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Genetic Testing for Autosomal Dominant Hypercholesterolemia

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Published: December 2008

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Dr. Baudhuin discusses the use of Mayo’s recently introduced reflex panel for diagnosis of autosomal dominant hypercholesterolemia (ADH). Use of this genetic testing panel to identify the mutations in some of the various genes involved in ADH provides a cost-effective strategy for a definitive diagnosis of a genetic disease.

Presenter: Linnea Baudhuin, MD

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Introduction

Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussions of current topics and may be helpful to you in your practice.

Our presenter for this program is Dr. Linnea Baudhuin, Co-Director of the Nucleotide Polymorphism Laboratory and Co-Director of Cardiovascular Laboratory Medicine, in the Division of Clinical Biochemistry and Immunology at Mayo Clinic. Dr. Baudhuin will be discussing the use of Mayo's recently introduced reflex panel for diagnosis of autosomal dominant hypercholesterolemia (ADH). Use of this genetic testing panel to identify the mutations in some of the various genes involved in ADH provides a cost-effective strategy for a definitive diagnosis of a genetic disease.

Autosomal Dominant Hypercholesterolemia

Autosomal Dominant Hypercholesterolemia or ADH is characterized by elevated total and LDL cholesterol. Classic clinical signs of ADH include lipid deposits, such as skin and tendinous xanthomas, and atheromas. Arcus corneae, a white ring of lipid deposition around the cornea, occurs in about 50% of those with ADH who are over the age of 30.

Premature atherosclerosis and premature cardiovascular disease are common in ADH with a mean age of onset of cardiovascular disease (CVD) at 40 – 45 years in men and 50 – 55 years in women. If ADH is left untreated, 75% of individuals will have CVD by the age of 60.

ADH is one of the most common genetic diseases and occurs in all populations with a worldwide frequency estimated to be at 1 in 500 individuals. ADH occurs at a higher frequency in some populations especially populations in South Africa, Lebanon, and French Canada.

As implied by the name, ADH is an autosomal dominant genetic disease meaning that individuals who are affected usually have one affected parent and they may also have affected siblings. They also usually have a family history positive for premature CVD.

First-degree relatives of affected individuals, including offspring, have a 50% chance of having ADH.

There are three categories of specific genetic conditions caused by mutations and specific genes that fall under ADH.

First is Familial Hypercholesterolemia (FH). FH is due to mutations in LDLR gene. Second is Familial Defective ApoB-100 (FDB). FDB is due to mutations in APOB gene. Third is the rarer condition Autosomal Dominant Hypercholesterolemia type 3, which is due to gain of function mutations in PCSK9 gene. As a side note, loss of function mutations in PCSK9  leads to hypercholesterolemia. For our purposes today, we will focus on FH and FDB.

Familial Hypercholesterolemia

FH has been termed autosomal codominant, meaning that the condition can occur in the heterozygous or homozygous state. As many of you know, heterozygous means that the individual has a mutation on only one copy of their LDLR gene, whereas homozygous means that there are mutations in both copies of the LDLR gene.

While LDL levels can be variable, individuals with heterozygous FH generally have LDL levels greater than 200 mg/dL, while those who are homozygous have LDL levels as high as 1,000mg/dL, sometimes higher. It is important to note that the cholesterol levels rise with age, so individuals who are younger or who have less penetrant mutations, may not demonstrate such highly elevated levels of LDL.

FH is associated with premature mortality from CVD. Heterozygous FH individuals tend to have atherosclerotic lesions in their 30s and 40s which are associated with a premature coronary artery disease (CAD). Homozygous FH individuals may have lesions as early as 6 years old. MIs can occur sometimes even in the infant stage and they often have death by 20.

FH-Primary Defect in LDLR

As mentioned earlier, mutations in the LDLR gene, which encodes through the Low Density Lipoprotein Receptor (LDLR), are the underlying cause of FH. The LDLR protein is present on the cell surface of most cell types. As the receptor for LDL, LDLR binds LDL and mediates its uptake into cells. Defects in the receptor result in lower uptake of LDL and increased levels of plasma LDL. The excess LDL is stored in cells and deposited as skin and tendinous xanthomas and atheromas.

LDLR Gene

The LDLR Gene is located on chromosome 19p and contains 18 exons, and is 45 kilobases in length. There have been over 500 mutations, most of them familial, reported across the gene and new mutations are described on a regular basis. Mutations can occur anywhere in the gene, but about 20% of all LDLR mutations occur in exon 4. Mutations have also been described in the transcription regulatory regions of the promoter of LDLR.

LDLR mutations are most commonly missense mutations, meaning that they result in a change from one amino acid to a different amino acid. It is important to note that a significant percentage of LDLR mutations are due to genomic rearrangements. This is caused by a recombination between Alu-repeat elements within and near the LDLR gene. These genomic rearrangements lead to deletions and duplications of exons. It has been demonstrated that different mutations will have different clinical phenotypes and the same mutation can lead to variability in phenotypic severity, both within and between families. Thus there is a significant level of clinical variability among individuals with genetically defined FH.

Familial Defective ApoB-100

A second genetic disease that falls under the ADH umbrella is Familial Defective ApoB-100, or FDB. It’s responsible for about 15% of ADH. FDB is caused by mutations in the APOB gene, which encodes apolipo-protein B-100. This is the major protein component of LDL. FDB-causing mutations in APOB result in defective binding of LDL to LDLR, leading to elevated plasma levels of LDL.

The most common APOB mutation is R3500Q, and it occurs at a frequency of about 1 in 500 to 1 in 700 in the Caucasian population. A rarer mutation that has been shown to occur repeatedly in individuals with FDB is the R3500W mutation. This has been shown mainly in individuals of Scottish and Asian ancestry and it accounts for about 2% of FDB. While the frequency of APOB mutations is relatively high, it is important to note that the penetrance of APOB mutations are not as high as the penetrance of LDLR mutations, thus explaining the lower phenotypic mutation of FDB compared to the frequency of the APOB mutations. In addition, the severity of FDB is generally not as high as FH, and only about 40% of male and 20% of female APOB mutation carriers are predicted to develop CAD.

How is ADH Diagnosed?

Now that we have covered some background on FH and FDB, I’d like to discuss how ADH is diagnosed. Elevated cholesterol is an important sign of ADH. The cut points for elevated cholesterol are age-dependent and it is important to note that there are several different cholesterol cut points for establishing ADH. It is also important to note that there is a considerable range in severity of hypercholesterolemia among individuals with ADH.

In addition to hypercholesterolemia other classical clinical signs include skin and tendinous xanthomas. However, many patients will not have classical clinical signs and often times, these signs, including elevated cholesterol, do not appear until later in life.

Individuals with FH oftentimes have a family history of premature CVD and elevated cholesterol. However, not all patients, especially ones with de novo or low penetrant mutations will have a family history. Taking all of this into account, it is apparent that it can sometimes be difficult to properly diagnose ADH. In fact, it is thought that ADH is highly under-diagnosed.

Impact of Failure to Diagnose

Without a clear diagnosis of ADH, it is more difficult for clinicians to provide genetic counseling to the individual, genetic counseling to the family, and proper therapy at an early stage of the disease.

Why is Genetic Testing for ADH Useful?

This is where genetic testing comes into play so now I would like to discuss why genetic testing is useful for ADH.

First, it provides for a definitive diagnosis of a genetic disease and allows for the opportunity to rule-out other causes of high cholesterol, including environmental causes.

Second, once a mutation is identified in the family, other at-risk family members can be tested to determine if they have ADH.

Third, once a mutation is identified in an individual or family, genetic counseling can take place.

Fourth, genetic identification of ADH in an individual can lead to more optimal treatment of that individual including instituting proper and potentially aggressive treatment at an early stage of the disease. Furthermore with the knowledge of being positive for a genetic disease, the drug regimen might be more strictly adhered to by the patient. Studies have shown that when individuals realize they have a genetic rather than an environmental cause of high cholesterol, they tend to be more adherent to long-term statin therapy. Taken together, early treatment and better adherence lead to improved outcomes for these patients.

Statin Treatment-How Early?

While I’m on the topic of treatment for ADH, I’d like to discuss statin treatment in ADH a little further. Statins are the most common drug used to treat ADH and studies have demonstrated that statins should be given as early as possible in the course of ADH. In fact, atherosclerotic disease begins in childhood and is progressive. 50% of children and 85% of young adults have fatty streaks which are the earliest pathological abnormality in atherosclerosis.

In addition, it has been shown that 45% of 22-year-olds have evidence of atherosclerosis. Studies have been performed on the evaluation of statin treatment in children and adolescents and these studies have supported the short-term safety and efficacy of such treatment. However, studies examining the long-term treatment of statin treatment on children are lacking at this time.

ADH-Genetic Testing Methodology

Given all the information provided here thus far, it is apparent that genetic testing for ADH is important in many cases. Now I would like to take the opportunity to describe the genetic testing methodology that we employ at Mayo as well as take you through our ADH genetic test reflex algorithm.

The first part of our test involves targeted mutation analysis of the two common APOB mutations. The second part of our test involves sequencing of all 18 exons and the promoter of LDLR.  The sequencing assay will allow us to detect point mutations and small insertion/deletion-type mutations. The final aspect of our ADH genetic testing involves a gene dosage method to identify the LDLR gene rearrangements or exonic deletions and duplications that were discussed earlier. As mentioned, these types of mutations account for about 10-15% of all LDLR mutations. It is important to note that we require a method alternative to sequencing in order to detect this type of mutation.

Familial/Autosomal Dominant Hypercholesterolemia Diagnostic Algorithm

This slide gives a depiction of our ADH diagnostic algorithm by reflex genetic testing. Over the next few slides I will briefly take you step-by-step through the algorithm.

APOB-Targeted Genotyping Performed First

If there is a clinical suspicion of ADH, test number 83375 should be ordered. Once the test is ordered and our clinical genetics molecular laboratory receives whole blood in an EDTA tube from the patient, we will extract the DNA and then perform the first component of the test, which is APOB genotyping for the two common APOB mutations.

As mentioned previously, APOD mutations account for about 15% of ADH. While LDLR mutations are more common than APOB mutations in ADH, targeted genotyping of APOB is more cost-effective than full gene sequencing of LDLR, which is why APOB analysis is performed initially in our reflex panel.

APOB Mutation-Positive

If an APOB mutation is identified, testing is stopped and an interpretive report is provided. Because a mutation has been identified in a patient, genetic counseling should be pursued and it may be important to consider ordering test 89097 to investigate the possibility of the same APOB mutation in an at-risk family member.

APOB Mutation-Negative

If an APOB mutation is not identified, then testing is automatically reflexed to LDLR sequencing. This involves bidirectional, fluorescent sequencing of all 18 exons and the promoter of LDLR. Sequencing of LDLR will detect approximately 85-90% of all LDLR mutations.

LDLR Sequence Mutation-Positive

If a mutation in LDLR is identified, testing is stopped and an interpretive report is provided. Because a mutation has been identified in a patient, genetic counseling should be pursued. It may be important to consider ordering test number 81183 to investigate the same LDLR mutation in at-risk family members. Note that this test involves sequencing of only the LDLR exon involved in the family and thus is more cost-effective than full gene sequencing.

LDLR Sequence Mutation-Negative

If an LDLR mutation is not identified, then testing is automatically reflexed to LDLR large deletion/duplication analysis. As mentioned earlier, this method will detect approximately 10-15% of all LDLR mutations.

LDLR Large Del/Dup Mutation-Positive

If an LDLR large deletion/duplication mutation is identified, testing is stopped and an interpretive report is provided. Because a mutation has been identified in a patient, genetic counseling should be pursued. It may be important to consider ordering test number 89073 to investigate the possibility of the same LDLR mutation in at-risk family members.

LDLR Large Del/Dup Mutation-Negative

If an LDLR large deletion/duplication mutation is not identified, testing is stopped and an interpretive report is provided.

Summary

To summarize what has been discussed today, ADH is a common genetic disease with an incidence of approximately 1 in 500 worldwide. ADH is highly under-diagnosed for several reasons, including that classical signs may not appear until later in life and criteria and cut points for hypercholesterolemia are variable. The definitive diagnosis of the genetic disease FH or FDB is important because other causes, including environmental causes, of hypercholesterolemia can be ruled out. Early and potentially more aggressive treatment can be instituted and may be more likely to adhered to, and at-risk family members can be ruled-in or ruled-out as having the disease. Finally, again because of the variability in clinical presentation and diagnostic criteria, genetic testing provides for a definitive diagnosis of a genetic disease.

Conclusion

In conclusion, the FH/ADH Genetic Reflex Panel that Mayo offers is comprehensive in that it involves testing for common mutations in APOB, sequencing of all exons and the promoter of LDLR to identify point mutations and small insertion/deletion mutations, and gene dosage analysis of LDLR for large deletion/duplication mutations.

In addition, Mayo’s FH/ADH Genetic Reflex Panel was designed to be more cost-effective than ordering all 3 tests at the same time.


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