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XSense, Fragile X with Reflex

XSense, Fragile X with Reflex

Test Summary

XSense®, Fragile X with Reflex

  

Clinical Use

  • Identify fragile X syndrome (FXS) carriers

  • Determine an individual's risk of having a child with FXS

  • Diagnose FXS postnatally

Clinical Background

Fragile X syndrome is the most common inherited cause of developmental delay and intellectual disability, occurring in approximately 1 in 2,500 to 3,700 males and 1 in 7,000 females.1

Affected males usually have moderate to severe intellectual disability, learning disabilities, attention deficit hyperactivity disorder (ADHD), depression, and difficult peer relationships.2 In addition, boys with FXS often exhibit autism-spectrum disorders beginning in the 2nd or 3rd year of life.3 Affected females have a variable phenotype that can range from normal intelligence to severe intellectual disability (depending upon X chromosome inactivation), with or without learning disabilities or behavioral concerns. Women who are premutation (Table 1) carriers may have fragile X-associated primary ovarian insufficiency (POI), including premature ovarian failure (POF), for which fertility evaluation and early intervention are important.4

In more than 99% of cases, FXS is caused by a loss-of-function variant of the FMR1 gene located on the X chromosome.4 Loss of function is caused by an expansion of a polymorphic CGG trinucleotide repeat in the 5′ untranslated region of the FMR1 gene, resulting in hypermethylation of the FMR1 promoter.5 Hypermethylation causes silencing of the FMR1 gene and therefore correlates negatively with the level of protein expression (absent in affected males and substantially reduced in affected females), which plays a role in brain synaptic development. The severity of the phenotype is related to the extent of expansion and hypermethylation (Table 1).4,6

Determining the extent of hypermethylation can help determine premutation or full mutation status in rare cases in which the number of CGG repeats is near the borderline value of 200.6 It can also detect a mosaic condition in which the CGG repeat number is in the full mutation range but subpopulations of cells have no hypermethylation (known as methylation mosaicism).6 A person with methylation mosaicism may have less severe symptoms than a person with a hypermethylated full mutation.6

Other rare FMR1 mutations associated with FXS include large deletions, point mutations, and missense mutations.

Table 1. Number of CGG Repeats in FMR1 and Associated Phenotype

Approximate Number of CGG Repeatsa

Hypermethylation

Classification

Gene Function

Phenotype

5 to 44

Not present

Normal

Normal

Not affected

45 to 54

Not present

Intermediate (“gray zone”)

Normal

Not affected

55 to 200

Not present

Premutation

Larger premutations may have decreased gene expression

Males:  ~40% incidence of FXTAS after age 50 years2
Females:~20% incidence of POI4; ~21% incidence of POF7

>200

Usually present in most or all cells

Full mutation

Loss of gene expression

Fragile X syndrome

FXTAS, fragile X-associated tremor/ataxia syndrome (ie, progressive cerebellar ataxia and intention tremor); POF, premature ovarian failure; POI, primary ovarian insufficiency.

a

Cutoffs are approximate and based on current research.6

FMR1-related disorders are inherited in an X-linked manner with variable penetrance, and inheritance is affected by the number of CGG repeats present (Table 2).8 Individuals with CGG repeats in the intermediate and premutation range are carriers.

Table 2. Inheritance Pattern of FMR1 by Parental CGG Repeat Size

Repeat Size in Parent

Result in Offspring

Women

Intermediatea
(“gray zone”)

Number of CGG repeats may increase to premutation size in offspring

Premutation

Premutation may expand during meiosis in oocytes; thus, mother may give birth to a child with a full mutationb

Full mutation

Full mutation

Menc

Intermediatea
(“gray zone”)

Intermediate status passed to daughters

Premutation

Premutation passed to daughters

Full mutation

Males with full mutation unlikely to reproduce

a

Individuals with an intermediate allele size are considered carriers because of the potential for offspring to inherit the premutation.

b

The greater the number of repeats, the greater the chance of expansion to a full mutation.

c

Sons are not affected because they inherit only the paternal Y chromosome.

Polymerase chain reaction (PCR) can accurately measure repeat numbers in the normal and small premutation ranges. To quantify larger CGG repeats, triplet-primed PCR is used.9 A unique amplicon containing stutter peaks is produced when the individual is at least a fragile X carrier. The absence of stutter peaks indicates absence of an expanded allele. If stutter peaks are detected, methylation-specific PCR can better establish the size and hypermethylation status of the expanded allele (≥85 CGG repeats).

Individuals Suitable for Testing

  • Individuals with a family history of FXS or undiagnosed intellectual disability, including those seeking reproductive counseling

  • Women with a personal history of primary ovarian insufficiency10

  • Symptomatic children and adults

Method

  • PCR and capillary electrophoresis used to determine sex and number of CGG repeats

  • Triplet-primed PCR and capillary electrophoresis used to detect stutter peaks
  • Methylation-specific PCR confirmation of FMR1 expansions and determination of hypermethylation status performed reflexively if PCR indicates an expanded allele
  • Results reported as number of CGG repeats and hypermethylation status of any expanded alleles, with interpretation

Interpretive Information

Individuals with <45 CGG repeats are considered negative for FXS, while those with 45 to 54 CGG repeats are considered to be gray zone allele carriers. No FXS-associated phenotype is expected for individuals with negative or gray zone allele carrier results.

If an expanded allele (>85 CGG repeats) is detected, both CGG repeat number and hypermethylation status are reported. Premutation allele carriers (repeat lengths of 55−200) are at risk of FXS-associated syndromes (Table 1). Individuals with a full mutation (>200 CGG repeats) and hypermethylation are predicted to be affected by FXS.

The associated risk of having a child with a premutation or a full mutation depends on the sex and mutation status of the parent and the sex of the child (Table 2).

This assay does not detect other mutations (eg, deletions and point mutations) that disrupt the function of the FMR1 gene or protein. Results should be interpreted in conjunction with other laboratory and clinical findings.

Additional assistance in interpretation of results is available from our Genetic Counselors by calling 1.866.GENE.INFO (1.866.436.3463).

 

References

  1. Hersh JH, Saul RA; Committee on Genetics. Health supervision for children with fragile X syndrome. Pediatrics. 2011;127:994-1006.

  2. Centers for Disease Control and Prevention. Fragile X syndrome. Related concerns. https://www.cdc.gov/ncbddd/fxs/relatedconcerns.html. Updated June 30, 2016. Accessed February 9, 2017.

  3. Belmonte MK, Bourgeron T. Fragile X syndrome and autism at the intersection of genetic and neural networks. Nat Neurosci. 2006;9:1221-1225.

  4. Saul RA, Tarleton JC. FMR1-related disorders. In: Pagon RA, Adam MP, Ardinger HH, et al., eds. GeneReviews® [Internet]. Seattle, WA; Copyright, University of Washington, Seattle; 1993-2017. https://www.ncbi.nlm.nih.gov/books/NBK1384/. Updated April 26, 2012. Accessed March 13, 2017.

  5. Crawford DC, Acuna JM, Sherman SL. FMR1 and the fragile X syndrome: human genome epidemiology review. Genet Med. 2001;5:359-371.

  6. Monaghan KG, Lyon E, Spector EB. ACMG Standards and Guidelines for fragile X testing: a revision to the disease-specific supplements to the Standards and Guidelines for Clinical Genetics Laboratories of the American College of Medical Genetics and Genomics. Genet Med. 2013;15:575-586.

  7. Sherman SL. Premature ovarian failure in the fragile X syndrome. Am J Med Genet. 2000;97:189-194.

  8. Hagerman PJ, Hagerman RJ. The fragile-X premutation: a maturing perspective. Am J Hum Genet. 2004;74:805-816.

  9. Hantash FM, Goos DG, Tsao D, et al. Qualitative assessment of FMR1 (CGG) triplet repeat status in normal, intermediate, premutation, full mutation and mosaic carriers in both sexes: implications for fragile X syndrome carrier and newborn screening. Genet Med. 2010;12:162-173.

  10. American College of Obstetricians and Gynecologists. ACOG Committee Opinion. Number 690, March 2017. Carrier screening in the age of genomic medicine. Obstet Gynecol. 2017;129:e35-e40.


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Content reviewed 06/2017

 
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