Grand Rounds: Neurofibromatosis type 1

Our Grand Rounds series highlights an interesting case with an in-depth look at Invitae’s technical and interpretative expertise. By involving referring clinicians, we showcase the collaborative efforts that are used to provide the most accurate and clinically meaningful genetic test reports.

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.
Panel ordered:

Invitae Multi-Cancer Panel

Past medical history

  • Clinical diagnosis of NF1 with:
    • optic glioma
    • pilocytic astrocytoma resected from corpus callosum
    • plexiform neurofibroma in left thoracolumbar paraspinal region
    • ADHD in childhood
  • Positive for familial BRCA1 pathogenic variant: c.2457delC
  • Multifocal colon cancer diagnosed at 24 years of age
  • Total abdominal colectomy with ileorectal anastomosis for polyposis and sigmoid colon cancer

Family history

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.

Invitae results

  • Known BRCA1 familial pathogenic variant: c.2475delC
  • De novo NF1 pathogenic variant: c.129_130ins568(p.Ile44GLyfs*27)
  • Variant of uncertain significance (VUS) in AXIN2: c.911G>T(p.SER304Ile)
BRCA1, exon 10, c.2457delC (p.Asp821Ilefs*25), heterozygous, pathogenic
This sequence change deletes 1 nucleotide from exon 10 of the BRCA1 mRNA (c.2457delC), causing a frameshift at codon 821. This creates a premature translational stop signal (p.Asp821Ilefs*25) and is expected to result in an absent or disrupted protein product.
Truncating mutations in BRCA1 are known to be pathogenic. This particular truncation has been reported in individuals affected with breast and ovarian cancer (PMID: 8807330, 23269703, 24504028, 26718727, 26681312, 24728189). This variant is also known as 2576delC, S819fs, and 819delC in the literature.
For these reasons, this variant has been classified as pathogenic.
NF1, exon 2, c.129_130ins568 (p.Ile44Glyfs*27), heterozygous, pathogenic
This sequence change inserts 568 nucleotides in exon 2 of the NF1 mRNA (c.129_130ins568), causing a frameshift at codon 44. This creates a premature translational stop signal (p.Ile44Glyfs*27) and is expected to result in an absent or disrupted protein product.
While this particular variant has not been reported in the literature, loss-of-function variants in NF1 are known to be pathogenic (PMID: 10712197, 23913538).
For these reasons, this variant has been classified as pathogenic.
AXIN2, exon 3, c.911G>T (p.Ser304Ile), heterozygous, uncertain significance
This sequence change replaces serine with isoleucine at codon 304 of the AXIN2 protein. The serine residue is highly conserved and there is a large physicochemical difference between serine and isoleucine.
This variant is not present in population databases (ExAC no frequency) and has not been reported in the literature in individuals with AXIN2-related disease.
Algorithms developed to predict the effect of missense changes on protein structure and function do not agree on the potential impact of this missense change (SIFT: "deleterious"; PolyPhen-2: "probably damaging"; Align-GVGD: "class C15").
In summary, this is a novel missense change with uncertain impact on protein function. It has been classified as a variant of uncertain significance (VUS).

Confirming the clinical diagnosis of NF1

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.).

Finding two pathogenic variants in proband

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).

Explanation of colon cancer etiology

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.

Variants of uncertain significance

To understand whether the variant of uncertain significance in AXIN2 was significant, we confirmed with the clinician that the patient does not have oligodontia, as AXIN2 gene variants may be associated with this disorder. We believe it is vital to communicate with providers to obtain the most accurate clinical information.

Implications for clinicians

Our case highlights several issues that are important for providing the most accurate genetic test results. Technologic advances in next-generation sequencing, including identification of complex genomic rearrangements, allow us to offer the most complete sequencing results. We need to be open to continuing diagnostic inquiries because a change in one gene may not provide all the answers, and communication between laboratories and clinicians provides the best way for us to elucidate the etiology of genetic disease.


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.


PMID: 8807330, 23269703, 24504028, 26718727, 26681312, 24728189, 10712197, 23913538, 27978560, 25195694.

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.

*Split-read analysis:
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.