Genetics

What is Rett syndrome?

It is a neurodevelopment disorder mainly seen in females. Children with Rett are born and develop normally. Usually between 6-18 months they start regressing, specifically in language and purposeful hand skills. The head circumference decreases, gait is impaired and stereotypic hand movements are developed.

What is MECP2?

MECP2 is the name of the gene that causes Rett syndrome. This gene is important in brain development.

What is a gene?

Genes are structures in our body that carry information for development. They make proteins in and are made of specific building blocks. The codes for these building blocks are C, G, A, and T. A change in the building blocks of a gene is called mutation. A mutation could be replacement of one bulding block for another or deletion of one or several building blocks. Mutations affect the production of proteins that are needed for normal body functions.

Where are the genes located?

They are located on the chromosomes and chromosomes are located in each cell of our body. We have 46 or 23 pairs of chromosomes. One copy of each pair comes from each parent. We have one pair sex chromosomes. Males have one X and one Y chromosome. Females have two X chromosomes. MECP2 is located on the X chromosome.

Are there males with Rett syndrome?

There have been a few reported cases of males with Rett syndrome that survived through infancy. Males who have a mutation that causes Rett syndrome are severely affected because they have only one X chromosome in contrast to females.

What is X inactivation?

Females have two X chromosomes but one of the X chromosomes is turned off (inactivated). The inactivated X could be the maternal or the paternal one in different tissues. This is a random event. If the X chromosome inactivated is always paternal or maternal in origin it’s called skewed inactivation.

How are X inactivation and Rett related?

We know that females who have the exact same MECP2 mutation show different symptoms. This is because each girl with Rett has a specific pattern of X-inactivation in her brain. Depending on which X is inactivated in which part of the brain the symptoms may vary in two individuals with the same mutation.

Is Rett syndrome genetic?

Yes. Children with Rett syndrome are born with the change in the gene that causes Rett syndrome. The change in the gene is not acquired after birth.

Is Rett syndrome inherited?

No. An inherited disorder is a disorder that is passed on through the parents. 99% cases of Rett syndrome are sporadic. It means that the change in the gene (mutation) occurred by accident only in the one egg or sperm that made the child. This is called a new mutation. So the parents are not affected. We know that mutations mostly occur in sperms, in other words the mutations are paternally derived. Since girls have two X chromosomes, they always receive one of her X chromosome form their fathers. A mutation in the sperm, therefore, will lead to an affected girl.

In what situation Rett syndrome is inherited?

There are mothers who carry a mutation silently. In other words they have skewed X-inactivation. So they show little to no symptoms. They have 50% risk of passing on the mutation to their offspring.

Is there a test for Rett syndrome?

Yes. There is a blood test for Rett syndrome. The test looks for mutation in the MECP2 gene.

My child was tested but she does not have an MECP2 mutation. Could she still have Rett?

Yes. The blood test is not perfect. It detects 80% of the mutations for the children who have classic Rett syndrome. So about 20% of the cases are missed. The diagnosis of Rett syndrome is based on clinical criteria. If the child meets the criteria, regardless of a negative test, she/he has Rett syndrome.

I have a daughter with Rett and I’m pregnant what is the risk that this baby would be affected?

About 1%, unless the mother is a silent carrier. In that case the risk is 50%.

Where does the figure of 1% recurrence risk come from?

Sometimes one of the parents carries the mutation in more than one egg or sperm. In other words their eggs or sperms have a mosaic pattern of some being normal and some carrying the mutation. This is called gonadal mosaicism. There is no way to estimate what percentage of the eggs or sperms carry the mutation. Therefore, in general a 1% recurrence risk is given when a couple have a daughter with Rett syndrome.

Should the parents be tested if they have a child with Rett syndrome and an MECP2 mutation?

The fathers do not need to be tested. They have only one X and cannot carry the mutation silently. Carrier testing for mothers is feasible to give appropriate recurrence risk for future pregnancies. If the mother carries the mutation her risk of having an affected child is 50%. If she is negative the risk is about 1%.

Is prenatal diagnosis possible?

Yes. The affected child should be tested first to see if the mutation could be found in her. Regardless of the baby’s sex screening of the MECP2 gene is recommended.

Should the siblings of an affected girl with MECP2 mutation be tested?

No test is needed for normally developing brothers. Normally developing sisters might carry the mutation silently. They can be tested when they are of reproductive age.

Why should we wait until our normal daughter is of reproductive age to test her?

In general genetic testing should only be done if useful health information can be obtained from testing. Testing a normal daughter does not change her health status. Finding out that a normal daughter is carrying a mutation silently can cause anxiety in the parents on how and when to inform their daughter. This in turn might bring anxiety in the child and make her fearful of the future. At the reproductive age the information can be used to make informed decision about reproductive choices.

Is there any other test available if my daughter tested negative for MECP2?

No. At this point we do not have any other test for Rett syndrome. However, it is suspected that another gene might be involved in causing the disorder. Researchers are currently working on this issue.

My daughter tested negative for MECP2 and I am pregnant. What is the risk that my baby would be affected?

A precise risk is not available at this time but we can estimate a risk of as high as 25%. It is possible that both parents are carrying a hidden gene that we are not aware of yet. Although both parents are healthy, if they both pass on that hidden gene to the baby there is 25% risk that the baby would be affected.

Is MECP2 related to other neurological disorders?

Yes. Recently there have been reported cases of males with MECP2 mutations who do not have Rett syndrome. These individuals have been shown to suffer from psychosis, schizophrenia and mental retardation. The mothers of these individuals are carriers of the MECP2 and some showed borderline intelligence.

Should distant family members with non-specific mental retardation be tested for MECP2?

It is suggested that 2% of non-specific mental retardation is caused by an MECP2 mutation. Screening for these individuals should be considered after an evaluation by a geneticist.

Guidelines
Parents should remember that the above are given only as a general guideline. If they have a complicated family history and specific questions they need to consult a geneticist or a genetic counselor for details.

Information Regarding MECP2 Testing in Ontario

Please Contact:

Rett Syndrome Clinic
Children Hospital of Eastern Ontario,
Ottawa, Ontario
Director: Dr. Peter Humphreys
Nurse Coordinator: Rene Brannan
(613) 737-7600 ext 2159

Rett Syndrome Clinic
Hospital for Sick Children
Toronto, Ontario
Director: Dr. Shelly Weiss
(must have a referral)

New Method for Mutation Testing - Multiplex Ligation-Dependent Probe Amplification (MLPA)

Several laboratories are now using a new method to test for MECP2 mutations through a process called Multiplex Ligation-Dependent Probe Amplification (MLPA) technology. Large deletions in the MECP2 gene have been found in patients who initially tested negative for MECP2 mutations. Preliminary results confirm that a search for MECP2 deletions is important for the diagnosis of Rett Syndrome and that MLPA is a simple and fast technology to reveal these alterations.
If your daughter has tested negative for MECP2 mutations and you would like to have her tested for possible exonic deletions please the following Canadian centre:
Dr. Patrick MacLeod, MD, FRCPC, FCCMG, DABMG
Clinical Director, Division of Medical Genetics
Department of Laboratory Medicine
University of British Columbia
Victoria, British Columbia
MacLeod, Patrick
pmacleodcaphealth [dot] org

MECP2 Analysis

Background

The human body is made up of trillions of building blocks called cells. Within the body, cells specialize to perform different functions. Assemblies of similar types of cells form tissues, and cooperation between tissues in turn forms organs, which enable the body to function.
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FIG 1. Genetic information is transcribed from a DNA acid sequence of a protein
A cell is enclosed by the plasma membrane, which forms a barrier allowing nutrients to enter and waste products to leave. The interior is organized into many specialized compartments, each surrounded by a separate membrane. One major compartment, the nucleus, contains the genetic information necessary for cell growth and reproduction. The rest of the cellular compartments are contained in the cell cytoplasm.
Information in a cell is stored in DNA, in a manner similar to the sequence of letters in the words you are now reading. DNA is comprised of an alphabet with only four letters, known as nucleotides. The bases that make up the nucleotides are A (adenine), G (guanine), C (cytosine) and T (thymine).

Fig 2. Structure of the common amino acids
These nucleotides are grouped into areas along the DNA called genes. In order for a cell to get the information encoded by each gene, the DNA (which is stored in the nucleus) must first be translated into another dialect, called

mRNA (also called messenger RNA). The mRNA enables the information to be transported out of the nucleus. The process of translating DNA to mRNA is called transcription. mRNA's alphabet is very similar to DNA -- it also contains A, G, and C, but U (uracil) replaces T. The mRNA, which is in the cytoplasm of the cell, has its message expressed by a process called translation. Translation, which results in the formation of protein, allows the four-letter language of RNA to be translated into the 20-letter language of proteins. The 20 "letters" which make up proteins are called amino acids (Fig 2.).

How can the amino acids "read" the mRNA?

Well, the mRNA is grouped into three-letter words, called codons. For example, the three-letter codon UUA can be read by leucine, and so at that spot along the mRNA, UUA is translated to leucine. Each amino acid can read at least one of the codons -- some amino acids can read up to six different codons.
There are 64 possible codons, and these 64 codons comprise the genetic code.
Not all codons can be translated to amino acids. Some of these codons are called termination codons, or nonsense codons. These codons tell the translation machinery to turn off and release the protein. When these codons are in the proper spot at the end of the gene, a "normal" or wild type protein is produced; however, mutations in the DNA can result in nonsense codons within the gene. In these cases abnormally shortened proteins are produced. These abnormal proteins may not function as well as a typical MeCP2 protein would function

Your MECP2 Analysis Results

When you receive your results from the lab, you may be understandably confused. Your results will usually contain two pieces of information -- the mutational change in the nucleotide sequence (C,G,A,T, etc...), and the resulting change in the amino acid sequence. You may also be told that the mutation is heterozygous -- this simply means that your daughter has one normal copy of the MECP2 gene on one X chromosome, and one abnormal copy of the MECP2 gene on the other X chromosome.
IF YOU RECEIVE NEGATIVE RESULTS -- this does not necessarily mean that your daughter does not have a mutation in the MECP2 gene. At the present time, labs are testing only the coding region of the gene -- this is the region of the gene that gets translated into protein. All genes also include untranslated regions -- these areas are not translated into protein but are important in that they are involved in the regulation of the gene itself (i.e., these regions help determine whether the gene is translated into protein). So, a negative result does not rule out the presence of a mutation in the untranslated regions of the MECP2 gene.
Researchers are working at this time to complete the analysis of the entire gene. Presently, the consequences of mutations in the MECP2 gene are not understood. Ongoing research in this area will be the key to unlocking the mystery of Rett Syndrome.
Table 2 contains a recent summary of mutations identified in MECP2. Here is some information on each column to help you interpret your results:

Column 1: Nucleotide Change
For most mutations, the wild type or "normal" nucleotide is listed first, with an arrow pointing to the nucleotide present in the mutated DNA. This information is preceeded by a number indicating the location of the mutation in the DNA. For example, if you received the result 316C-->T this means that in this sample where cytosine should be at location 316, there is a thymine instead.
Not all nucleotide mutations result in a simple switch of one nucleotide for another. Here are some other possible sample mutations:
706delG At position 706 in the MECP2 gene, a guanine has been deleted and not replaced by another nucleotide.
620insT At position 620 in the MECP2 gene, an extra thymine has been improperly inserted.

Column 2: Amino Acid Change
This column describes the changes in the protein''''s amino acid sequence as a result of the nucleotide mutation/s.
Most are listed with the abbreviated letter describing the wild type (normal) amino acid, followed by it''''s location in the protein, and ending with the abnormal amino acid present in the sample.
In the example described above, a nucleotide mutation of 316C-->T would result in an amino acid change of R106W. This means that at position 106 in the MeCP2 protein, the sample contains a tryptophan instead of an arginine (the one letter abbrevations for the amino acids are listed in Table 1).
Of course, not all nucleotide changes result in the simple switch of one amino acid for another. Here are some examples of other possibilities:

R168X The X denotes a nucleotide mutation resulting in a nonsense codon. This means that the protein was abnormally shortened at position 168.
Fs This denotes a frameshift mutation, and is caused by either the abnormal insertion or deletion of one or more nucleotides. This deletion or insertion causes all codons downstream of the problem area to be misread, leading to many changes in the amino acid sequence.
803del4 This indicates that starting at base number 803, four bases have been deleted.

Column 3: Frequency
This column describes how frequently a particular mutation has occurred in all MECP2 samples tested.
For example, the mutation R106W has a frequency of 7.50. This means that 7.5% of the mutations found so far resulted in tryptophan substituting for arginine at position 106 in the protein. If you look at column 3 in Table 2 you can also determine that a frequency of 7.50 indicates that this mutation is relatively common among Rett patients tested so far. Because of the prevalence of this mutation, R106W would be called a hotspot mutation.

Table 1. Abbreviations for the Amino Acids

Amino Acid One Letter Abbreviation

Alanine A

Arginine R

Asparagine N

Aspartate D

Cysteine C

Glycine G

Glutamine Q

Glutamate E

Histidine H

soleucine I

Leucine L

Lysine K

Methionine M

Phenylalanine F

Proline P

Serine S

Threonine T

Tryptophan W

Tyrosine Y

Valine V

Table 2. Mutations identified in MECP2
(Adapted from Dragich et al., Hum. Mol. Genetics, 2000,9(16);2365-2375)

Nucleotide Change Amino Acid Change Frequency (%)

258delCA Fs 0.42

291C-->A D97E 0.42

299T-->G L100R

301C-->A P101T 0.42

302C-->A P101H 0.42

302C-->T P101L 0.42

316C-->T R106W 7.5

317G-->A R106Q 0.42

397C-->T R133C 3.75

398G-->A R133H 0.42

401C-->G S134C 0.83

411delG L138X 0.42