Correlation of Polymorphisms of Lipoprotein Lipase (LpL) and Apolipoprotein E (Apo E) With Lipid Profile of Children With Acute Lymphoblastic Eukaemia During Therapy With L - Asparaginase.
Correlation of Polymorphisms of Lipoprotein Lipase (LpL) and Apolipoprotein E (Apo E) With Lipid Profile of Children With Acute Lymphoblastic Eukaemia During Therapy With L - Asparaginase.
Haematological malignancies constitute the most common neoplastic disease in child
 population, with acute leukemia occupying the number one spot with a percentage of 32.8%. In
 children, leukaemia is primarily encountered in its acute form (97%) and in the majority of
 the cases it is presented as Acute Lymphoblastic Leukaemia - ALL (80%). Acute
 Non-Lymphoblastic Leukemia - ANLL is encountered less frequently (17%) and it includes Acute
 Myelogenous Leukaemia - AML (15%) and some other rare forms (2%), while the remainder 3%
 corresponds to chronic leukaemia.

 L-Asparaginase (L-ASP) is a fundamental component during the loading phase with regards to
 achieving remission of the disease and, likewise, during the maintenance phase with the
 intention of establishing that remission in both children and adults suffering from ALL. The
 cytotoxic effect of the exogenous administration of Asparaginase is caused by the depletion
 of the reserve of asparagine in the blood. Asparaginase (ASP) acts as a catalyst for the
 hydrolysis of asparagine to aspartic acid and ammonia. Asparagine is vital for protein and
 cell synthesis and, therefore, for their survival. The normal cells of the human body have
 the ability to produce asparagine from aspartic acid, with the assistance of the enzyme
 asparagine synthetase. However, the neoplastic cells either lack the enzyme completely or
 contain minute amounts of it resulting in their inability to synthesize asparagine de novo.
 The survival of these cells and their ability to synthesize proteins depends entirely on
 receiving asparagine from the blood. Thus, the administration of ASP leads to the inhibition
 of DNA, RNA and protein synthesis which, in turn, results in the apoptosis of these cells.

 Despite L-ASP's paramount importance in the chemotherapy treatment of leukaemia, it is
 responsible for a plethora of toxic adverse effects that sometimes even require the
 termination of its administration. A critical adverse event of ASP is a disorder in the
 metabolism of lipids. Specifically, it appears that the activation of the endogenous pathway
 that produces triglycerides through hepatic synthesis leads to hypertriglyceridaemia. The
 liver is capable of synthesizing VLDL (Very Low Density Lipoproteins) that are rich in
 triglycerides. Utilising the effect of the enzyme Lipoprotein Lipase (LpL), located on the
 vascular endothelium, the triglycerides detach from the VLDL causing the latter to transform
 into IDL (Intermediate Density Lipoproteins) and afterwards into LDL (Low Density
 Lipoproteins). The triglycerides are later extracted from the blood circulatory system and
 stored in the adipose tissue, while the LDL particles connect with tissue receptors or
 macrophage receptors. The final products of the breakdown (coming from the peripheral
 hydrolysis of triglycerides with the help of LpL) of chylomicrons, VLDL, the remnants of
 lipoproteins, will eventually be removed by hepatic receptors. Apolipoprotein E (Apo-E) plays
 an important role in this procedure, it binds these remnants in the presence of LpL and
 hepatic lipase. Along the duration of the treatment with ASP, reduced LpL functionality is
 recorded, resulting in impaired plasma clearance of triglycerides and an increase in their
 levels, while L-ASP appears to cause disorders in other lipid factors, such as cholesterol,
 HDL and apolipoprotein A. Disorders of lipid metabolism have been found to be associated with
 polymorphisms of the LpL and Apo-E genes, sometimes with positive and sometimes with negative
 effects on the lipid profile and more likely participation in cardiovascular complications.
 The current study will evaluate, the lipid profile of children with ALL, the effect of L-ASP
 on the lipid profile of the aforementioned patients, as well as the correlation between the
 polymorphisms of Lipoprotein Lipase (LpL) and Apolipoprotein E (ApoE) with the values of the
 lipids during chemotherapy. Both the universal and national bibliography that pertain to the
 effect of ASP on the potency of LpL and App E and to the values of the lipids in children
 that suffer from ALL during chemotherapy with L-ASP is limited, while there exists no
 bibliographic reference correlating the genetic background to LpL and Apo E and the relation
 of the lipid profile. The current study will examine for the first time gene polymorphisms of
 LpL and Apo E in children with ALL during treatment with ASP.
Introduction

 Malignancies during childhood constitute the 2nd cause of death, following injuries
 worldwide. According to epidemiological data, 300,000 new cases of neoplasia present
 themselves every year in children and teenagers under 19 years of age1, 160,000 of which
 concern children under the age of 15. Haematological malignancies constitute the most common
 neoplastic disease in child population, with acute leukemia occupying the number one spot
 with a percentage of 32.8%, followed by central nervous system tumors with a percentage of
 21% on the second spot and lymphoma with a percentage of 12% on the third spot.

 Definition - Pathophysiology

 Leukaemia (L) is defined as a group of perturbations characterized by uncontrolled
 proliferation of white blood cells, due to the bone marrow's primary divergence from normal
 and the its infiltration by immature undifferentiated stem cells. By definition, more than
 20% of the bone marrow is infiltrated by stem cells. Leukaemia's exact pathogenic mechanism
 has not been clarified as of yet, it appears, however, to be the result of genetic mutation
 and damage to the multipotential haematopoietic ancestral cells, during one of their distinct
 stages of differentiation. This fact leads to clonal expansion and inhibition of
 differentiation. The leukaemic cell's immunophenotype reflects the level of differentiation
 achieved by the neoplastic clone. The leukaemic cells divide at a slower pace and require
 additional time to synthesize DNA compared to normal haematopoietic cells causing anaemia,
 thrombocytopenia and neutropenia. At the time of the diagnosis, the leukaemic cells could
 have, not only replaced the normal cells of the marrow but also expanded into extra-medullary
 areas.

 The various types of leukaemia are exceptionally heterogeneous and, depending on the cells'
 origin, they are differentiated into lymphoblastic and non-lymphoblastic leukemias. Each one
 of the above mentioned subgroups can be encountered in its Acute Leukaemia (AL) or Chronic
 Leukaemia (CL) form. In the acute form, the immature cells of the haematopoietic tissue are
 dominant and the progression of the disease without any treatment leads quickly to death. In
 the chronic forms, the mature cells dominate the tissue and the development of the disease,
 generally, takes longer. In children, leukaemia is primarily encountered in its acute form
 (97%) and in the majority of the cases it is presented as Acute Lymphoblastic Leukaemia (ALL,
 80%). Acute Non-Lymphoblastic Leukaemia - ANLL is encountered less frequently (17%) and it
 includes Acute Myelogenous Leukaemia - AML (15%) and some other rare forms (2%), while the
 remainder 3% corresponds to chronic leukaemia. AL occurs more often between the ages of 2 and
 6 years old while the incidence is higher in boys compared to girls (1:1.3). 2.5% - 5% of
 children's ALL and 6% - 14% of children's AML is encountered in infants under 1 year old. The
 characteristic type of ALL is the B-cell ALL, which is additionally the most common in
 children (85%) compared to the T-cell ALL (15%) which is accompanied by masses in the lymph
 nodes of the mediastinum amongst other areas.

 Epidemiology

 In the United States of America (USA) and most of the countries of Western Europe, the
 frequency of leukaemia in children is 3.5 - 4.0 cases per 100,000 childhood population yearly
 and it is ranked as the 20th cause of death among malignancies of all ages. It is deduced
 from large-scale cohort studies that the incidence of childhood cancer in developed countries
 is rising 1% every year. The prevalence of ALL in children was detected for the first time in
 the 1930s in the United Kingdom (UK) and the USA. In the USA, ALL appeared first in children
 of European descent, and afterwards, in children of African descent in the 1960s. The
 frequency of ALL is lower in developing and underdeveloped countries, a fact that hints at a
 possible correlation between industrialization and leukaemogenesis.

 Asparaginase (ASP)

 The chemotherapy combination treatments used today to treat ALL have improved significantly
 the long-term course of the disease with the overall five-year-survival rate exceeding 80%,
 while the respective ratio in the 1960s was under 30%. One of the main reasons that
 contributed to this development is the intensive, extended and higher dosed administration of
 Asparaginase. L-Asparaginase (L-ASP) is a fundamental component during the loading dose with
 regards to achieving remission of the disease and, likewise, during the maintenance dose with
 the intention of establishing that remission in both children and adults suffering from ALL.
 The cytotoxic effect of the exogenous administration of asparaginase is caused by the
 depletion of the reserve of asparagine in the blood. Asparaginase acts as a catalyst for the
 hydrolysis of asparagine to aspartic acid and ammonia. Asparagine is vital for protein and
 cell synthesis and, therefore, for their survival. The normal cells of the human body have
 the ability to produce asparagine from aspartic acid, with the assistance of the enzyme
 asparagine synthetase. However, the neoplastic cells either lack the enzyme completely or
 contain minute amounts of it resulting in their inability to synthesize asparagine de novo.
 The survival of these cells and their ability to synthesize proteins depends entirely on
 receiving asparagine from the blood. Thus, the administration of asparaginase leads to the
 inhibition of DNA, RNA and protein synthesis which, in turn, results in the apoptosis of
 these cells. The low levels of asparagine only affect the survivability of the cancer cells,
 leaving the healthy cells undisturbed.

 The ability of L-ASP to suppress the proliferation of the leukaemic cells was first observed
 in clinical studies during the 1970s, and from that moment on it has remained the cornerstone
 of childhood ALL treatment. The natural forms of the enzyme, produced by the bacteria Erwinia
 chrysanthemi and Escherichia coli (E.Coli), are being used for treatment, much like L-ASP's
 product created from the conjugation of E. Coli. with polyethylene glycol
 (Pegylated-Asparaginase, PEG-ASP) with prolonged half-life of 5.5 days instead of the 26 hour
 half-life of the natural forms. Chemotherapy protocols for ALL recommend a dosage of 1,000 -
 2,500 IU/m2 of PEG-ASP and 5,000 - 12,000 IU/m2 of the natural form.

 Adverse Reactions of Asparaginase

 Despite L-ASP's paramount importance in the chemotherapy treatment of leukaemia, it is
 responsible for a plethora of toxic adverse effects that sometimes even require the
 termination of its administration. Studies have shown that the omission of administration of
 doses due to the emergence of toxic adverse effects in previous administrations is linked to
 poor prognoses in children with ALL. The most common adverse effects are hypersensitivity
 reactions, as studies indicate their rate occurrence is as high as 75%. The ASP that is being
 utilized is a large molecule of bacterial origin (mainly E. Coli) and thus is able to induce
 an immune response which grows more intense after repeated exposure and, mainly, after a
 delay of the drug administration. Aside from the clinical hypersensitivity reactions that
 signal an immediate response from the attending medical staff, there also exist subclinical
 reactions that can lead to a decrease in the potency of the drug that is difficult to detect
 in order to act swiftly. Pancreatitis is reported at a rate as high as 18% of the patients
 with ALL during treatment with ASP. The pathogenic mechanism is not yet known, however it
 appears that the fault lies with the decrease of protein synthesis from organs with
 significant protein turnover, like the pancreas. Moreover, it is considered that this
 mechanism is also responsible for the hepatotoxicity detected during treatment with
 asparaginase. Additionally, the ASP induced decrease in protein synthesis is involved in the
 processes of coagulation and fibrinolysis which in turn leads to thrombotic episodes of the
 central nervous system and deep vein thrombosis of the extremities. Other adverse reactions
 are hyperglycaemia, as a result of decreased insulin production and myelosuppression, mainly
 due to enhancing the suppression effect of the other chemotherapy drugs. Furthermore, there
 have been reports of incidents of encephalopathy during therapy with ASP that could be
 related with the elevated levels of ammonia that result from the breakdown of ASP.

 A critical adverse event of ASP that could result in additional complications if it is not
 promptly identified is a disorder in the metabolism of lipids. Specifically, it appears that
 the activation of the endogenous pathway that produces triglycerides through hepatic
 synthesis leads to hypertriglyceridaemia. Some studies indicate that the rate of high values
 of triglycerides is 67% 8 - 14 days after administering the medicine. The liver is capable of
 synthesizing VLDL (Very Low Density Lipoproteins) that are rich in triglycerides. Utilizing
 the effect of the enzyme Lipoprotein Lipase (LpL), located on the vascular endothelium, the
 triglycerides detach from the VLDL causing the latter to transform into IDL (Intermediate
 Density Lipoproteins) and afterwards into LDL (Low Density Lipoproteins). The triglycerides
 are later extracted from the blood circulatory system and stored in the adipose tissue, while
 the LDL particles connect with tissue receptors or macrophage receptors. The final products
 of the breakdown (coming from the peripheral hydrolysis of triglycerides with the help of
 LpL) of chylomicrons, VLDL, the remnants of lipoproteins, will eventually be removed by
 hepatic receptors. Apolipoprotein E (apo-E) plays an important role in this procedure, it
 binds these remnants in the presence of LpL and hepatic lipase.

 Lipoprotein Lipase (LpL)

 The LpL gene is located on the chromosome 8p22 and is comprised of 10 exons. There have been
 recorded roughly 100 mutations, with 3 of them being the most common, 2 of which are
 connected with reduced functionality of LpL and increased cardiovascular risk. The mutation
 known as polymorphism N291S is present in 2-5% of the general population and its existence is
 associated with decreased functionality of LpL and an imbalance in the VLDL metabolism
 [increase of triglycerides, decrease of HDL (High Density Lipoproteins)]. In addition, it
 appears that the effect of this mutation is amplified by the coexistence of other genetic
 factors (familial hypercholesterolaemia, apolipoprotein E2/E2). The second mutation, known as
 polymorphism D9N, is related to increased risk of atherosclerosis, even if the mutation
 causes a light decrease in the functionality of LpL. As of late, it was observed that the
 combination of small nucleotide polymorphisms (SNP) on the genes of Apo E and of LpL
 denominate a high-risk group for cardiovascular disease. Lastly, the third mutation,
 encountered in the 20% of the general population, polymorphism S447X is linked with slightly
 elevated HDL levels and low levels of triglycerides and thus has a positive impact on the
 lipid profile.

 Along the duration of the treatment with ASP, reduced LpL functionality is recorded, likely
 caused by the altogether reduced protein synthesis from the liver, as is apparent from the
 decreased concentrations of other proteins, such as albumin and fibrinogen. Studies have
 demonstrated, in particular, that the suppression of the LpL's effects is connected with an
 increase in the ratio of ApoCIII/ApoCII, where ApoCII is an essential cofactor of LpL and it
 promotes the hydrolysis of triglycerides and ApoCIII acts as an inhibitor of LpL while
 simultaneously appears to be able to regulate the levels of triglycerides in the serum
 independently from LpL. Hence, a problem is created in the triglyceride cleansing from the
 plasma as their levels rise. Another suggested mechanism for the high levels of triglycerides
 during treatment with asparaginase is the increase in VLDL synthesis, which is also reflected
 in the high apolipoprotein ApoB100 concentration, a component of VLDL.

 Apolipoprotein E (Apo E)

 The gene of Apo E is situated on chromosome 19 and it is comprised of 4 exons. Apolipoprotein
 E (Apo E) is one of the structural components included in lipoproteins, which are rich in
 triglycerides, and it plays an important role in the hepatic protein intake. Apolipoprotein E
 (Apo E) has 3 alleles ε2, ε3, ε4 [APOE-ε2 (cys112, cys158), APOE-ε3 (cys112, arg158) and
 APOE-ε4 (arg112, arg158)]. ε3 is the most common in the general population and it contains
 cysteine at position 112 and arginine at position 158. ε4 differs from ε3 at position 112,
 where instead of it substitutes the cysteine for arginine55. The isomorph ε2 has a weaker
 connection with the lipoprotein receptors of the liver which results in the accumulation of
 remnants from chylomicrons and VLDLs.

 ApoE4 attaches to the lipoprotein receptors in the liver in vitro having the same affinity as
 ApoE3, however the effect of ApoE4 in the cleansing of lipoproteins rich in triglycerides in
 vivo is undefined as of yet. The ε2 allele is connected with higher triglyceride levels after
 fasting, while the ε4 allele is connected with hypercholesterolaemia and high LDL
 cholesterol. The individuals with the genotype E3/E4 display slower cleansing of lipoproteins
 rich in triglycerides in comparison with individuals with the genotype E3/E3. The ε4 allele
 looks to be associated with an affected triglyceride metabolism. The individuals with the ε2
 allele have lower cholesterol levels compared to the individuals with ε3 and ε4 alleles.
 Studies indicate, further, that individuals with genotype E2/E4 have higher triglyceride
 levels after fasting in relation to the individuals with genotype E3/E3.

 Alterations in the ApoE gene lead to changes in the amino acids of the coded protein, which
 results in a modified structure of the normal protein. The latter results in a slower
 cleansing of lipoproteins rich in triglycerides. In one study, it was revealed that there was
 a higher prevalence of the phenotype E4/E3 in patients with hypertriglyceridaemia compared to
 patients with normal triglyceride levels. Additionally, no significant statistical difference
 was observed between triglyceride levels and the phenotypes of ApoE after treatment with
 asparaginase.

 Asparaginase and Lipid Profile

 With regard to the effect of asparaginase on cholesterol levels, the studies do not allow to
 reach any reliable conclusion, as some studies found an increase in a small percentage of the
 patients after administering the medicine, others indicated a significant increase, while
 others yet presented a small but not significant decrease. Regarding the HDL levels, studies
 have discovered that they are lower in children suffering from malignancies, specifically of
 a haematological type. Another study focusing on children with ALL, during and after
 treatment with ASP the HDL levels rose with a simultaneous drop Apo-A1 levels. HDL plays a
 crucial role in the process of reverse transfer of cholesterol from the peripheral tissue to
 the liver, a mechanism that imparts on HDL the properties of an anticoagulant agent.
 Functioning in tandem with the aforementioned properties, other parts of HDL have
 antioxidant, anti-inflammatory, anti-apoptotic and anticoagulant properties that contribute
 to safeguarding against atherogenesis. Lipoprotein Apo-A1 comprises the main structural
 protein for HDL. It is apparent that asparaginase as an inhibitory factor of protein
 synthesis could possibly modify the ratio of lipids/ proteins in favor of the first on the
 molecules of HDL. Low HDL and Apo-A1 levels have been noticed even in free of disease
 survivors of ALL after the end of treatment. This observation is of particular importance if
 one thinks about the danger of atheromatosis and coronary disease due to low levels of HDL.

 Hypertriglyceridaemia is able to become the sole contributor to the development of acute
 pancreatitis. When the levels of triglycerides rise above 900 mg/dl they form chylomicrons,
 molecules big enough in size that they can obstruct the pancreatic capillaries leading to
 ischaemia and release of pancreatic lipase. The latter will reinforce the lipolysis with an
 increase of free fatty acids which will, in turn, allow the release of free radicals and
 pro-inflammatory derivatives causing, in the end, inflammation and necrosis. As seen above,
 asparaginase is capable of taking part in the pathogenesis of pancreatitis through two
 routes, an independent and a dependent upon triglycerides. Hypertriglyceridaemia could also
 promote a predisposition for thrombosis in patients suffering from ALL and who are under
 treatment with ASP, as on one hand, the medicine is connected to modifications of the
 fibrinolytic system, as was already mentioned, meanwhile on the other hand the increase of
 triglycerides causes hyper-viscosity of the blood raising the risk of thromboembolic
 complications. Drawing from the cases documented in the universal bibliography, it is made
 clear that the majority of the thromboses involves the central nervous system (CNS), while on
 second place are cases of deep vein thrombosis of the upper extremities.

 Lastly, it is significant to add that administration of ASP to children with haematological
 malignancies in multiple phases of the treatment is co-administrated with corticosteroids,
 either prednisone or dexamethasone. It can be easily perceived that corticosteroids can
 synergize and intensify the adverse events, such as hyperlipidaemia, pancreatitis and
 thrombosis. Furthermore, asparaginase can contribute to some adverse events of the
 corticosteroids, such as osteonecrosis through the hyper-coagulant state that it creates.
 Since both of these medications are pivotal therapeutic factors, a very close and attentive
 monitoring of the patient's condition and emergence of adverse events is required in order to
 intervene if necessary.

 Originality

 Both the universal and national bibliography that pertain to the effect of asparaginase on
 the potency of LpL and Apo E and to the values of the lipids in children that suffer from ALL
 during chemotherapy with ASP is limited, while there exists no bibliographic reference
 correlating the genetic background to LpL and Apo E and the relation of the lipid profile.
 The current study will examine for the first time gene polymorphisms of LpL and Apo E in
 children with acute ALL during treatment with ASP.

 Importance

 In this particular study investigators will focus on researching the lipid profile of
 children with ALL, on the effect of ASP on the lipid profile of these patients, as well the
 correlation between the polymorphisms of lipoprotein lipase (LpL) and Apolipoprotein E (ApoE)
 with the values of lipids for the duration of the chemotherapeutic protocol. The hypothesis
 that is going to be tested is whether recording the genotypes of the under-study children
 with ALL will be able to constitute a preemptive indicative factor for the outcome of their
 treatment.

 In this manner, vital conclusions will be extracted for the probable contribution of
 genotypes of children that are being administered L-ASP to the laboratory demonstration of
 lipid disorders during treatment, the closer supervision during the corresponding phases of
 treatment and the timely therapeutic intervention in order to prevent any complications.

 Methodology

 Study Type: Prospective case-control study The characterization epidemiologic study is in
 complete accord with the aim of our study, a study of the effect of ASP on the lipid profile
 of children and adolescents with ALL (Patient Group) as well as the correlation of the
 polymorphisms of LpL and ApoE with the lipid levels duration of the chemotherapeutic
 protocol. The existence of two additional children groups that will act as population-control
 (children and adolescents of the same age group that do not manifest the disease and
 participants of the similar ages that have manifested the disease and have completed
 chemotherapy as per protocol for at least 6 months prior) specialises our study type into a
 case-control study.

 Study Site: The study will be conducted in the Paediatric and Adolescent Haematology -
 Oncology Unit of the 2nd Department of Paediatrics, Aristotle University of Thessaloniki
 (AUTH), at University General Hospital AHEPA.

 Description of the recorded parameters

 During the disease's diagnosis, the lipid profile of the patients' will be determined by
 measuring the following parameters: total cholesterol, triglycerides, HDL-cholesterol,
 LDL-cholesterol, apolipoprotein A1, apolipoprotein B100, lipoprotein α [Lp(α)], glucose,
 Serum Glutamic Oxaloacetic Transaminase (SGOT), Serum Glutamic Pyruvic Transaminase
 (SGPT),Thyroid-Stimulating Hormone (TSH), Free Thyroxine (FT4), amylase, lipase.
 Additionally, a genetic analysis for polymorphisms of LpL and Apo E will be conducted.
 Similar measurements will be done to all participants of all groups.

 The participants of group A will be immediately subjected to the chemotherapy protocol ALLIC
 BFM 2009. During the loading dose, these children will receive treatment with prednisolone
 for 33 consecutive days and asparaginase will be added on days 12, 15, 18, 21, 24, 27, 30,
 33. During the maintenance phase the children will be subjected anew to treatment with
 corticosteroids, dexamethasone, and asparaginase will be added on days 8, 11, 16, 18. The
 lipid identity will be checked before administration of ASP on day 0 and 11 as well as after
 the administration of ASP and before the administration of the next dose at the loading dose,
 meaning days 15, 24, 33. Similarly, the lipid identity will be checked on the maintenance
 phase, meaning on days 8, 16, 21 as well as after the end of every phase of chemotherapy
 protocol in which ASP is not administered.

 Moreover, an examination of LpL polymorphisms (the three most common polymorphisms p.N291S,
 p.D9N, p.S447X) and of Apo E polymorphisms [ε2(rs7412-T,rs429358-T), ε3(rs7412-C, rs429358-T)
 and ε4 (rs7412-C, rs429358-C)] will be performed after isolating the DNA from the peripheral
 blood and analyzing it with molecular techniques.

 Participants that meet all the inclusion criteria for this study will be examined and undergo
 treatment with ASP (Patient group) and will be compared to the corresponding teams of healthy
 children and children that have ailed and are off chemotherapy for at least 6 months
 (Controls group).
Inclusion Criteria:

 Children and adolescents newly diagnosed with acute lymphoblastic leukaemia. A complete
 personal and family history will be recorded for each participant from every group with
 particular emphasis on the presence of dyslipidaemia, early cardiovascular disease or
 cerebrovascular accident in the individual or in the family. A thorough clinical
 examination will be performed and the vital signs, blood pressure, weight (kg), height (m)
 and a Body/Mass Index (BMI) will be recorded and calculate.

 The Exclusion criteria are the following:

 -  Thyroid Disorder (Abnormal thyroid function)

 -  Familial Hypercholesterolemia

 -  High Body/ Mass Index (BMI)

 -  Administration of corticosteroids some time during the past 2 weeks before diagnosis

 -  Administration of total parenteral nutrition before the diagnosis

 -  Administration of plasma before the diagnosis

 -  Changes or variation of the parameters under study before the start of treatment and
 the 11th day (before the start of asparagine administration).