Familial HDL deficiency due to ABCA1 gene mutations with or without other genetic lipoprotein disorders

Atherosclerosis 172 (2004) 309–320

Familial HDL deficiency due to ABCA1 gene mutations with or without other genetic lipoprotein disorders Livia Pisciotta a , Ian Hamilton-Craig b , Patrizia Tarugi c , Antonella Bellocchio a , Tommaso Fasano c , Paola Alessandrini d , Gabriele Bittolo Bon d , Donatella Siepi e , Elmo Mannarino e , Luigi Cattin f , Maurizio Averna g , Angelo Balassare Cefalù g , Alfredo Cantafora h , Sebastiano Calandra c,1 , Stefano Bertolini a,∗ a

c

Department of Internal Medicine, University of Genoa, Viale Benedetto XV 6, I-16132 Genoa, Italy b North Adelaide Cardiac Clinic, Adelaide, Australia Department of Biomedical Sciences, University of Modena and Reggio Emilia, Via Campi 287, I-41100 Modena, Italy d S. Giovanni e Paolo Hospital, Venice, Italy e Department of Clinical and Experimental Medicine, University of Perugia, Perugia, Italy f Deparment of Medical Sciences, University of Trieste, Trieste, Italy g Deparment of Internal Medicine, University of Palermo, Palermo, Italy h National Institute of Health, Rome, Italy Received 18 July 2003; received in revised form 4 November 2003; accepted 7 November 2003

Abstract Mutations in ABCA1 have been shown to be the cause of Tangier disease (TD) and some forms of familial hypoalphalipoproteinemia (HA), two genetic disorders characterized by low plasma HDL levels. Here we report six subjects with low HDL, carrying seven ABCA1 mutations, six of which are previously unreported. Two mutations (R557X and H160FsX173) were predicted to generate short truncated proteins; two mutations (E284K and Y482C) were located in the first extracellular loop and two (R1901S and Q2196H) in the C-terminal cytoplasmic domain of ABCA1. Two subjects found to be compound heterozygotes for ABCA1 mutations did not have overt clinical manifestations of TD. Three subjects, all with premature coronary artery disease (pCAD), had a combination of genetic defects. Besides being heterozygotes for ABCA1 mutations, two of them were also carriers of the R3500Q substitution in apolipoprotein B and the third was a carrier of N291S substitution in lipoprotein lipase. By extending family studies we identified 17 heterozygotes for ABCA1 mutations. Plasma HDL-C and Apo A-I values in these subjects were 38.3 and 36.9% lower than in unaffected family members and similar to the values found in heterozygotes for Apo A-I gene mutations which prevent Apo A-I synthesis. This survey underlines the allelic heterogeneity of ABCA1 mutations and suggests that: (i) TD subjects, if asymptomatic, may be overlooked and (ii) there may be a selection bias in genotyping towards carriers of ABCA1 mutations who have pCAD possibly related to a combination of genetic and environmental cardiovascular risk factors. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Tangier disease; Familial HDL deficiency; ABCA1 gene; Familial defective Apo B (FDB); Lipoprotein lipase; Premature coronary artery disease

1. Introduction Hypoalphalipoproteinemia (HA), otherwise designated HDL deficiency and defined by plasma level HDL-C below ∗ Corresponding author. Tel.: +39-010-3537992; fax: +39-010-3537797. E-mail addresses: [email protected] (S. Calandra), [email protected] (S. Bertolini). 1 Co-corresponding author. Tel.: +39-059-2055423; fax: +39-059-2055426.

the 10th percentile of the distribution in the population, is the most prevalent lipoprotein abnormality found in patients with premature coronary artery disease (pCAD) [1]. The coronary risk associated with HA seems independent of the concentrations of LDL-C and triglycerides [2]. The protective effect of HDL against atherosclerosis is probably due to many factors such as: (i) reverse cholesterol transport, the process whereby cholesterol is transported by HDL from the peripheral tissues (including arterial intima) to the liver for excretion; (ii) protection of LDL from oxidation; (iii) preservation of endothelial cell function; (iv) reduction

0021-9150/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2003.11.009

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of platelet aggregation; (v) increase of fibrinolysis; (vi) reduction of inflammation [3–5]. HA may be due to environmental factors and life style (e.g. overweight, physical inactivity, smoking, medications), as well as to genetic defects (primary HA). Although in many cases of primary HA the underlying genetic defect is not clearly understood, mutations in some genes (such as ABCA1, LCAT, Apo A-I and LPL) are associated with low plasma HDL. Some mutations of these genes are also associated with an increased risk of pCAD [6]. The most severe form of HDL deficiency is Tangier disease (TD), a recessive disorder characterized by the accumulation of cholesteryl esters (CE) in macrophages, orange-yellow tonsils, hepato-splenomegaly, peripheral nerve neuropathy, corneal opacifications and pCAD in 50% of cases [7]. TD is due to mutations of the gene encoding the “ATP-binding cassette transporter 1” (ABCA1) [8], a 2261 amino acid peptide, which promotes the efflux of phospholipids and unesterified cholesterol (UC) from cell membrane to lipid-poor Apo A-I particles (pre-␤-HDL) [9]. Since 1999 more than 30 mutations of the ABCA1 gene have been identified in TD patients [7,10–23]. These patients were either homozygotes or compound heterozygotes depending on whether they carry two identical or two different mutant alleles of ABCA1 gene. Carriers of a single ABCA1 mutation (simple heterozygotes) have a variable reduction of plasma HDL level which is associated with a defect of ABCA1-mediated cell cholesterol efflux and an increased risk of pCAD. The latter condition, designated familial HDL deficiency (FHD), is more common than TD and has a co-dominant transmission [11,12,24–28]. Little is known about the factors which may modify the phenotype in subjects heterozygous for ABCA1 mutations, apart from age; increasing age is associated with a marked decrease of plasma HDL in these subjects as compared to unaffected controls [12]. No data have been reported so far on the effect of common polymorphisms of genes affecting lipoprotein metabolism (e.g. Apo E, LPL, Apo B) on the phenotype of subjects carrying ABCA1 mutations. In this work, we describe five patients with pCAD carrying mutations of ABCA1 gene. Three of them were found to be heterozygous for an ABCA1 mutation associated with a mutation in Apo B (R3500Q) in two cases and a mutation in LPL (N291S) in the other. The fourth patient was found to have two ABCA1 mutations without the classical manifestations of TD; the fifth patient was a carrier of a single ABCA1 mutation. In addition, we report a severe HDL deficiency in a young female with a family history of pCAD, who was discovered to be a compound heterozygote for two ABCA1 mutations. Furthermore, in four of these families we found several heterozygotes for ABCA1 mutations who carried the Apo E ε4 allele. Some of these families provide a unique opportunity to investigate the interaction between mutations in different genes affecting lipoprotein metabolism.

2. Methods 2.1. Kindreds 2.1.1. Family 1 (Fig. 1) The proband (II.1) was a 52-year-old Caucasian female from Australia who has been suffering from angina pectoris since the age of 41. Coronary angiography performed at 49 showed 60% stenosis of the left main coronary artery and 80% stenosis of the anterior descending artery. She had double-vessel CABG at 49 and PTCA with stent to the right coronary artery at 50. At the age of 23 years, she had been found to be affected by Crohn’s disease, which had been treated with various medications (salazopyrin and occasionally corticosteroids). At the age of 51 years, she underwent a right hemicolectomy for Crohn’s disease. She was a non-smoker and had normal blood pressure. She had no clinical evidence of cholesterol deposition in the form of arcus senilis, xanthelasma or xanthomas. In view of the elevated plasma total cholesterol and LDL-C associated with low HDL she has been treated with various statins and niacin. The proband’s 71-year-old mother (I.2) was apparently healthy. No information about the proband’s father (I.1) was available, apart from a family history of pCAD. 2.1.2. Family 2 (Fig. 1) The proband (I.2) was a 65-year-old Caucasian female from Slovenia who had myocardial infarction at the age of 58 years. She had no family history of pCAD and had normal blood pressure; she was a heavy smoker up to the coronary event. Coronary angiography showed a total occlusion of the right coronary artery and 50% stenosis of the left anterior descending and circumflex. Ultrasound evaluation of the carotid arteries (CAs) showed bilateral atherosclerotic plaques without significant stenosis. The proband’s 39-year-old son (II.1) was apparently healthy; he was a moderate smoker and had normal blood pressure. The proband’s 9-year-old grand-daughter (III.1) was affected by Friedreich ataxia due to GAA trinucleotide repeat expansion (920/950 repeats) in intron 1 of the frataxin gene (9q13). 2.1.3. Family 3 (Fig. 1) The proband (II.1) was a 47-year-old Caucasian female from Australia who was referred to the Lipid Clinic because of a long-standing personal history of low HDL and a family history of pCAD. Both paternal grandparents and her father (I.1) died from acute coronary disease at 54 (grandfather), 49 (grandmother) and 61. She was a non-smoker and had normal blood pressure. Physical examination did not reveal tendon or planar xanthomas or any signs of TD. Ultrasound examination of CAs showed minor plaques of the left internal CA and intimal thickening of the right CA. Helical computer tomography revealed a total coronary artery calcification (CAC) score of 41.0 units, placing the proband in the 92nd percentile for an apparently healthy person of the same age and gender. In view of the very low plasma HDL,

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Fig. 1. Pedigrees of Families 1, 2 and 3 with familial HDL deficiency. The probands are indicated by an arrow. The proband of Family 1 was a carrier of an ABCA1 mutation (E284K) and an Apo B mutation (R3500Q). Only the Apo B mutation was transmitted to the offsprings. The proband of Family 2 was a carrier of an ABCA1 mutation (N1800H) and a common LPL variant (N291S). Both mutations were transmitted to the proband’s son, but only LPL variant was present in proband’s grand-daughter. The Proband of Family 3 was a compound heterozygote for two ABCA1 mutations (Y482C and N1800H); the N1800H mutation was transmitted to her son.

she was separately treated with niacin and gemfibrozil (each, for 3 months)—without success. 2.1.4. Family 4 (Fig. 2) The proband (II.1) was a 62-year-old Caucasian male from Italy suffering from angina pectoris since the age of 59

years. Coronary angiography showed 99% stenosis of the left anterior descending. He underwent PTCA at the age of 61 years. Vascular ultrasound evaluation revealed plaques in both carotid and femoral arteries with 25–30% stenosis. He was a non-smoker and had normal blood pressure. The proband’s parents (I.1 and I.2) died at an old age. The other

Fig. 2. Pedigrees of Families 4, 5 and 6 with familial HDL deficiency. The probands are indicated by an arrow. The proband of Family 4 (the only family member available for the study) was a carrier of an ABCA1 mutation (Q2196H). The proband of Family 5 was a carrier of an ABCA1 mutation (R557X) and an Apo B mutation (R3500Q). The Apo B mutation was transmitted to the eldest daughter and the ABCA1 mutation to the twin daughters. The proband of Family 6 was a compound heterozygote for two ABCA1 mutations (H160FsX173 and R1901S). The frameshift mutation was inherited from the mother and the missense mutation from the father.

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members of the family refused to grant their consent to the study; however, the proband’s brother (II.2) had a history of severe peripheral arterial disease. 2.1.5. Family 5 (Fig. 2) The proband (II.1) was a 59-year-old Caucasian male from Italy with myocardial silent ischaemia, revealed during ECG stress testing. Coronary angiography showed a three-vessel disease. He underwent thyroidectomy for substernal goiter at 47 years. Since the age of 47 years, he has been treated with thyroid hormone. He was a smoker up to 48 years and has been treated with ACE inhibitors and ␤-adrenergic receptor blockers for arterial hypertension for 10 years. During the last 3 years, he has been treated with statins for hypercholesterolemia. Ultrasound examination of CAs showed plaques with 30–50% stenosis. Both the proband’s parents died at an old age, but they suffered from myocardial infarction; the proband’s father at 68 years and the mother at 72 years.

and sequenced as reported previously [21]. The mutations were designated according to the recommendations of the Nomenclature Working Group for human gene mutations [32]. 2.4. Genotyping for genetic variants of lipoprotein lipase (LPL) and Apo E polymorphism The probands and available members of the five families were also genotyped for D9N and N291S variants of LPL [33,34] and for Apo E polymorphism [35]. 2.5. Search for mutations in LDL-R and Apo B genes The LDL-R gene was sequenced [36] in the two hypercholesterolemic probands of Families 1 and 5. The region of Apo B gene encompassing the mutations known to cause FDB was sequenced directly as previously reported [37]. 2.6. Screening of ABCA1 gene mutations

2.1.6. Family 6 (Fig. 2) The proband (III.1) was a 16-year-old Caucasian female from Italy. She had a mild mental retardation and congenital bilateral radial-ulnar synostosis. Since the age of 11 years she has been under treatment with thyroid hormone for congenital hypothyroidism. Despite the presence of very low HDL level she had no clinical manifestations of TD. However, at the age of 12 years she had undergone adeno-tonsillectomy. The proband’s 10-year-old sister (III.2) was affected by dwarfism due to hGH deficiency and had low HDL levels. The proband’s parents (II.3 and II.4), who were unrelated, were apparently healthy and had normal blood pressure; the proband’s father was a heavy smoker. The proband’s mother had a family history of pCAD in the maternal line. In fact, proband’s maternal grandmother (I.1) suffered from angina pectoris and had coronary bypass surgery at 56 years; her sister (I.2) was obese, a heavy smoker, had normal blood pressure and no clinical or ECG signs of CAD. Informed consent was obtained from all subjects investigated. The study protocol was approved by the institutional human investigation committee of each participating institution. 2.2. Biochemical analyses Plasma lipids and apolipoproteins were measured as previously reported [21].

2.6.1. Del.c.479 (A) in exon 6 (H160FsX173) The screening for this mutation was performed by heteroduplex analysis. A 284 bp fragment encompassing exon 6 was amplified using the following primers: 5 -ATG ATA ACG TTT CTC CAC TGT CCC-3 (forward primer) and 5 -TTC TTT CCA GTA GCT GCA CAA CG-3 (reverse primer). The experimental conditions were those previously reported [21]. 2.6.2. Transition c.850 G > A in exon 9 (E284K) A 181 bp fragment of exon 9 was amplified using a forward mismatched primer: 5 -AAG CTG GAG TGA CAT GCG AT C G-3 and a canonic reverse primer: 5 -GTC TCC AAA GAG GGC TTT GTA G-3 . The amplification conditions were: 94 ◦ C for 3 min; 30 cycles at 94 ◦ C for 15 s, 58 ◦ C for 30 s, 72 ◦ C for 1 min; final extension at 72 ◦ C for 7 min. Since in the presence of the mutation the mismatched primer introduces a Taq I restriction site, the PCR fragment of exon 9 was digested with Taq I (MBI Fermentas GmbH, St. Leon-Rot, Germany). The digestion products were separated on 3% agarose gel. 2.6.3. Transition c.1445 A > G in exon 12 (Y482C) As this mutation introduces an Apa LI restriction site, a 325 bp PCR fragment encompassing exon 12 was digested with Apa LI (Amersham Pharmacia Biotech, Cologno Monzese, Italy). The digestion products were separated on 2% agarose gel.

2.3. Sequence analysis of candidate genes Genomic DNA was extracted from peripheral blood leukocytes by a standard procedure [29]. The Apo A-I and LCAT genes were sequenced using the primers previously described [30,31]. The promoter and the 50 exons, as well as the splice junctions, of the ABCA1 gene were amplified

2.6.4. Transition c.1669 C > T in exon 13 (R557X) As this mutation introduces a Bgl II restriction site, a 333 bp PCR fragment encompassing exon 13 was digested with Bgl II (Amersham Pharmacia Biotech, Cologno Monzese, Italy). The digestion products were separated on 2% agarose gel.

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2.6.5. Transversion c.5398 A > C in exon 40 (N1800H) As this mutation introduces a new Nla III restriction site, a 144 bp PCR fragment encompassing exon 40 was digested with Nla III (Amersham Pharmacia Biotech, Cologno Monzese, Italy). The digestion products were separated on 8% polyacrylamide gel. 2.6.6. Transversion c.5703 A > C in exon 42 (R1901S) As this mutation introduces a Bsm I restriction site, a 223 bp PCR fragment encompassing exon 42 was digested with Bsm I (Roche Diagnostics GmbH, Mannheim, Germany). The digestion products were separated on 2% agarose gel. 2.6.7. Transversion c.6588 G > C in exon 49 (Q2196H) A 219 bp fragment of exon 49 was amplified using a canonic forward primer: 5 -CCT GCT TTC AGG TTT GGA GAT G-3 and a mismatched reverse primer: 5 -CTA TGT GGA GTC GCT TTC AGC T-3 . The amplification conditions were: 95 ◦ C for 2 min; 35 cycles at 95 ◦ C for 45 s, 57 ◦ C for 45 s, 72 ◦ C for 1 min; final extension at 72 ◦ C for 7 min. Since in the presence of the mutation the mismatched primer introduces a Pvu II restriction site, the PCR product was digested with Pvu II (Amersham Pharmacia Biotech, Cologno Monzese, Italy). The digestion products were separated on 3% agarose gel.

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mother. No carriers of D9N or N291S variants of LPL were found in this family. 3.2. Family 2 (Fig. 1) 3.2.1. Plasma lipids The proband (I.2) had low levels of HDL-C and Apo A-I. The proband’s son (II.1) had low level of HDL and moderate increase of LDL-C and Apo B; the proband’s grand-daughter (III.1) also had low HDL (Table 1). 3.2.2. DNA sequence analysis The sequence of LCAT and Apo A-I genes did not reveal any mutation in the proband (I.2); the sequence of ABCA1 gene revealed a heterozygous transversion c.5398 A > C (N1800H) in exon 40 (Fig. 3). In addition, the proband carried the LPL variant N291S. The proband’s son (II.1) inherited the two mutations from his mother and one ε4 allele from his father. The proband’s grand-daughter (III.1) carried the LPL variant only. 3.3. Family 3 (Fig. 1) 3.3.1. Plasma lipids The proband (II.1) had very low levels of HDL-C and Apo A-I. The proband’s son (III.3) had low level of HDL. The proband’s brother (II.2) had a normal lipoprotein profile (Table 1).

2.7. Statistical analysis The statistical analysis was performed by Kruskal–Wallis and Mann–Whitney tests using SPSS 11.0 statistical software package (SPSS Inc., Chicago, IL).

3. Results 3.1. Family 1 (Fig. 1) 3.1.1. Plasma lipids The proband (II.1) had high levels of LDL-C and Apo B and very low levels of HDL-C and Apo A-I. The proband’s son (III.1) had hypercholesterolemia and low HDL level; one of the proband’s grandchildren (IV.1) also had hypercholesterolemia. The other members of the family had a normal lipoprotein profile (Table 1). 3.1.2. DNA sequence analysis The sequence of LDL-R, LCAT and Apo A-I genes did not reveal any mutation in the proband (II.1). The sequence of the Apo B gene region harbouring mutations causing FDB showed that the proband was heterozygous for R3500Q mutation. The sequence of ABCA1 gene revealed an heterozygous transition c.850 G > A (E284K) in exon 9 (Fig. 3). The proband’s son (III.1) and grandson (IV.1) were carriers of Apo B R3500Q. In spite of the low HDL level, the proband’s son did not carry the ABCA1 gene mutation found in his

3.3.2. DNA sequence analysis The sequence of LCAT and Apo A-I genes did not reveal any mutation in the proband (II.1). The sequence of ABCA1 gene showed that the proband was a compound heterozygote, as she carried a transition c.1445 A > G (Y482C) in exon 12 (Fig. 3) and a transversion c.5398 A > C (N1800H) in exon 40 (Fig. 3). The proband’s son (III.3) inherited the N1800H mutation from his mother. No members of this family carried LPL variants. 3.4. Family 4 (Fig. 2) 3.4.1. Plasma lipids The only lipoprotein abnormality observed in this patient was a low level of HDL and slight elevation of triglycerides (Table 1). 3.4.2. DNA sequence analysis The sequence of LCAT and Apo A-I genes was found to be normal and the LPL common variants were absent. The sequence of ABCA1 gene showed that the proband was heterozygous for a transversion c.6588 G > C (Q2196H) in exon 49 (Fig. 4). 3.5. Family 5 (Fig. 2) 3.5.1. Plasma lipids The proband (II.1) had high levels of LDL-C and Apo B and low levels of HDL-C and Apo A-I. The proband’s eldest

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Table 1 Plasma lipid and apolipoprotein concentrations in members of the six families Subject

ABCA1 gene alleles

Age (years)

Sex

BMI (kg/m2 )

TC (mmol/l)

LDL-C (mmol/l)

HDL-C (mmol/l)

TG (mmol/l)

Apo A-I (mg/dl)

Apo B (mg/dl)

Apo E genotype

Family 1 I.2 II.1a II.2 III.1a III.2 IV.1a IV.2

W/W M1/W W/W W/W W/W W/W W/W

71 52 43 34 29 4 2

F F M M F M F

24.0 22.4 23.5 24.0 22.0 – –

4.42 7.11 5.12 7.27 3.80 7.16 3.59

2.40 6.26 3.05 6.00 2.00 5.74 1.99

1.40 0.20 1.24 0.90 1.40 1.20 1.10

1.37 1.40 1.82 0.80 0.91 0.50 1.09

130 30 130 90 145 124 115

78 168 84 156 70 119 52

ε3ε3 ε3ε3 ε3ε3 ε3ε3 ε3ε3 ε3ε3 ε3ε3

Family 2 I.1 I.2b II.1b II.2 III.1b

W/W M2/W M2/W W/W W/W

69 65 39 38 9

M F M F F

26.0 24.2 21.0 22.0 –

6.26 4.50 6.10 6.36 4.96

4.14 2.97 4.63 3.90 3.72

1.21 0.72 0.77 1.86 0.85

1.97 1.75 1.51 1.30 0.88

128 80 85 160 92

100 85 115 88 83

ε4ε4 ε3ε3 ε3ε4 ε3ε3 ε3ε3

Family 3 II.1 M2/M3 II.2 W/W III.3 M2/W

47 43 9

F M M

24.7 28.0 –

4.50 5.10 3.00

3.68 3.70 1.94

0.40 0.96 0.75

0.93 0.80 0.70

57 104 92

102 98 52

ε3ε4 ε3ε4 ε3ε4

+ (?)

Family 4 II.1 M4/W

62

M

23.3

4.45

2.71

0.72

2.21

102

92

ε3ε3

+++

Family 5 II.1a III.1a III.2 III.3

59 33 31 31

M F F F

36.7 21.8 22.8 24.4

7.16 7.52 4.68 4.00

6.02 5.02 3.28 2.74

0.52 1.99 0.85 0.90

1.71 1.13 1.18 0.78

81 162 92 97

133 112 82 72

ε3ε4 ε4ε4 ε3ε4 ε3ε4

+++

53 41 39 37 37 16 10

F M M F M F F

40.2 27.5 26.2 21.3 18.8 25.4 14.2

4.76 6.54 3.57 4.44 3.67 3.33 2.66

3.00 4.35 2.44 2.63 2.43 2.45 1.34

1.16 1.19 0.77 0.76 1.00 0.18 0.98

1.31 2.20 0.77 2.30 0.50 1.55 0.76

104 141 93 85 89 12 103

81 148 71 89 57 102 38

ε3ε3 ε3ε4 ε3ε4 ε3ε4 ε3ε3 ε3ε3 ε3ε3

M5/W W/W M5/W M5/W

Family 6 I.2 M6/W II.1 W/W II.2 M6/W II.3 M6/W II.4 M7/W III.1 M6/M7 III.2 M7/W

Premature CAD

+++

+++

W, ABCA1 wild-type allele; M, ABCA1 mutant allele: M1 (E284K); M2 (N1800H); M3 (Y482C); M4 (Q2196H); M5 (R557X); M6 (H160FsX173); M7 (R1901S); ND: not determined. The presence of overt clinical manifestations of CAD is indicated by +++; features of coronary lesions in the absence of clinical manifestations is indicated by +. a Apo B (R3500Q) carriers. b LPL (N291S) carriers.

daughter (III.1) had elevated LDL-C and Apo B; HDL-C and Apo A-I levels were in the upper quartile of the distribution in the age and sex matched population. The proband’s twin daughters (III.2 and III.3) had normal LDL-C and Apo B but low HDL levels (Table 1). 3.5.2. DNA sequence analysis The sequence of LDL-R gene did not reveal any mutation in the proband (II.1). The sequence of the Apo B gene showed that the proband and his hypercholesterolemic daughter (III.1) were heterozygous for R3500Q mutation. The sequence of ABCA1 gene revealed that the proband was heterozygous for a transition c.1669 C > T (R557X) in exon 13 (Fig. 4); this mutation was also present in proband’s twin daughters. The proband and his daughters were not carriers of D9N or N291S variants of LPL gene or mutations in Apo A-I and LCAT genes.

3.6. Family 6 (Fig. 2) 3.6.1. Plasma lipids The proband (III.1) had extremely low levels of HDL-C and Apo A-I. Her parents (II.3 and II.4), her sister (III.2) and one of the maternal uncles (II.2) had low levels of HDL. The sister of the proband’s maternal grandmother (I.2) had only moderately reduced levels of HDL-C and Apo A-I (Table 1). 3.6.2. DNA sequence analysis The sequence of LCAT and Apo A-I genes did not reveal any mutation in the proband (III.1). The sequence of ABCA1 gene showed that the proband was a compound heterozygote carrying the following mutations: (i) c.479(A) deletion in exon 6 predicted to cause a frameshift starting from codon 160 leading to a premature termination codon at

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315

Fig. 3. Analysis of ABCA1 gene in Families 1, 2 and 3. The panels on the left show the partial sequence of exon 9 in the proband of Family 1, exon 40 in the proband of Family 2 and exon 12 in the proband of Family 3. The panels on the right show the result of the screening for these mutations based on PCR amplification and enzymatic digestion of exons 9, 40 and 12, respectively (see Section 2 for details). M: carrier of the mutant allele; W: wild-type allele.

position 173 (c.479 del A, H160FsX173); (ii) c.5703 A > C transversion (R1901S) in exon 42 (Fig. 5). The father (II.4) and the sister (III.2) of the proband carried the missense mutation in exon 42. The nucleotide deletion in exon 6 was present in proband’s mother (II.3) and in other members of the maternal line (I.2 and II.2). No members of this family carried LPL variants. 3.7. Screening of ABCA1 mutations in a population sample Screening for the seven ABCA1 mutations found in our families was carried out in 75 healthy subjects (150 chromo-

somes) randomly selected from the medical personnel that we assume to represent a sample of our general population. No carriers of these mutations were found.

4. Discussion In this study, we report the characterization of seven mutations of ABCA1 gene identified in subjects with familial HDL deficiency. One of these mutations (N1800H) had been reported previously [10,38] in a TD patient of Italian origin (case 52 in ref. [39]), who was homozygous for this mutation. Since we have found N1800H in two unrelated subjects

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Fig. 4. Analysis of ABCA1 gene in Families 4 and 5. The panels on the left show the partial sequence of exon 49 in the proband of Family 4 and exon 13 in the proband of Family 5. The panels on the right show the result of the screening for these mutations based on PCR amplification and enzymatic digestion of exons 49 and 13, respectively (see Section 2 for details). M: carrier of the mutant allele; W: wild-type allele.

of our series (Families 2 and 3), we are tempted to suggest that N1800H might be a recurrent mutation. The novel mutations we identified included: (a) one non-sense (R557X) and one frameshift (H160FsX173) mutation, both predicted to encode short peptides presumably devoid of any function; (b) four missense mutations (E284K, Y482C, R1901S and Q2196H) resulting in non-homologous amino acid substitutions. These missense mutations, which were not detected in 150 normal chromosomes of randomly selected subjects, are likely to be the cause of ABCA1 dysfunction, since they involve amino acids which are highly conserved in mouse, rat and chick ABCA1 (Genbank accession no. X75926 and NM 013454, NM 178095 and AF362377, respectively). The E284K and the Y482C are located in the first extracellular loop of ABCA1, where they may interfere with the binding to Apo A-I and/or the membrane release of phospholipids, as it has been demonstrated for other mutations (R587W and Q597R) located in the same extracellular domain [40,41]. It is also possible that these mutations interfere with the intracellular transport of ABCA1 resulting in very little ABCA1 reaching the plasma membrane [42]. The other missense mutation we found in the first extracellular loop (Y482C) introduces a new cysteine residue which might induce the formation of a new disulfide bridge with cysteine residues present in the same loop or in the other large extracellular loop of the C-terminal half of ABCA1. The formation of a new disulfide bridge

may disrupt the structure of ABCA1 on the cell surface, thus preventing either Apo A-I binding or phospholipid release. Interestingly, a cysteine for arginine substitution in the first extracellular loop (R230C) was reported in three Oji-Cree subjects with very low plasma HDL levels [11]. The other two missence mutations (R1901S and Q2196H) we found in our patients are located in the C-terminal cytoplasmic region close to the NBD-2 domain. In view of this localization these mutations might interfere with ATP-binding to NBD-2 or its activity as an ATPase [23]. With regard to the severity of plasma HDL deficiency (≤5th percentile for age and sex), our probands can be separated into two groups: one with extremely low HDL-C levels (≤0.4 mmol/l), which includes the probands of Families 1, 3 and 6 (Figs. 1 and 2, and Table 1); the other with low HDL-C levels (0.52–0.72 mmol/l), which includes the probands of Families 2, 4 and 5 (Figs. 1 and 2, Table 1). The probands of Families 3 and 6 were carriers of two different mutant alleles (compound heterozygotes) of ABCA1 gene. Strictly speaking we would have expected them to show some of the clinical manifestations of Tangier disease [39], which in fact were not detected in either of them. As a matter of fact these probands came to medical attention for other reasons, such as positive family history for pCAD (proband of Family 3) or mental retardation and congenital hypothyroidism (proband of Family 6). These observations, along with our previous reports of two other patients

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Fig. 5. Analysis of ABCA1 gene in Family 6. The panels on the left show the partial sequence of exons 6 and 42 in the proband of Family 6. The panels on the right show the result of the screening for these mutations based on heteroduplex analysis of exon 6 and PCR amplification and enzymatic digestion of exon 42, respectively (see Section 2 for details). M: carrier of the mutant allele; W: wild-type allele.

with severe HDL deficiency due to homozygosity [18] or compound heterozygosity [21] for ABCA1 mutations, suggest that a genotype setting consistent with the diagnosis of Tangier disease (i.e. the presence of two mutant ABCA1 alleles) might be “relatively” more frequent than presently assumed and not necessarily associated with overt clinical manifestations suggesting Tangier disease. The extremely low plasma HDL-C observed in the proband of Family 1 (0.20 mmol/l), who was a carrier of a single ABCA1 mutation, has no simple explanation. We excluded the presence of rare mutations in Apo A-I and LCAT genes and the common variants of LPL gene, associated with reduced plasma HDL. However, we can not rule out mutations in other genes, such as the genes encoding the transported ABCC6 [43], the enzymes ␤-glucocerebrosidase (GBA) [44] and the sphingomyelin phosphodiesterase-1 (SMPD1) [45]. In this patient, however, the low HDL level may be part of the biochemical feature of the chronic inflammation present in Crohn’s disease [46,47]. It is well established that chronic inflammation is associated with reduced plasma HDL level and changes in its lipid and protein constituents [48], and that inflammatory markers (CRP, ceruloplasmin and leukocyte count) are inversely related to plasma HDL-C concentration [49]. The probands of the second group (Families 2, 4 and 5), found to carry a single ABCA1 mutation, had plasma HDL

levels comparable to those in subjects with familial HDL deficiency as reported by other authors [12,20,24–27]. During this study we discovered that three of our probands were carriers of other genetic disorders of plasma lipoprotein (FDB in Families 1 and 5, and partial LPL deficiency in Family 2). The probands of Families 1 and 5 and family members who inherited the Apo B R3500Q mutation had high levels of LDL-C (5.81 ± 0.48 mmol/l), which are comparable to those reported by other authors in subjects with FDB [50,51] and to those observed by our group in Italian FDB individuals (no. 11; 5.45 ± 0.98 mmol/l). These LDL-C levels were significantly lower than those reported in patients with heterozygous FH due to LDL-R gene mutations [36,50]. In keeping with the lower LDL-C levels, the prevalence of pCAD (before 60 years of age) is lower in FDB than in FH patients [51]. The presence of severe pCAD in our probands with FDB might be related to the concomitant presence of very low level of HDL, which is considered a powerful predictor of pCAD in subjects with FH [52–54]. The proband of Family 2 and her son were found to carry the N291S substitution in LPL, a common mutation that leads to slightly reduced enzymatic activity [55]. N291S has been estimated to increase fasting plasma triglycerides by 31% and to reduce HDL-C by 0.12 mmol/l [56]. This

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mutation does not seem to increase coronary heart disease in the general population [56,57] although women may be more affected than men [57,58]. In the proband of Family 2 and her son the HDL-lowering effect of N291S may have been masked by the more pronounced effect of the ABCA1 mutation (N1800H). The effect of N291S, however, is clearly detectable in the 9-year-old proband’s grand-daughter who carried only this mutation and had a plasma level of HDL-C (0.85 mmol/l) strongly below the 5th percentile (0.99 mmol/l) of the distribution we found in 276 randomly selected girls of similar age. Another aspect of our family studies which deserves comment is the possible contribution of ε4 allele on plasma HDL. The ε4 allele (ε3ε4 genotype) was present in eight subjects carrying ABCA1 mutations (Table 1). When we compared the lipid values (adjusted for gender, age and BMI) of subjects heterozygous for ABCA1 mutations with and without the ε4 allele (no. 6 versus no. 5), excluding Apo B R3500Q carriers, we observed a significantly lower level of HDL-C (0.78 ± 0.04 mmol/l versus 0.93 ± 0.11 mmol/l, P 0.03) in ε4+ group. A possible explanation for this finding is given by recent animal studies which showed that plasma from Apo E4-expressing mice was less efficient at transferring Apo A-I from VLDL to HDL and at generating HDL in vitro than that from Apo E3-expressing mice [59]. During our survey of ABCA1 mutations ([18,21], present work) we have identified 17 carriers of ABCA1 mutations. The mean plasma HDL-C and Apo A-I levels of these subjects (0.79±0.22 mmol/l and 88.5±20.0 mg/dl, respectively) were significantly lower than those observed in non-affected relatives (1.28 ± 0.22 mmol/l and 140.2 ± 13.3 mg/dl, respectively; P < 0.0001) and were similar to those found in carriers of c.85 del C mutation of Apo A-I gene (a mutation with prevents the synthesis of Apo A-I) [60], thus indicating that these parameters cannot distinguish carriers of ABCA1 mutations from carriers of asynthetic Apo A-I mutations. In striking contrast, plasma HDL-C and Apo A-I levels in carriers of missense mutations of Apo A-I located in repeats 5 and 6 of this peptide (such as L141R substitution) [60] were significantly lower (dominant negative effect) than those found in carriers of ABCA1 mutations. In conclusion we report six novel mutations of ABCA1 gene found in subjects with severe HDL deficiency with and without premature coronary artery disease and belonging to six unrelated families. We discovered that two ABCA1 mutation carriers were also affected by FDB and another had a common LPL mutation. Moreover we found that in carriers of ABCA1 mutations the presence of ε4 allele is associated with a more pronounced reduction of plasma HDL levels. These family studies emphasize the complex interplay of genetic factors which, together with environmental factors (such as chronic inflammation and smoking), may affect the lipid profile and the development of premature cardiovascular disease in carriers of ABCA1 mutations.

Acknowledgements This work was supported by grants from the Italian Ministry of Education and Research (MM06178194 001 and 006) to S.B. and S.C. The authors thank Mr. Stan Sobecki, IMVS Lipid Laboratory-Adelaide, for technical assistance.

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