Dried Blood Spot Testing

Methodology

Real time polymerase chain reaction (PCR)

Patient Preparation

None

Preferred Specimen

1.0 mL whole blood collected in EDTA (Lavender Top)

Alternate Specimen

• Buccal swab
• Dried Blood collected on an AdvanceDx 100 card

Transport Temperature

Refrigerated (ship on frozen cold packs)

1. LIPOPROTEIN (A) OR LPA GENOTYPE (CVD RISK & ASPIRIN BENEFIT)

• Lipoprotein(a) or Lp(a) is a low-density lipoprotein (LDL)-like particle that has another protein known as apolipoprotein(a) or apo(a) attached to its apolipoprotein (apo) B-100 or protein component. Elevated plasma or serum levels of Lp(a) >50 mg/dL have been associated with significantly increased CVD risk. About 20% of families with premature CVD have significantly elevated Lp(a) levels (1,2). Your serum Lp(a) levels can be measured with Boston Heart Diagnostics testing.
• Lp(a) levels are genetically determined mainly due to variation in the LPA gene. Rs3798220 is an important LPA gene variant that causes isoleucine to be replaced by methionine at amino acid position 4399 of the apo(a) protein within the active protease-like domain (3). This serine proteinase inhibits tissue-type plasminogen activator I activity and can affect clotting. This variant causes a form of apo(a) that has increased binding to atherosclerotic lesions and also promotes clot formation. This variant (T/C or C/C) is present in about 4% of the population and results in significantly elevated serum Lp(a) levels and a two-fold increased risk of CVD. This risk can be normalized with low dose daily aspirin (81-100 mg/day) (4-6). In addition, in both subjects with the C/T, and C/C variant all efforts should be made to normalize all other CVD risk factors .

 

2. APOLIPOPROTEIN E (APOE) GENOTYPE (CVD & DEMENTIA RISK)

• ApoE is a protein that plays an important in the breakdown of triglyceride-rich particles in the bloodstream, as well as in the breakdown and removal of β-amyloid protein in the brain. β-amyloid protein is known to accumulate in the brains of patients with all-cause dementia and Alzheimer’s disease (7).
• There are three forms of the apoE protein. The most common form is apoE3, which has a cysteine at amino acid position 112 and an arginine at position 158 in the 299 amino acid chain. The next most common form is apoE4, with an arginine at residue 112 and an arginine at position 158. The least common form is apoE2, with a cysteine at position 112 and a cysteine at residue 158. ApoE3 is associated with normal breakdown of β-amyloid in the brain, while apoE2 is associated with increased breakdown and apoE4 is associated with decreased breakdown, resulting in excess β-amyloid in the brain and increased dementia risk (7).
• Subjects with the APOE3/3 genotype (about 60% of the population) have a normal risk of CVD and all-cause dementia, mainly Alzheimer’s disease (8-13).
• Subjects with the APOE2/2 genotype (about 1% of the population) or the 2/3 genotypes (about 11% of the population) have about a 50% reduction in the risk of all-cause dementia. APOE2/2 subjects are sometimes at increased risk of combined hyperlipidemia with elevations of both triglycerides and intermediate density lipoproteins (dysbetalipoproteinemia) if they also have combined hyperlipidemia (8-13).
• Subjects with the APO4/4 genotype (about 2% of the population) have about a 10-fold increased risk of dementia (greatly increased risk) (6). Subjects with the APOE3/4 genotype (about 24% of the population) or the APOE2/4 genotype (about 2% of the population) have about a 3-fold increased risk of dementia and an increased risk of CVD. ApoE3/4 and 4/4 subjects are also at increased risk for high LDL-C and are more responsive to diet low in saturated fats and less responsive to statins in terms of LDL-C lowering as compared to APOE3/3 subjects (8-15). Subjects of Chinese, Japanese, and Korean ancestry have even greater dementia risk associated with APOE4 genotypes and less risk reduction associated with APOE2 genotypes than Caucasians (13). African Americans are less affected by APOE genotypes in terms of dementia risk (13).
• All subjects should do everything they can to decrease their ASCVD and dementia risk (see below), and this is especially true for those having APOE4 genotypes.

 

3. METHYLENE TETRAHYDROFOLATE REDUCTASE (MTHFR) GENOTYPE

• MTHFR is the rate-limiting enzyme in the methylation of folate, necessary for the breakdown of homocysteine to methionine. Elevated serum homocysteine levels (>14 µmol/L) have been shown to be an important risk factor for CVD and all-cause dementia (doubles the risk) and have been associated with MTHFR variants, as well as with deficiencies of folate (<12 ng/mL) and vitamin B12 (<500 pg/mL), and vitamin B6. Lowering homocysteine with supplementation lowers risk of stroke and dementia (17-23).
• The normal form of MTHFR protein has an alanine at amino acid position 222 with the normal 677C/C genotype. About 30% of the population have a genetic heterozygous variant 677C/T of the MTHFR gene leading to a valine at position 222 associated with modestly decreased MTHFR activity and folate methylation, and modestly increased serum homocysteine levels. About 9% of the population have the homozygous 677T/T genotype associated with significantly decreased MTHFR activity and folate methylation, and usually increased serum homocysteine levels. These latter subjects may require methyl folate to normalize their serum homocysteine levels to <10 µmol/L if they cannot do so with normal folate, vitamin B12 and vitamin B6 supplementation (17-23).
• The normal form of MTHFR protein has a glutamic acid at amino acid position 429 with the MTHFR 1298A/A genotype. About 25% of the population has the heterozygous MTHFR 1298A/C genotype leading to an alanine at position 429 and modestly decreased MTHFR activity and folate methylation, and modestly increased serum homocysteine levels. About 8% of the population have the homozygous 1298C/C genotype associated with decreased MTHFR activity and folate methylation and increased serum homocysteine levels (17-23). These latter subjects may require methyl folate to normalize their serum homocysteine levels to <10 µmol/L if they cannot do so with normal folate, vitamin B12 and vitamin B6 supplementation (17-23).

 

4. FACTOR V LEIDEN (CLOTTING RISK)

• Factor V is a clotting factor that forms prothrombinase with factor X, which then converts prothrombin to thrombin. Thrombin converts fibrinogen into fibrin, which forms the clot.
• Factor V Leiden is the most common genetic hypercoagulability disorder among Caucasians being found in about 5% of the population. This genetic variant is due to an amino acid substitution in which arginine is replaced by glutamine at position 506 causing a decreased ability of protein C to inhibit the pro-clotting activity of factor V because of lack of binding. This circumstance leads to a hypercoagulable state with an increased risk of blood clots, especially deep vein thromboses (DVT), pulmonary emboli (PE), and strokes (24-26).
• Having factor V Leiden significantly increases the risk of: 1) having a first DVT or PE prior to age 50 years, 2) having recurrent DVT and PE events, 3) having a family history of DVT and PE events, and 4) having a DVT or PE during pregnancy. Up to 30% of subjects presenting with a first DVT or PE have Factor V Leiden.
  Use of hormones, such as oral contraceptive pills (OCPs) in pre-menopausal women and hormone replacement therapy (HRT), including estrogen and estrogen-like drugs in post-menopausal women increases the risk of developing DVT and PE. Healthy women taking OCPs have a three- to four-fold increased risk of developing a DVT or PE compared with women who do not take OCP.
• Women with factor V Leiden (-/+) who take OCPs have about a 35-fold increased risk of developing a DVT or PE compared with women without factor V Leiden and those not taking OCPs. Likewise, postmenopausal women taking HRT have a two- to three-fold higher risk of developing a DVT or PE than women who do not take HRT, and women with factor V Leiden who take HRT have a 15-fold higher risk.
• Women with heterozygous or homozygous factor V Leiden who are making decisions about OCP or HRT use should take these statistics into consideration when weighing the risks and benefits of treatment.
• Subjects with heterozygous factor V Leiden (-/+, 5.0% of the population) should be treated for at least 3 months with anticoagulant therapy after a DVT or PE. Thereafter chronic low dose aspirin therapy should be considered.
• Subjects with homozygous factor V Leiden (+/+, 0.05% of the population) are at 80 times the risk of developing a DVT or PE and should be considered for lifelong oral anticoagulation therapy (24-26).

 

5. PROTHROMBIN OR FACTOR 2 VARIANT (CLOTTING RISK)

• Prothrombin or Factor 2 (F2) is converted to thrombin by prothrombinase (formed by factor X and V). Thrombin in turn converts fibrinogen into fibrin, which forms the clot. Prothrombin is encoded by the F2-gene.
• About 2% of the Caucasian population have the F2 variant in the 3’ untranslated region (nucleotide 20210) which causes increased prothrombin production and a hypercoaguable state.
• Being heterozygous (-/+) for the F2 variants increases the risk of a DVT or PE 2.5-fold, while being homozygous (+/+) increases the risk 20-fold.
• Heterozygous carriers (-/+) who take OCP or HRT are at a 5-fold increased risk of VTE and PE, while carriers who are also heterozygous for factor V Leiden have a 20-fold increased risk.
• Subjects with the heterozygous F2 variant (-/+) should be treated for at least 3 months with anticoagulant therapy after a DVT or PE. Thereafter chronic low dose aspirin therapy should be considered. Subject heterozygous for both the F2 variant and Factor V Leiden as well as homozygotes (+/+) should be considered for lifelong therapy (12).

 

6. 4Q25 ATRIAL FIBRILLATION RISK GENOTYPE TEST

• The 4q25 gene locus is on chromosome 4, adjacent to the paired-like homeodomain 2 (PITX2) gene, which plays a major role in the development of the heart including the sinus node (which plays a critical role in the regulation of heart rhythm and rate).
• Two 4q25 variants rs2200733 and rs10033464, found in about 20% of the population, have been associated with about a 50-90% increase in the risk of atrial fibrillation (27-29). This association has been observed in multiple studies and is especially apparent in subjects over age 60 years.
• Atrial fibrillation causes the heart to beat irregularly and often rapidly, with resulting palpitations, heart failure, and blood clots in the heart, which can go up into the brain causing strokes. Atrial fibrillation should be treated with anticoagulation therapy (such as apixaban) in order to prevent this latter problem (27-29).

 

7. SLCO1B1 STATIN INDUCED MYOPATHY TEST

• Statins are the most widely prescribed lipid-lowering therapies for the treatment and prevention of cardiovascular disease. They lower LDL cholesterol by inhibiting cholesterol production. Statins have been shown to significantly decrease risk of CVD events and mortality in many placebo-controlled trials and are generally safe and well tolerated in most individuals (30).
• However, some patients may develop statin-associated muscle symptoms (SAMS), observed in 7-29% of patients in registries (30). In some cases very significant muscle inflammation can be observed, associated with marked increases in serum creatine kinase levels. These symptoms frequently lead to statin non-adherence and discontinuation, leading to higher rates of CVD events and mortality (31). Risk factors for SAMS include hypothyroidism, vitamin D deficiency, use of amiodorone, and co-enzyme Q10 deficiency (32).
• The organic anion-transporting polypeptide 1B1 (encoded by the solute carrier anion transporter 1B1 (SLCO1B1) gene) is responsible for the uptake and breakdown of statins in the liver. It was noted in a large placebo-controlled statin trial that a SLCO1B1 variant rs4149056 (found in about 13% of the population) had a 4.3-fold increase in risk of SAMS and that those that were homozygous for this variant had a 17.4-fold increased risk of SAMS (30).
• The SLCO1B1 variant is almost always associated with another variant rs4363657 which causes an amino acid substitution of valine by alanine at position 174 in the amino acid sequence of the organic anion transporter 1B1. This substitution results in decreased statin liver uptake, excess statin plasma levels, and increased SAMS risk (30,31). Simvastatin and atorvastatin at high doses are most likely to cause SAMS in affected patients (32,33). Patients with abnormal genotypes should be given low doses of rosuvastatin (10 mg/day) and in our view they should be supplemented with 200 mg/day of co-enzyme Q10 (32-34).