Discovery

Fragile X Factor

More is not necessarily better in the genetic code.

Greg Walker

Fragile X syndrome is the most common inherited genetic condition causing mental retardation in humans but one that has received limited publicity due to the lack of understanding of its cause or an accurate means of diagnosis until recent times.

In New Zealand, the incidence of fragile X matches that of countries overseas. Diagnosis of the condition is done by hospital laboratories in the main centres and fragile X testing is highly recommended in situations where there is unexplained mental retardation, late onset of speech or autistic or hyperactive behaviours. Work has been carried out in this country to establish a DNA-based test for detecting fragile X, as well as studies of the nature of the mutation and the detection of carriers in families. Intervention therapies for fragile X children are available but there is a lack of public awareness of the condition and limited genetic counselling services available.

Caused by an abnormal gene on the lower end of the long arm of the X chromosome, fragile X affects about 1 in 1,500 males but can also affect females at a rate of about 1 in 2,500. Approximately 80% of males that have fragile X have some degree of mental impairment.

Fragile X has a unique inheritance pattern, being neither fully recessive nor fully dominant. There can be affected females and normal carrier males despite it being on the X chromosome. Being on the X chromosome makes fragile X a sex-linked or X-linked trait and it is therefore passed easily from one generation to the next.

Mothers can be carriers (heterozygous) of the condition and their sons are at most risk of being affected. Daughters, on the other hand, can be carriers or are at a risk of being slightly affected. Due to the X-linked nature of the gene, it cannot be passed from father to son as the son inherits the Y chromosome from his father.

In males, fragile X may or may not be characterised by mental retardation along with physical symptoms such as:

• long or prominent ears
• a long narrow face
• enlarged testicles

Behavioural characteristics include:

• hyperactivity
• repetitive speech or sounds
• shy behaviours and avoiding eye contact when speaking
• unusually fast speech or speech that fluctuates in rate
• hand biting or hand-flapping
• autistic-like behaviours
• right-left disorientation
• motor delays

Women that have significant mental disadvantages often also show associated physical features similar to males, including the prominent ears and narrow face, but also flat feet and very loose finger joints.

About 30% of women who inherit the fragile X gene will be affected intellectually and may have below-average IQ scores, but it is more common for females to exhibit learning difficulties and attention span problems. Many also have difficulty with mathematical concepts. Because females have two X chromosomes -- a healthy one which can mask the abnormal gene -- girls are often protected from fragile X and simply act as carriers, although the degree of protection varies from individual to individual. In sex-linked inheritance, a carrier or heterozygous female has a 50% chance of passing on to her children the X chromosome with the fragile X gene.

Male carriers of the disease are non-penetrant individuals. They carry "premutations" which dispose them towards producing fragile X offspring, without exhibiting the full signs of fragile X themselves. Such "non-penetrant" fathers will pass on an X chromosome to their daughters, who will be carriers. Males pass the premutation on unchanged to their daughters. Mothers on the other hand can pass on a modified or amplified version of the premutation which leads to the disease.

What Causes Fragile X?

The gene implicated in fragile X has been known since 1991 and is called FMR-1. The cause of the disease is an unusual mutation in which there is a repetition of a particular sequence, the CGG triplet, in the DNA.

Most people have 6-60 repeats of the CGG sequence, but when more than 200 repeats arise then the gene is switched off and the person suffers from fragile X syndrome. It may be that the gene is not expressed at all and that it is the absence of the protein products of the FMR-1 gene that lead to the abnormalities. The role of the protein product is relatively unknown but it does occur in the nucleus and binds to RNA.

It appears that as many as one in 260 people carry the alleles known as fragile X premutations that, when transmitted by the mother, expand into full mutations and lead to fragile X. When the repeats are between 60 and 200, the effects of the FMR-1 gene tend not to be seen. Carriers of fragile X have the premutation which is an intermediate-sized piece of the CGG-repeating DNA, of 60-200 repeats.

Unfortunately there is a risk of the premutation repeat lengthening when ova are being formed which affects the resulting offspring. There is some evidence to suggest that the repetition occurs once the sperm has fertilised the egg, rather than during egg formation. In some cases, the lengthening will remain under the 200 "no-show" limit; in others amplified repeats go over this limit and result in the genetic information becoming faulty and the syndrome being expressed in the child.

This increase in length does not seem to occur in sperm production, so men pass on the same-sized repeats as they themselves possess.

Detection

Fragile X traditionally has been detected by examining white blood cells for the presence of a fragile site on the X chromosome. This unfortunately is highly inaccurate. Now a variety of tests enable detection of both the premutations and full fragile X mutations pre-natally, using foetal tissue or cells collected from the amniotic fluid that surrounds a foetus.

Mutations detected during pregnancy pose difficult ethical questions, especially if the foetus is female because the degree of retardation can be highly variable. Males on the other hand are usually more severely affected.

The expansion of the DNA causing the mutation is commonly detected by Southern Blotting. Southern Blotting involves collecting a blood sample -- 5ml is enough -- and extracting the leucocytes, the white blood cells. The leucocytes are broken open to release their DNA which is then digested using restriction enzymes. These enzymes cut the DNA at specific sites, chopping it into different sized fragments. Electrophoresis, where the fragments are forced through a gel, is used to separate the fragments based on their relative sizes. The smaller fragments, which have a lower molecular weight, move further in the gel compared to the larger fragments of higher molecular weight.

A nylon membrane is put on top of the gel with a weight on top. A liquid is poured around the gel and this soaks up through the gel and onto the membrane taking with it the fragments of DNA. These DNA blots are then hybridised overnight with a radioactive probe which searches for the FMR-1 site. The radioactive probe binds to the FMR-1 site. The membrane is then placed in a film canister with a piece of photographic film and left for several days. The DNA appears as bands on the photographic film. This is known as autoradiography. Diagnosis can then take place based on the size of the bands.

Fragile X males tend to show an increase in band size in the fragment of affected DNA. Normal individuals show a band of about 1kb (kilobases) or 1,000 bases. Affected males tend to show increases greater than 1.5kb (200 copies of the repeat), whereas transmitting males tend to have less than 1.5kb. Normal negative females show less than 1.5kb while positive carrier females have more than 1.5kb. Carrier females with less than 1.2 kb may produce transmitting and/or unaffected sons while those carriers with more than 1.2kb tend to produce more affected sons.

Treatment

Treatment for fragile X is particularly difficult but can involve drug therapies which help improve attention and concentration problems, aggressiveness and hyperactivity. Further treatments can use individualised programs of speech therapy, education and physical therapy.

Sensory Integrative Therapy can be used to make a person calmer and less sensitive to noises, sights and movement. This form of therapy involves the patient in movement, tactile, visual and motor activities that help the person to feel that they can interact with their environment more easily. Educational activities usually involve features such as teaching counting and number skills, play, and number games. Genetic counselling for the extended family is especially important as the discovery of one fragile X individual can easily lead to the discovery of further carriers within the family despite there being no family history of mental retardation. Parents that are carriers of the condition need to be made aware of the "risks" involved in having children.

Recent treatment research has concentrated on gene therapy or protein replacement therapies. It may be that any treatment would have to occur before birth to have the greatest benefit.

Dr Martin Kennedy of the Cytogenetic and Molecular Oncology Unit, Christchurch School of Medicine and the staff of Canterbury Health Laboratories, Christchurch Hospital gave invaluable assistance in the production of this article.

Greg Walker, NZSM