Objective: We present a family with initial false negative commercial testing for Huntington Disease (HD) and subsequent whole genome sequencing (WGS) that revealed an expanded repeat in HTT consistent with HD diagnosis.

Background: Commercial genetic testing for HD commonly utilizes PCR-based assays sizing the HTT CAG repeat [1-2]. Discrepancies in CAG repeat lengths are found in 4% of patients and PCR methods remain fallible to genomic variants that cause issues with template amplification or primer annealing [2,3].  Errors in HTT testing, as illustrated in this case, lead to additional testing, delayed diagnosis, and stress.

Method: The proband presented at age 47 with six year history of progressive chorea after myocardial infarction. By age 49 he had significant psychiatric and cognitive decline. Family history included a paternal cousin with HD and uncle with bipolar disorder. MRI brain showed global cerebral atrophy, ventricular enlargement, and flattening of the bilateral caudate. Commercially available HD PCR testing detected apparently homozygous alleles with 17 CAG repeats. Labs otherwise showed elevated hemoglobin/hematocrit. Due to concern for a contributory hyper-viscosity syndrome, therapeutic phlebotomy was initiated, but with continued symptom progression.

The patient’s father had symptom onset a year after his son at age 72. He developed choreiform movements, joint pain, and difficulties with gait and concentration. MRI brain showed mild generalized atrophy with slight flattening of bilateral caudate. Commercial duo whole exome analysis returned negative.

Results: Due to the initial false negative genetic results, the proband underwent additional testing over the next two years for suspected HD-like syndromes, all unrevealing. Onset of choreiform movements in his father again raised concern for paternally inherited HD and prompted in-house WGS. Expansion Hunter testing revealed heterozygosity for an expanded CAG repeat in HTT (45/18 in proband, 42/18 in father) consistent with a diagnosis of HD. Pathologic expansion was confirmed using a PCR primer designed to avoid variants in the proband’s .bam alignment.

Conclusion: False negative genetic testing has significant impact for HD patients and families. Accurate diagnosis is essential and companies should provide confirmation of homozygous genotypes using alternative primers or orthogonal methods, and make the primer sequences used in their assay’s available upon request.

References: 1. Jama, M., Millson, A., Miller, C. E. & Lyon, E. Triplet Repeat Primed PCR Simplifies Testing for Huntington Disease. The Journal of Molecular Diagnostics 15, 255–262 (2013).
2. Quarrell, O. W. et al. Discrepancies in reporting the CAG repeat lengths for Huntington’s disease. Eur J Hum Genet 20, 20–26 (2012).
3. S, Y., A, F., D, F. & RJ, T. Polymorphisms in the CAG repeat–a source of error in Huntington disease DNA testing. Clin Genet 58, 469–472 (2000).
4. Andrew, S. E., Goldberg, Y. P., Theilmann, J., Zeisler, J. & Hayden, M. R. A CCG repeat polymorphism adjacent to the CAG repeat in the huntington disease gene: Implications for diagnostic accuracy and predictive testing. Hum Mol Genet 3, 65–67 (1994).

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