Fragile X Syndrome via FMR1 CGG Repeat Expansion

Fragile X syndrome is the most common genetic cause of intellectual disability in males and the second most common cause in females, with a prevalence of 1:4000 males and 1:6000 females. Fragile X syndrome is characterized by developmental delay, moderate to severe intellectual disability and autistic behaviors. Fragile X syndrome is typically diagnosed at around three years of age when developmental delay becomes pronounced. Physical features of Fragile X syndrome include an elongated face, prominent jaw, broad forehead, prominent ears, macroorchidism in males, flat feet and hyperextensible finger joints (Gallagher and Hallahan 2012). Common behaviors associated with Fragile X syndrome include attention deficit hyperactivity disorder (ADHD), trouble sleeping, anxiety, mood disorders and aggression. Autistic-like behaviors seen in Fragile X syndrome patients include perseveration of speech, motor stereotypies such as hand flapping, restricted interests and poor eye contact. Approximately 30-50% of Fragile X syndrome patients meet the diagnostic criteria for autism, although symptoms tend to be less severe than in patients with idiopathic autism (McCary and Roberts 2013; McDuffie et al. 2015).

Premature Ovarian Failure (POF type 1) or Primary Ovarian Insufficiency (POI) refers to ovarian dysfunction that results in infertility or menopause before the age of 40 in women. Other indications of POF are increased serum FSH and low estradiol levels, indicative of hypoestrogenism. POF can result from follicular depletion or follicular dysfunction (Rafique et al. 2012). Approximately 20% of women who have an FMR1-premutation develop premature ovarian failure (Sullivan et al. 2005; Mailick et al 2014).

Fragile X Tremor/Ataxia Syndrome (FXTAS) is a progressive neurodegenerative disorder. It presents between 60 and 65 years with intention tremor and gait ataxia. MRI studies reveal a white matter hyperintensity of the middle cerebellar peduncle (MCP) in patients with fragile X tremor/ataxia syndrome and this MCP sign is associated with more severe disease progression (Hagerman and Hagerman 2013). Another key finding is the presence of ubiquitin positive intranuclear inclusions in neurons and astrocytes throughout the brain (Hagerman 2013). Both males and females can develop fragile X tremor/ataxia syndrome, but penetrance is higher and disease progression is more severe in males.

Fragile X syndrome is inherited in an X-linked recessive manner and is caused by a repeat expansion in the 5'UTR of the FMR1 gene. FMR1 CGG expansions fall into four classes: normal (~5-44 repeats), intermediate (~45-54 repeats), premutation (~55-200 repeats) and full mutation (>200 repeats) (Monaghan et al. 2013). Only individuals with >200 CGG repeats in the FMR1 gene have Fragile X syndrome. More than 99% of Fragile X syndrome cases result from expansion of the CGG repeat located in the 5'-UTR of the FMR1 gene (Monaghan et al. 2013). Large CGG repeats (>200) are generally hypermethylated which leads to transcriptional silencing of the FMR1 gene. Penetrance in males with a full mutation is nearly 100%. Females heterozygous for a full mutation can have phenotypes ranging from mild to severe Fragile X Syndrome depending upon patterns of X-inactivation (Monaghan et al. 2013). Premutation alleles do not result in a Fragile X syndrome phenotype, but they have the ability to expand to a full mutation in a single generation. Females with a premutation allele are at risk for having a child with Fragile X syndrome.

Premature Ovarian Failure (POF1) or Primary Ovarian Insufficiency (POI): The premutation allele is found in roughly 1:200 females (Hagerman and Hagerman 2013). Females with a premutation allele are at risk for developing premature ovarian failure. Approximately 20% of females that carry an FMR1 premutation allele will experience menopause before 40 years of age. Females with a premutation allele of ~80-99 CGG repeats have the highest risk for developing premature ovarian failure (Sullivan 2005).

Fragile X Tremor/Ataxia Syndrome: The premutation allele is found in roughly 1:200 females and 1:400 males worldwide (Hagerman and Hagerman 2013). Both male and female carriers of an FMR1 pre-mutation allele are at risk for Fragile X tremor/ataxia syndrome. The age of onset and severity of this syndrome is correlated with the number of CGG repeats; carriers with >100 repeats are more likely to develop symptoms of Fragile X tremor/ataxia syndrome (Hunter et al. 2012).

FMR1 encodes the fragile-X mental retardation protein (FMRP) which is an RNA binding protein highly expressed in the brain. FMRP binds RNAs and transports them to synapses for local translation (Liu-Yesucevitz et al. 2011). FMRP is predicted to bind to as much as 4% of all mRNA in the brain and acts as a translational repressor. Fragile X syndrome is caused by loss of FMRP and improper translation of neuronal mRNAs (Bagni et al. 2012). FMRP mRNA targets are enriched for genes implicated in intellectual disability and autism spectrum disorders, suggesting a common molecular mechanism (Ascano et al. 2012). Loss of FMRP results in long, thin dendritic spines in the cortex which reduces neuronal contacts and impairs synaptic plasticity. The molecular cause of Fragile X related premature ovarian failure and Fragile X tremor/ataxia syndrome is proposed to be the elevated levels of FMR1 mRNA seen in individuals with premutation alleles. It is hypothesized that either repeat associated non-ATG (RAN) translation of FMR1 mRNA or sequestering of RNA binding proteins by excess FMR1 mRNA underlies disease pathogenesis (Todd et al. 2010; Sellier et al. 2013).

FMR1 CGG repeat expansions of >200 are found in 1-2% of patients with developmental delay (Strom et al. 2007). FMR1 CGG repeat expansions are estimated to explain ~99% of FXS cases, with point mutations or deletions in the FMR1 locus accounting for the remaining 1%. However, careful studies which analyze both CGG repeat size and FMR1 gene sequence in FXS patients are lacking (Peprah 2012; Myrick et al. 2014; Monaghan et al. 2013). FMR premutation alleles were found in 2-4% of men with cerebellar ataxia who tested negative for SCA expansions (Brussino et al. 2005; Van Esch et al. 2005). FMR1 premutation alleles were found in 2-8% of idiopathic POI cases (Ferrarini et al. 2013; Bachelot et al. 2009).

Repeat-primed PCR is used as a screening method for the presence or absence of a pathogenic CGG trinucleotide repeat expansion located in the 5' UTR of the FMR1 gene. Of note, this test is not designed to determine the precise number of CGG repeats in pathogenic expansions (CGG repeats >200). A methylation-sensitive PCR assay will be performed on all full mutation alleles (>200 CGG repeats) to determine methylation status.