Although genetic testing is available for congenital adrenal hyperplasia (CAH) by having a blood test, it is not as simple or routine as it may first sound. Genetic testing for CAH is complicated by the location of the gene and by the different types of mutations or changes that can occur in the gene.

Approximately 90 to 95% of cases of CAH are due to 21-hydroxylase (21-OH) deficiency. The gene that makes 21-OH is called CYP21A2. The 21-OH deficiency form of CAH is inherited in an autosomal recessive manner. This means that parents of a child with CAH each have one working copy of the gene responsible for CAH and one non-working copy, and are referred to as carriers.

About 1 in 60 people in the general population are carriers. When two carriers achieve a pregnancy, each pregnancy has a 1 in 4, or 25% chance to be affected with CAH because the developing baby did not inherit a working copy of the gene from either parent.

The CYP21A2 gene responsible for 21-hydroxylase deficiency is located on chromosome 6. Chromosomes are the packages that contain our genes, and genes are the instructions that direct how our bodies grow, develop, and function. Each of our chromosomes come in pairs, so we each have one chromosome 6 inherited from our mother, and another chromosome 6 inherited from our father.

Another gene, called CYP21A, is located very close to CYP21A2 on each chromosome 6. CYP21A is a pseudogene. A pseudogene is a copy of a gene that is no longer working. The most common genetic situation seen on each chromosome 6 in people who do not have CAH is one copy of CYP21A located close to CYP21A2 as shown in “A” of the diagram below. This means that the person has two pseudogenes and two functional, or working, genes in total. The pseudogene no longer works because it has acquired multiple alterations, or mutations, along its length as shown under “D” in the diagram. Although the pseudogene has many mutations that make it different from the functional gene, the two are 98% identical overall. Because both gene and pseudogene are so similar, genetic testing is more difficult and the gene is more likely to acquire changes that prevent it from working.

Mutations in the functional gene occur because one or more mutations have moved from the pseudogene to the functional gene. Sometimes this involves little bits and pieces of the pseudogene and other times it involves major rearrangements of the gene and pseudogene. The major rearrangements can delete or add an entire copy of the gene or pseudogene, or can leave behind changed structures that are part gene and part pseudogene as illustrated in “C” and “D” of the diagram.

Labs that do testing for CAH use some or all of the following genetic techniques in order to provide results. Genes are made of DNA, so the first step of the process is isolating DNA (deoxyribonucleic acid). Usually a blood sample is used as the DNA source, but other bodily tissues and fluids can be used as well. Then, different methods are used to detect small genetic changes, or large alterations such as whole gene deletions. Labs that offer testing for CAH should be able to detect 90% to 95% of the mutations causing the classic form of the disease.

Glossary of Genetic Testing

The polymerase chain reaction (PCR) is a common method used for genetic testing that can tell one gene apart from another. PCR can be thought of as a “genetic Xerox machine.” By using PCR, the laboratory can make up to a million copies of a specific gene or piece of a gene from a DNA sample for studies.

Some laboratories use a technique called a Southern blot. In this method, extracted DNA is cut at specific sites near or within the gene and pseudogene. A specially made piece of DNA, referred to as a probe, is used to detect the specific DNA pattern of the gene and pseudogene. A Southern blot will usually find large gene deletions and rearrangements. After a lab performs a Southern blot, it will also need to perform PCR and some other method, like sequencing, in order to find those smaller changes.

PCR can also be used to distinguish the pseudogene and functional gene, and in most cases can determine the number of functional genes and pseudogenes a person has in their DNA without using the Southern blot technique. Unique landmarks outside the gene and pseudogene are used to separately copy and identify the gene, the pseudogene, as well as any rearranged copies of gene and pseudogene that might be present.

The copies of the gene and pseudogene that have been made by PCR can be closely looked at using a technique called sequencing. Through sequencing, labs look for the presence or absence of the most common “small” genetic alterations seen in individuals affected with CAH. These are known as P30L, Intron 2 ”G”, G110del8, I172N, Exon 6 cluster mutations, V281L, F306+1, Q318X, R356W, and P453S. Different labs may choose to look for some or all of these small mutations. This technique can also be used to carefully review the entire gene for less common mutations; this is generally referred to as “full gene sequencing” or “sequencing the entire coding region.” This approach would be valuable for individuals with a confirmed diagnosis of CAH due to 21-OH deficiency who do not have one of the common mutations. Full gene sequencing is labor intensive, generally takes longer to obtain results, and might cost significantly more than the tests for the common mutations.

In families where the diagnosis of CAH due to 21-hydroxylase deficiency has been proven but a mutation cannot be identified, another test called linkage can be used to determine if a relative has the mutation or if a pregnancy is affected. Linkage cannot be performed without testing multiple family members. At least both parents must be tested, as well as a previously born affected child. Unique genetic landmarks either within the gene or pseudogene, or close to, or “linked” to the CYP21A2 gene are used to identify and follow copies of the mutated gene causing CAH from generation to generation without actually knowing the exact mutation involved. Sequencing small areas of the pseudogene and functional gene can provide good genetic information for linkage analysis. Linkage can be used to predict whether or not a subsequent child will be affected with a high degree of accuracy. Because linkage does not test for the actual mutation, however, there is a small risk that the linked genetic landmark will become unlinked and thus an incorrect result will be obtained.

Who Might Consider Genetic Testing?

Genetic testing can be used to confirm the diagnosis of CAH and identify the mutations present in a person who is suspected of having classic or non-classic CAH. For example, genetic testing can help confirm the diagnosis in infants that have a positive newborn screen for CAH. Adults with suspected CAH due to infertility problems or women who have symptoms of androgen excess might also have their diagnosis confirmed through genetic testing.

Parents of a child with CAH may want to know the genetic alterations present in their affected child. This information can be used, early in pregnancy, to determine whether a subsequent child has CAH by testing the baby through amniocentesis or chorionic villus sampling. The information could help direct prenatal treatment with dexamethasone or help families and doctors anticipate and prepare for the birth of an affected child. Genetic information can also be used for preimplantation genetic diagnosis in future pregnancies in order to significantly reduce the chance of a having another child affected with CAH (see “Preimplantation Genetic Diagnosis: Another Alternative” Winter 2003-04 newsletter).

Finding the mutations present in an affected child allows testing of other family members, such as the aunts and uncles of an affected child, for the specific mutation(s) identified. This allows other relatives to know if they are carriers so that they can use the information in the way that is best for themselves and their own family planning. If an affected child has not been tested and a relative decides to pursue their own testing, problems with interpretation of the results can occur. It is always best to test the affected individual. For example, if the relative is negative, it is still possible that the family carries a mutation that cannot be detected with current methods, and that would not be known unless the affected individual was also tested and was negative.

Genetic testing can also be used to screen an unaffected person who has no family history of CAH to determine if they carry CAH. This can be especially useful for the partners of individuals who are either affected by CAH, or are known carriers of CAH, for the purpose of family planning and pregnancy management. If both members of a couple are known carriers, they can consider the option of starting dexamethasone treatment early in the pregnancy which would reduce the degree of masculinization of the female genitalia in an affected female infant.

For couples that have no known personal or family history of CAH, but are currently pregnant with a female fetus that has ambiguous genitalia detected by prenatal ultrasound, genetic testing for CAH may be appropriate after other causes have been ruled out. The advance knowledge can help the family and physicians prepare for the medical, social and emotional issues related to the diagnosis and birth of an affected child.

Prenatal Testing for CAH

Prenatal testing can be performed by two methods. Chorionic villus sampling (CVS) is a procedure that obtains fetal cells by sampling cells from the developing placenta. The procedure is usually done with ultrasound guidance to see the physical structures of the patient and fetus. CVS is typically offered at 10-12 weeks from the last known menstrual period. As with any prenatal procedure, CVS carries with it a small risk of miscarriage. The advantage of CVS is that the procedure takes place in the first trimester and genetic test results can be obtained early in the pregnancy.

The cells obtained are taken to a laboratory and grown so that the DNA can ultimately be obtained and tested.

Amniocentesis is another technique that can be used to obtain a sample of fetal cells for genetic testing. This method is typically performed at 15-20 weeks from the last menstrual period. This procedure requires that a thin needle is passed through the abdomen, under ultrasound guidance, into the fluid filled sac that surrounds the fetus. A few tablespoons of the fluid are taken from the sac. This fluid contains cells from the baby that are then grown in a laboratory. As is the case with CVS, amniocentesis is not a risk-free procedure. With amniocentesis, there is a small risk of about 1 in 200 that the procedure will cause a miscarriage and loss of the pregnancy. The risks and benefits must be taken into consideration when considering prenatal testing. Your doctor and genetic counselor are good resources for additional information regarding these procedures.

If prenatal genetic testing for CAH is to be accurate and reliable, it is very important to think about genetic testing in advance. Due to the complexities in testing previously described, it is recommended that both parents as well as the affected child be tested prior to any prenatal testing of a subsequent child. If the fetus alone is tested first, it may not be possible for a lab to issue a final report without requesting blood specimens from the parents and the previously affected child. This may delay the results by a few weeks while the lab compares the genetic pattern present in the fetus with that of the rest of the family.

Some laboratories recommend that another genetic test called maternal cell contamination studies be ordered at the same time as the prenatal test. Maternal cell contamination studies ensure that the fetal DNA being tested is in fact from the fetus and it is not contaminated with mom’s DNA, thus ensuring an accurate analysis and result. For example, if the DNA being tested were actually the mother’s DNA and not the baby’s, then the result would be incorrectly reported that the baby was a carrier just like mom, when in fact the baby could be affected, not a carrier at all, or a carrier of dad’s mutation.

Anyone considering genetic testing for CAH, for whatever reason, may benefit from meeting with a genetic counselor to discuss the various options available. You can find a genetic counselor near you at www.nsgc.org. A genetic counselor can also assist in coordinating your testing and can help explain and interpret your results.

How CAH Genes Translate Into Symptoms: The Genotype/ Phenotype Connection from our newsletter.

Genetic Nondiscrimination Bill Stalled In Congress

The 108th Congress failed to pass legislation protecting against genetic discrimination. Although the Senate passed a bill (S.1053) 95-0 in October 2003, and President Bush supported such legislation, the House has not moved any such bill to the floor for consideration.

Without protective legislation in place, some patients fear having their conditions known to insurers, who might use it as a reason to deny coverage. As a result, these patients might forego testing that would be useful in making decisions related to their health and family planning. Other may seek anonymous genetic testing, according to a recent article in the San Jose Mercury News, which reported that some patients are being tested under assumed names, with their doctor’s help.

To call your Senator or Representative to urge their support of this legislation, you can call the Capitol Switchboard at (202) 224-3121. Ask to be transferred to your Representative or Senator's office. If you do not know who your representative is, you can go to www.congress.org, enter your zip code, and find a list of your federally elected officials.