This case focuses on the benefits of confirming a clinical diagnosis with technologically advanced next-generation sequencing and bioinformatics. In addition, the case demonstrates the increasingly common identification of two pathogenic variants in the same patient and the unanswered questions we continually face in explaining disease etiology.
A patient was tested for confirmation of a clinical diagnosis of neurofibromatosis type 1 (NF1). He had been previously tested by two other laboratories in an attempt to find an explanation for his latest diagnosis of colon cancer, but the results were negative for NF1 variants.
Indication for testing
A 27-year-old Caucasian male was referred for genetic testing because of optic glioma and multifocal colon cancer diagnoses. Previous testing was negative for pathogenic changes in NF1 and positive for familial BRCA1 pathogenic variant.
Patient’s mother was diagnosed with breast cancer at 46 years of age and found to be positive for a BRCA1 pathogenic variant. Patient’s unaffected sister is BRCA1 positive as well. His maternal aunt had a colectomy at 55 years of age due to Crohn’s disease. Two maternal great-aunts also had breast cancer. Maternal great-uncle was diagnosed with bone cancer and leukemia. Maternal great-grandfather was diagnosed with bile duct cancer, and maternal cousin had stomach cancer diagnosed at 51 years of age.
There is no family history of NF1. Family is of Irish/German/Greek descent. There is no parental consanguinity.
In this case, the patient had two previous negative NF1 test results. At Invitae, our rigorous approach to deletion and duplication (del/dup) testing in every single gene and panel test led to the positive NF1 test result. Our next-generation sequencing approach to del/dup testing uses a customized, validated set of computer algorithms in conjunction with optimized biochemical methods (see our white paper for details).
We detected a 568-nucleotide insertion from chromosome 3 to the NF1 gene exon 2 on chromosome 17, which caused a frameshift and resulted in a stop signal and protein truncation. Critical to the detection of this event is an approach that we refer to as split-read analysis.* This method detects events with breakpoints in or near an exon, which are often missed by traditional approaches.
Across our testing, we find that deletions and duplications account for a significant percentage of positive results. For NF1 specifically, we find that deletions and duplications account for approximately 12% of positive results (Trudy et al.).
The proband has pathogenic variants in both NF1 and BRCA1. With the utilization of panel testing and whole exome sequencing, it is increasingly common to identify more than one pathogenic variant in an individual patient. In one cohort of over 2,000 individuals who underwent molecular diagnostic testing, 5% had more than one pathogenic variant identified (PMID: 27959697).
One of the main reasons for pursuing further genetic testing in this case was the onset of multifocal colon cancer at a young age. Confirming the NF1 clinical diagnosis and identifying the BRCA1 pathogenic variant in this patient does not completely explain the new onset of colon cancer, as this cancer type is not common in NF1 patients. Also, a recent study of patients with early-onset colorectal cancer, found that only 2% of individuals with BRCA1 pathogenic variants had colorectal cancer (PMID: 27978560). However, the Hereditary Breast and Ovarian Cancer study group reported that BRCA1 carriers under the age of 50 years had a fivefold increase in the risk of developing colorectal cancer (PMID: 25195694).
It is also of interest that AXIN2 variants have been identified in colorectal cancer tumors and in families with the oligodontia-colorectal cancer syndrome. AXIN2 may play a role in tumorigenesis but its identification in our patient remains of uncertain clinical significance (PMID: 25236910).
Is BRCA1 the reason for the colon cancer in this patient? Could we postulate that gene interaction is responsible in this case? In a time of rapid technologic advances and expanding knowledge, we continue to seek the answers to explaining genetic disease etiology.
We would like to thank Kate McReynolds, APRN, MSc, ANP-BC, AGN-BC, Genetic Nurse Practitioner, from Vanderbilt Hereditary Cancer Clinic, and her patient and his family for their participation. Also thank you to the Invitae team: Michael Kennemer, PhD, Karen Ouyang, PhD, FACMG, Paige Taylor, PhD, and Jackie Tahilani, MS, CGC.
Trudy FC et al. Tracing the dark matter: prevalence of copy number and structural variants across mendelian disorders. American College of Medical Genetics and Genomics Annual Meeting. March 2017; Phoenix, Arizona. Platform presentation.
We use hybridization capture with densely-tiled single-strand oligonucleotide baits. As a part of our standard finishing procedure, our analysis pipeline identifies and scores loci with excessive soft-clipped read alignments, which we call a “split-read” signal. The soft-clipped sequences, along with discordant mate pairs, are then re-aligned to assess support for either precise breakpoints of called copy-number changes or for otherwise undetected large insertions, deletions, or genomic rearrangements. A customized confirmation assay is used to identify the breakpoints and compare the sample to a negative control.