The specialist concluded that Sean was having developmental problems. Most children begin to take their first steps between 9 and 12 months old so Sean started to walk slightly later than most children. Ruth described that Sean’s walk is floppy which could be due to low muscle tone. After assessing his fine motor skills (such as picking up small objects with a thumb and finger), it is clear that Sean’s fine motoring is impaired with poor control of muscles in the finger and hand which is why he has difficulty holding objects. Although ear infections are not uncommon, children with Fragile X Syndrome are at increased risk of suffering from inner ear infections (otitis media). As these symptoms are suggestive of Fragile X Syndrome, the CDC specialist he decides to collect information about the family. The pedigree concludes that the disease has been inherited from Ruth’s side of the family. At first the pedigree looks like it could show X-linked inheritance, often where only women are carriers and only males are affected. However, the pedigree shows unusual features, where both genders in the family are affected. It is also clear that the risk of expressing mental retardation increases through the generations which is why the affected male children have a greater degree of symptoms than their mothers. This is known as anticipation, where signs and symptoms become more severe in each successive generation. The mutation has been passed from Brian to his both of his two daughters, but not his son. On the other hand, the female carriers have passed the mutation to either sons or daughters. Based on these features, the specialist makes a preliminary diagnosis of Fragile X Syndrome (FXS). In Fragile X Syndrome, a mother who carries the mutation has a 50% chance of passing the mutated gene to each of her children who can be carriers or have the disease. This explains why only some of Elizabeth and Ruth’s children are affected. Sean is referred to a clinical geneticist who examines the notes made by the paediatrician and the pedigree chart. She concludes that they are consistent with Fragile X syndrome. She explains to Ruth and David that Fragile X syndrome is a genetic condition the most common known cause of learning disabilities, affecting around 1 in 4000 males and 1 in 5000 to 8000 females. Ruth and David are made aware of how Fragile X syndrome is inherited. They are told that it is caused by a mutation (change) in a gene called FMR1 which is found on the X chromosome. This gene makes a protein called Fragile X mental retardation protein (FMRP) which is required for normal brain development. The FMR1 gene can appear in four forms depending on the number of repeats of a pattern in the non-coding DNA called CGG repeats. The geneticist shows Ruth and David the diagram in Figure 2. They are told that Sean has Fragile X Syndrome because he has over 200 CGG repeats, which is the full mutation. > 200 CGG repeats in the 5′ untranslated region of the FMR1 gene results in methylation of a region (CpG island) upstream. Methylation turns the gene off due to transcriptional silencing, loss of FMRP protein and therefore Fragile X syndrome. She explains that females with Fragile X syndrome show varying degrees of severity and usually are affected less severely than males. Females have two X chromosomes so may have one faulty copy of FMR1 and one working copy, which may protect them from some of the effects of Fragile X syndrome because cells randomly choose which X chromosome is used. Males inherit one X chromosome from their mother and one Y chromosome from their father. This means if a male has the full mutation, they will suffer from FXS.The geneticist says that to confirm the diagnosis it is possible to carry out a genetic test. The two main tests for fragile X syndrome are Southern blot and analysis and Polymerase Chain Reaction (PCR). PCR can be used to determine the number of CGG repeats in the FMR1 gene. However PCR is not the test of choice for diagnosis, due to the high GC content of the gene and it does not work well for repeat sizes above 120 units. The Southern blot is used to determine if the gene has the full mutation (>200 repeats), premutation (50-200 repeats) and if it has been methylated in the CpG island. The clinical geneticist explains to Ruth and David that a leukocyte DNA will be needed from a blood sample and says that it would be useful to have DNA samples from Ruth, her parents and her sister tested as well, if they are all willing to be tested. She explains that the DNA sample will undergo Southern Blot analysis, which involves using a methylation-sensitive enzyme that does not cut methylated sites, distinguishing between methylated and unmethylated alleles (as shown in Figure 3). The fragments will be separated via agarose gel electrophoresis and then transferred to a membrane which contains a specific radioactive probe that will identify a fragment containing the CGG repeats upstream of the FMR1 gene. In Fragile X syndrome, there is hypermethylation of CGG repeats in the promoter region of FMR1 which leads to repression of FMR1 and loss of Fragile X mental retardation protein. Laura (A) has Fragile X Syndrome Figure 4 shows a complete mutation fragment (>200 CGG methylated) and also two other fragments corresponding to either the inactive or active normal X chromosome. Premutation females Angela (B), Ruth (C) and Vicky (F) show 4 restriction fragments which correspond to the normal active X (<55 CGG unmethylated), normal inactive X chromosome (<200 CGG methylated), premutated active X (55-200 CGG unmethylated) and permutated inactive X (<200 CGG methylated). Sean (E) has only one band as he only has one X chromosome, which is >200 CGG repeats and is methylated which confirms his diagnosis of Fragile X syndrome. The methylation due to the mutation leads to no restriction by EclX1. The smearing represents the variability in the size of the CGG repeat from leukocyte DNA. Tom (D) and David (G) have normal band sizes of <55 CGG unmethylated from EclX1 + EcoR1 digestion, corresponding to their 1 active X chromosome. Ruth and David arrange an appointment with a genetic counsellor to have the results explained. Looking back at the pedigree, the genetic counsellor realises that Ruth's grandfather's illness and her sister's early menopause are consistent with Fragile X inheritance. Vicky's early menopause is a result of Fragile X-associated primary ovarian insufficiency (FXPOI). It causes premature ovarian failure and female premutation carriers (55-200 repeats) like Vicky are at ~20% increased risk of developing FXPOI. Brian's illness is a result of Fragile X-associated tremor/ataxia syndrome (FXTAS) which usually occurs after the age of 50 and worsens with age, explaining why he required care towards the end of his life. FXTAS occurs more frequently in males than females. It damages the white matter of the brain in the cerebellum which can lead to intention tremors (limb shaking when moving voluntarily) and ataxia which develops later. 40% of males and 8% of females with premutation (55-200 repeats) develop FXTAS. In both FXTAS and FXPOI, there is increased transcription which can lead to 8x the level of normal RNA. Increased levels of the repeat containing RNA sequesters several RNA binding proteins. This results in dysregulation of proteins whose expression usually regulated by those RNA binding proteins why may lead to FXTAS/FXPOI. The geneticist explains that Brian passed on his X chromosome (carrying premutation) to both Angela and Elizabeth without expanding, however his son inherited the working copy of the FMR1 gene from Brian's wife. The genetic counsellor then goes through the test results and explains that the southern Blot shows that Angela is has the premutation (55-200 repeats), as she is a genetic carrier but not intellectually affected. The geneticist explained that women have two X chromosomes, so have a 50% chance of passing on the mutation in each pregnancy. The premutation has been passed on to Ruth and Vicky without expanding (55-200 repeats), so are also carriers like their mother. She also explains that premutations are considered "unstable", where the CGG repeat has been expanded to the full mutation (>200 repeats) when passed on from Ruth to Sean and Laura, which is why they display the classic Fragile X phenotype. Ruth and David want to know what will happen to Sean and Laura as they grow older and the challenges that the family will face. The counsellor explains that Fragile X syndrome results in a spectrum of developmental and behavioural complications and are usually more severe in males. They may have intellectual problems, affecting their ability to reason learn and think. They may have attention deficit, hyperactivity, anxiety and an unstable mood. FXS sufferers can also show autistic behaviour such as lacking eye contact and hand-flapping. They may become hypersensitive to bright lights and loud noises, and also suffer speech delay. The physical features may become more apparent with age, including a long face, large prominent ears, flat feet, high broad forehead and hyperextensible joints. After puberty, males may have large testes. The geneticist told the parents that people with FXS also often have a good visual memory, sense of humor and an empathetic nature. Ruth and David are made aware that there is no single treatment for FXS but early intervention to help manage the symptoms is important. This includes specialized help from a speech and language therapist. In some cases, medication can be used to treat hyperactivity however behavioural therapy is more commonly used. It is also important that they receive appropriate education tailored to their strengths and weaknesses. Some children can attend mainstream schools depending on degree if intellectual disability. The geneticist also explains that because Laura has the full mutation, there is a 50% chance that she will pass on the affected X chromosome to each of her offspring. This means that there is a 1 in 4 chance that she will have an unaffected daughter (without full mutation), a 1 in 4 chance that she will have a daughter with the full mutation (50% of these will have Fragile X syndrome, 50% will be a carrier), a 1 in 4 chance of an unaffected son (without full mutation) and a 1 in 4 chance of having an affected son (full mutation).