July 2023 – Molecular Pathology

A 3-year-old boy with no clinical information is referred for FMR1 testing. Results of the repeat primed PCR revealed two alleles, both with normal repeat size (as shown below). The test was repeated, and the results were the same.

Figure 1

What further testing is recommended?

  • Cytogenetic studies
  • Alelle-specific PCR
  • Methylation PCR
  • Southern blot

The correct answer is ...

Cytogenetic studies.

FMR1 disorders include fragile X syndrome (FXS), fragile X-associated tremor/ataxia syndrome (FXTAS), and fragile X-associated primary ovarian insufficiency (FXPOI). FXS is nearly always characterized by developmental delay and intellectual disability along with a variety of behavioral issues. FXTAS is characterized by late-onset, progressive cerebellar ataxia and intention tremor followed by cognitive impairment. Age of onset is typically between 60 and 65 years. FXPOI is characterized by hypergonadotropic hypogonadism before 40 years of age.

FMR1 disorders are the result of CGG trinucleotide repeat expansion in the 5' UTR of FMR1 with abnormal gene methylation for most alleles with >200 repeats. FXS requires the presence of a full-mutation repeat size (>200 CGG repeats), while FXTAS or FXPOI is associated with a premutation-sized repeat (55-200 CGG repeats).

Polymerase chain reaction (PCR) is used to detect expanded CGG trinucleotide repeats of FMR1. Early PCR techniques failed to amplify full mutations. Currently, PCR techniques such as repeat primed PCR can be utilized to identify large FMR1 expansion mutations. Methylation status of a full mutation can be assessed by PCR-based methods independent of measuring the number of CGG repeats. Southern blot analysis detects all FMR1 alleles including normal, larger-sized premutations, and full mutations. In addition, it determines methylation status of the FMR1 promoter region.

In this case, repeat primed PCR has revealed this patient has two alleles with normal range repeats size. These results are not consistent with an expansion variant of the FMR1 gene. A diagnosis of FXS or other FMR1-related disorders is highly unlikely. No further testing is needed to interrogate the expansions in the FMR1 gene or methylation status. However, the results reveal two unique alleles of FMR1 (which is located on the X chromosome) in an individual reported to be male. As this may indicate a sex chromosome aneuploidy or duplication, cytogenetic studies are recommended.

Two possibilities to consider is that this individual is XX or XXY. In this case, microarray analysis at an outside institution confirmed that this individual is XXY. Klinefelter syndrome is the result of two or more X chromosomes in a phenotypic male. The clinical phenotype includes tall stature, small testes, gynecomastia, and azoospermia. Individuals may also have mild learning disability and psychosocial adjustment issues. Importantly, Klinefelter syndrome has an estimated prevalence of between 1:500 to 1:1000 males. Testing for FMR1 or other genes on the X chromosome may incidentally detect sex chromosome aneuploidy consistent with Klinefelter syndrome.

Further cytogenetic studies for this case were not received at our institution and this patient was lost to follow-up.


  1. Hunter JE, Berry-Kravis E, Hipp H, et al. FMR1 Disorders. 1998 Jun 16 [Updated 2019 Nov 21]. In: Adam MP, Everman DB, Mirzaa GM, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of
  2. Washington, Seattle; 1993-2022. Available from: https://www.ncbi.nlm.nih.gov/books/NBK1384/ 2. Los E, Ford GA. Klinefelter Syndrome. [Updated 2022 Jul 18]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK482314/

Sara Cook, M.D., Ph.D.

Fellow, Molecular Genetic Pathology
Mayo Clinic

Ande Rumilla, MD

Consultant, Laboratory Genetics and Genomics
Mayo Clinic
Assistant Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science

MCL Education

This post was developed by our Education and Technical Publications Team.