What do the genetic tests look for?

   Genetic tests for CLS involve reading the genetic code from cells in the patient’s blood or other type of sample. The genetic code is made up of four chemical structures, which are represented by the letters A, T, C and G. Each gene is made up of a unique sequence using these four letters of the DNA alphabet. Just as the words on this page have meaning because of the order of the letters, so do the letters in a gene. Each gene serves a purpose, which is to provide instructions for a unique protein to be made by the body. It’s as if the DNA in genes is a biological recipe. Once the DNA recipe makes a protein, the protein has a specific function. Proteins must work properly and be in the right amount to prevent genetic disease.
   A specific gene, known as RSK2, was identified by a group of researchers studying patients with CLS in France, Philadelphia and England (Trivier, et al. 1996). When they compared the DNA from patients who have CLS to people who do not have CLS, they found differences in the DNA code. These differences are called genetic mutations. The researchers wanted to see if the mutations they found in the RSK2 gene cause CLS, so they studied how the mutations affect the way the RSK2 protein, called a kinase, works. What they found was that patients with CLS did not have any normally functioning RSK2 kinase. This led them to conclude that mutations in the RSK2 gene cause the RSK2 kinase to be made improperly, and the creation of an abnormal kinase leads to all of the symptoms associated with CLS. When a DNA laboratory does the test for CLS on a patient, they are looking for mutations in the RSK2 gene. Amazingly, the RSK2 gene is made up of thousands of DNA letters and yet it is not uncommon for just one letter to be changed in a patient with CLS. The genetic test requires reading the DNA code from a patient’s sample, letter-by-letter, to find these small changes.

My doctor isn’t sure that my child has CLS. Will doing genetic testing on him help to make the diagnosis?

   Genetic testing may indeed be helpful, especially for younger children. Many young children do not have all of the symptoms of CLS and it is more difficult for the doctors to make the diagnosis in them. There may be other genetic conditions that the doctors are considering, many of which have some of the same symptoms. If the RSK2 gene test shows a mutation, then the result is “positive,” and the diagnosis of CLS can definitively be made. If it is negative, or no mutation is found, there may be other things to consider, but it doesn’t necessarily mean that the diagnosis is not CLS.
   There are some symptoms that become apparent later on in CLS patients, and these can be anticipated if the diagnosis is known early. In particular, scoliosis (curvature of the spine) is common in adolescents, particularly boys, with CLS. Because it may not be present when the child is first evaluated, the doctors will know that a child with a positive DNA test for CLS has a higher chance than other children to develop scoliosis. The fact that they can anticipate this potential medical complication allows them to treat it earlier. The learning problems associated with CLS can also be approached with early intervention if the diagnosis is known. Lastly, there may be a social and emotional benefit to having the diagnosis early, because it allows newly diagnosed children and their families to connect with other parents. Other parents and families can be invaluable and it is often helpful to talk to others who understand about the problems, and the joys, unique to CLS.

I’ve been told by my doctor that my child definitely has CLS. What will a genetic test tell us that we don’t already know?

   In addition to confirming the diagnosis and determining the inheritance pattern (discussed later), genetic testing can also provide important information to family members. For example, the mother, sisters, aunts or cousins of a boy or man with CLS may want to have children now or some day in the future. When a child with any genetic condition is born into a family, other family members may want to know if they need to consider the possibility that they may have a child with that condition as well. The most helpful approach to determining the chances of having a baby with CLS is to test the person who has CLS first. If that person shows an RSK2 gene mutation, then other family members can also be tested. This may be difficult to bring up with family members, but it is important to discuss and is something your relatives might be wondering about. Sometimes getting it out in the open is a relief to everyone. If they can find out their chances of bearing a child with CLS, it helps them to prepare for the possibility and consider all of their options for having a family. Anyone with a family history of CLS who wants more information about risks, testing and their reproductive options can meet with a genetic counselor. Genetic counselors can be found by going to http://www.nsgc.org or by requesting a referral from a gynecologist or family doctor.

I understand that CLS is a genetic condition. How is it inherited? Is it always inherited?

   We’ve already established that CLS is caused by a mutation in the RSK2 gene. Genes, and the DNA that makes them up, are packaged into structures called chromosomes. Each of us has 23 pairs of chromosomes, and the important chromosome in CLS is the X chromosome. Females have two X chromosomes and two copies of the RSK2 gene. Males have only one X chromosome and only one RSK2 gene. If a boy has a mutation in his RSK2 gene, he will not make a functional RSK2 kinase. However, if a female has a mutation in one of her two RSK2 genes, she will make normal RSK2 kinase from the other copy of the gene. The normal copy of the RSK2 gene may compensate, totally or in part, for the deficiency caused by the mutation. There are many females with some mild features of CLS, however, and these girls and women don’t have enough normal RSK2 kinase being produced to completely prevent the CLS symptoms. One thing to note is that it is rare for a female with an RSK2 mutation to have all of the same features as a male with the condition.
   The inheritance of CLS is X-linked, because it is on the X chromosome. Females with an RSK2 mutation are called “carriers” and have a 50% chance, with each pregnancy, of passing on the mutation. If the mutation is inherited by a female, she will be a carrier. If the mutation is inherited by a male, he will have CLS. Overall, before a pregnancy is conceived, there is a 25% chance to have a son with CLS and a 25% chance to have a carrier daughter. As mentioned, female carriers may have some of the symptoms associated with CLS although they are usually more mildly affected.
   CLS is not always inherited, however. Based on a relatively small number of cases in which there was no family history of CLS, the medical literature suggests that over half of the mothers of most boys with RSK2 mutations are not carriers (Jacquot, et al. 1998). If the mother of a child with CLS has no symptoms and no family members with CLS, genetic testing may be the only way to determine if she is a carrier or not. Mothers who are not carriers still have a small chance of having a daughter who is a carrier or more than one affected son; however, this risk is likely to be low (on the order of 1%). If a woman who has a child with CLS is not a CLS carrier, her mother, sisters, cousins and nieces would also not be carriers and they do not have an increased chance of having a child with CLS. The family members of an unaffected father of a child with CLS also need not be concerned about an increased chance for having a baby with CLS, because of the X-linked inheritance pattern.

My child had the RSK2 gene testing done and it was negative. What could this mean?

   Genetic testing for CLS currently only looks at the DNA sequence of the RSK2 gene. If a mutation is present, the diagnosis of CLS is confirmed. If no mutation is found, there are a few possible explanations. For one, there is some evidence in mice that another gene exists which causes the same symptoms as CLS (Yang, et al. 2004). A negative result of RSK2 testing may be due to the fact that there is a DNA mutation, but it lies in another gene. At this point, the role of another gene or genes in CLS has not been confirmed in patients with CLS.
   A second possibility is that a mutation is present in the RSK2 gene, but was not picked up by the methods used in the lab. This is a limitation of the technology that is available to us at this time. Because CLS is a rare condition, it is difficult to know how many mutations are not being detected, but the estimate is that it is likely to be low. Certain types of rarely-occurring mutations cannot be detected in females, and testing an affected boy in the family (if one is available) may be recommended.
   Lastly, some doctors order RSK2 testing for patients that don’t completely fit the CLS phenotype, meaning that they may have only some of the symptoms associated with CLS and may even have other symptoms that are not typically seen in CLS patients. This is not uncommon, and many tests may be ordered to get to the bottom of the diagnosis. Sometimes one of the tests comes back positive, and then the diagnosis is known. Other times, we are limited by our current understanding of genetics and the available technology, and the tests are all negative. Because of our rapidly advancing knowledge in genetics, checking in with your geneticist or genetic counselor on an annual basis is a good idea, as they can discuss new discoveries with you as they are made.

How do I get the testing done?

   The testing is currently being offered at two laboratories in the US: GeneDx, Inc. (http://www.genedx.com) and Greenwood Genetic Center (http://www.ggc.org). RSK2 gene testing must be ordered by a physician or genetic counselor, who will discuss the results with the patient and family. The testing is slightly different between these two labs, and your doctor or genetic counselor can help you decide which lab is best for you. Testing can be done on a blood sample that can be shipped to the lab, so there is no need to travel if you want the testing done. The Greenwood Genetic Center (GGC) requires a special tube that the blood must be shipped in, which they will send to your doctor’s office free of charge. The special kit is only necessary for the first person tested in the family. After that, blood samples for carrier testing can be sent in the tubes available at any drawing station. If you’d prefer not to have blood drawn at all, GeneDx can send a brush kit to your doctor or your home to collect cheek cells for DNA testing. The accuracy, turnaround time and cost are the same for blood and cheek brush samples. Unfortunately, unlike the quick results the investigators seem to get on TV programs, the results of DNA testing for CLS usually take several weeks, and many steps go into the analysis. The time is well-spent, however, and the laboratory works hard to insure that the results they report to your doctor are accurate.
   Because of the methods they use, GGC prefers doing the initial testing on males with CLS while GeneDx has experience testing both affected males and females. Both labs require testing the person with CLS first, and can then offer carrier testing to other family members. Testing a sample from a pregnancy is also available when a mutation is known in a family. It is important to be aware of the billing policy of the lab before sending a sample, as the testing is expensive ($1,300 - $2,600, for the first person in the family; $250 - $1,050 for carrier testing and testing on a pregnancy). Insurance frequently covers RSK2 testing, but you will probably want to check with your insurance company to see if you will be responsible for all or part of the cost of testing.
   This article covers some commonly-asked questions about genetics and DNA testing. The next time you’re watching a crime show on TV or reading an article that describes DNA, you’ll have a better understanding of genetics and the methods used to examine genetic material. Of course, it’s especially nice to know that not only is this technology helpful in solving crimes, but it can also be used to help families like yours.
   If there are any questions about this article or genetic testing for CLS, please feel free to contact the author at cheryl@genedx.com.

Cheryl Scacheri, MS, CGC

Director of Genetic Counseling Services

GeneDx, Inc.

Gaithersburg, MD

http://www.genedx.com/services/dis_cls.php 

References:
Trivier, et al (1996) Mutations in the kinase Rsk-2 associated with Coffin-Lowry syndrome. Nature. 384:567-570.
Jacquot, et al (1998) Germline mosaicism in Coffin-Lowry syndrome. European Journal of Human Genetics. 6:578-582.
Yang, et al (2004) ATF4 is a substrate of RSK2 and an essential regulator of osteoblast biology: Implication for Coffin-Lowry syndrome. Cell. 117:387-398.