
Whole exome/genome sequencing studies have revolutionized the diagnosis of genetic disorders. However, typical high-throughput sequencing strategies require Sanger confirmation to meet the exacting demands of clinical diagnostic sequencing. To address this gap, we tested orthogonal next-generation platforms that employ complementary target capture and sequencing chemistries to improve call accuracy at a genome scale. DNA is captured using bait-based hybridization followed by Illumina NextSeq sequencing. In parallel, DNA is captured using amplification-based selection followed by Ion Proton semiconductor sequencing. Applying this to NA12878, we found our dual NGS approach to be as accurate as Sanger for orthogonally confirmed variants (~95% of total). Overall variant sensitivity improves as each method covered thousands of coding exons missed by the other. We conclude that orthogonal NGS offers improvements in sensitivity and specificity of variant calling, greatly reducing time and expense of Sanger and enabling physicians and patients to act on information more quickly.

511- Natalie Vena, MS, CGC: Variant Reanalysis and Rates of Reclassification: One Laboratory’s Experience and Practical Considerations for Practice
Over the past decade, the amount of genetic knowledge accessible to the public has grown rapidly, as new data repositories and tools for analysis have been introduced. The abundance of information, together with advances in knowledge have drastically changed how laboratories interpret existing and forthcoming clinical genetic data. As this knowledge grows, the ability of a clinical genetic testing laboratory to maintain variant classifications with the most up-to-date evidence is challenged. As noted in many laboratory reports, variant classifications are based on currently available literature and database resources and are subject to change with the advancement of knowledge.  Although laboratories typically have variant reanalysis policies, these policies and practices vary among laboratories. The current American College of Medical Genetics and Genomics (ACMG) standards and guidelines recommend, “laboratories should provide clear policies on the reanalysis of data from genetic testing and whether additional charges for reanalysis may apply;” Nonetheless, specific industry standards do not exist. Many factors, including time and resources, can impact a laboratory’s policy. However, there is limited data available regarding rates of reclassification and the factors influencing these reanalysis policies.  In considering the lack of standards for variant reanalysis in the clinical laboratory setting, we sought to investigate the rates of reclassification and the impact this process has on resources, policies, and practice.

In this pilot study, a subset of variants previously identified in our laboratory that had not been reviewed within the past six months were randomly selected for reanalysis. Our laboratory’s current practice is to initiate a reanalysis of a variant when observed again in clinical practice if it had not been reviewed within the most recent six-months. The selected variants were then segregated into two groups: variants last classified prior to October 2014 (group 1) and variants last classified in October 2014 or later (group 2).  This distinction was made due to the implementation of new classification guidelines in October 2014. These variants were then reanalyzed and classified according to standard lab practices, which follow the current ACMG standards and guidelines for the interpretation of sequence variants (Richards et al. Genet in Med. 2015;17:405-423).  Rates of reclassification and the evidence used to support reclassification were compared between the two groups.

A total of 45 variants were selected for reanalysis: 29 in group 1 and 16 in group 2.  The overall rate of reclassification was 24.4% (11/45); however, when the two groups were compared, the reclassification rate was 37.9% (11/29) for group 1 and 0% (0/16) for group 2. When further analyzed to determine what triggered a reclassification of the variant, we found that new evidence, specifically the use of ExAC data, triggered a reclassification in approximately half of the instances (6/11), whereas the implementation of new classification guidelines triggered the reclassification in the other half (5/11). Of the variants that were reclassified, the majority of changes (9/11) resulted in a clarification of an uncertain result (e.g., likely benign to benign or likely pathogenic to pathogenic). 

The landscape and knowledge regarding human genetic variation is changing rapidly. Implementation of a robust reclassification and provider notification system in the clinical genetic laboratory is necessary to provide the best care for patients. Although the number of variants evaluated was small, the low reclassification rate of variants last reviewed post-October 2014 found in our study suggests that our laboratory’s standard of a 6-month interval for reanalysis may be too frequent. Given the time and resources consumed by the process of variant review, it may be advisable for laboratories to strongly consider the criteria necessary for initiation of variant review. These policies should consider meaningful changes to the knowledgebase and tools available, in addition to time elapsed. 

FRIDAY, MARCH 11, 10:30a-12p

182- Ann Seman, MS, CGC: Mosaicism for SRY Positive Y Chromosome in Karyotype and Chromosomal Microarray Analysis in a Female with Turner syndrome Presentation.
Mixed gonadal dysgenesis is a sexual differentiation disorder typically caused by a chromosomal abnormality, usually a form of 45,X/46,XY mosaicism.  A combination of testing methodologies is often necessary in order to elucidate the finding.  We report on a three-year-old female who was assessed as a newborn due to intrauterine growth restriction, coarctation of the aorta, congenital hypothyroidism, and hypoglycemia.  Due to suspicion for Turner syndrome, a karyotype was ordered and was resulted as 45,X[6]/46,X,+mar[16].  The karyotype was reported as indicative of Turner syndrome (TS), or a TS variant.  FISH analysis using X and Y probes led to the identification of the marker chromosome as idic(Y)(q11.2), which includes the gene SRY, the sex-determining factor for males.  As a result of the presence of Y chromosome material, the patient underwent gonadectomy due to the increased risk for gonadoblastoma.  Pathologic examination revealed fallopian tube and epididymis on the left, but no definitive gonadal tissue.  The right gonad showed dysgenesis consistent with ovotestis containing Sertoli-only tubules, streak ovarian stroma, epididymis, and fallopian tube with no germ cells present.  This pathology is consistent with mixed gonadal dysgenesis.  Now at the age of 3 the patient was referred for the Claritas Genomics custom SNP chromosomal microarray analysis (CMA) to further refine the breakpoints of the isodicentric Y.  The CMA was run against reference DNA from a female then a male.  When run with the female control, the CMA identified the entire loss of X and a gain of Yp11.31-q11.221; with the male control the CMA detected loss of Yq11.221-q11.23.  In most cases of 45,X/46,XY with SRY present, the phenotypic presentation is male, however, this patient is phenotypically female, most likely as a result of the mosaicism, although her presentation could also be due to a sequence change within SRY.  Sequencing of SRY has not been performed, so the possibility that the female phenotype is due to a pathogenic variant has not been excluded.  However, studies have shown this to be a rare event (Hersmus R et al. BMC Med Genet. 2012; 13:108).  Since it can simultaneously identify the chromosomal origin of and assign fairly precise breakpoints to marker chromosomes as well as confirm the presence of individual genes, CMA can be used in place of FISH following karyotype findings such as those present in this case.  The full story of this patient’s chromosome abnormality could not have been learned without the combination of analyses, but even with the utilization of this sophisticated testing, the patient’s atypical presentation would not have been predicted.