- Email: [email protected]
95 Heterozygous mutation of LCAT gene causing severe hypoalphalipoproteinemia in two brothers with coronary artery disease
Abstracts XIXth National Congress, Italian SocieO, f o r the Stu&, o f Athelvsclelvsis
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NOVEL MUTATION OF APOA1 GENE IN A LARGE FAMILY WITH HYPOALPHALIPOPROTEIN EMIA
L. Pisciotta 1 , C. Grondona 1 , E. Pino 1 , A. Bellocchio 1 , R. Fresa 1 , V. Guido 1 , S. Calandra2, S. Bertolini 1 . 1Dpt. of hzte~Tml Med.. Univ. of Genoa; 2Dpt. of
Biomedical Sci.. Univ. of Modena. Italy E-mail: [email protected] During a survey of patients with primary hypoalphalipoproteinemia we identified a novel mutation ofAPOA1 gene. The proband was a 38 year-old male with mixed hyperlipidemia and low HDL level (T-C 276, LDL-C 185, HDL-C 28, TG 314, Apo A172, Apo B 133 mg/dl). The subject was apparently healthy, was a non-smoker, had e3e3 genotype and no mutations or pathogenic variants in LPL, APOA5, APOC3, LDLR, APOB, PCSK9, ABCA1 and LCAT gene. ECG stress test was negative, however, echographic evaluation o f the carotid arteries revealed fibrous plaques with 20 25% stenosis. Sequencing the APOA1 gene we found that the probaaad was heterozygous for a cytosine insertion in exon 3 (c.49 50 ins C). This mutation causes a frameshift of the reading frame and the predicted protein has a stretch o f 9 novel amino acids that follows Serl6 of the signal peptide and precedes a termination codon (p.Q17PfsX10). The mutation was inherited from the deceased mother, who had suffered from angina mad ischemic cerebrovascular disease, since the father, although affected by mixed hyperlipidemia, CAD and dramatic carotid atherosclerosis, was not the carrier o f the mutation. The mutation was also found in one o f the proband's sisters (41y, HDL-C 38 and Apo AI 86 mg/dl), in one o f his brothers (33y, HDL-C 22 and Apo AI 89mg/dl) and in the proband's daughter ( l l y , HDL-C 27 mad Apo A169 mg/dl). The probaaad's sister with APOA1 mutation was also a carrier o f the LPL N291S variant. The proband's son was unaffected (6y, HDL-C 62 and Apo AI 163 mg/dl).
NOVEL MUTATION OF LDLR GENE IN A PATIENT WITH [93]APSEUDOXANTHOMA ELASTICUM (PXE): IMPLICATIONS FOR THE TREATMENT L. Pisciotta 1, P. Tarugi 2, A. Bellocchio 1 , R. Fresa 1 , C. Fioravaaati1 , C. Grondona 1 , S. Calandra2, I. Ronchetti 2, S. Bertolini 1 . IDpt. of Inte~Tzal
Med.. Univ. of Genoa; 2Dpt. of Biomedical Sci.. Univ. of Modena. Italy E-mail: [email protected] PXE is characterized by lesions of the skin (yellowish papules), retina (angioid streaks, peripapillary atrophy, haemorrhages) and cardiovascular system (early atherosclerosis, vessel calcification and rupture, cardiomyopathy, fibrous thickening of endocaxdium and valves). We have studied a 60y female with PXE due to mutations in ABCC6 gene (16p13.1): c.951 C > G in exon 8, p.S317R and c.3421 C > T in exon 24, p . R l l 4 1 X . The patient had skin lesions, severe retinopathy with visual impairment and angina. In addition, she had hypercholesterolemia (HCH): T-C 473, LDL-C 384, Apo B 238 mg/dl. Coronary angiography showed a 2V-CAD with a critical stenosis in the proximal LCx. Since H C H is not a feature of PXE and it was present in the probaJnd's three sons, we sequenced the candidate genes (LDLR, APOB, PCSK9) and found that the patient was heterozygous for a novel mutation in exon 12 of LDLR (c.1721 G > A , p.R574H). Coronary revascularization in this patient was inadvisable for the risk ofhaemorrhages and blindness. On the other hand, since she had a history of adverse events with some statins her H C H had not been treated for a long period. After angiographic findings we started the treatment with ezetimibe 10 mg/day and subsequently added simvastatin, increasing step by step the daily dose from 5 to 60 mg/day in the space o f 6 months. With the last dosage we obtained a reduction o f LDL-C from 384 to 111 ± 9 mg/dl and o f CRP-HS from 9.7 to <3mg/L. The patient has become asymptomatic for angina and SPET imaging showed a substantial reduction of the perfusion defect.
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NOVEL MUTATION OF LCAT GENE IN A PAKISTANI PATIENT WITH LECITHIN:CHOLESTEROL ACYLTRANSFERASE DEFICIENCY
L. Pisciotta 1 , M Charlton-Menys 2, L. Calabresi 3, R. Fresa 1 , E. Pino 1 , E Masturzo 1 , S. Calandra4, S. Bertolini 1 . 1Dpt. ofInte~Tzal Med.. Unic~ of
Genoa. Italy; 2Dpt. of Medicine. (;~ildren's Univ. Hospitals. Manchester; UIw 3Dpt. of Pha~vzacol. Sci.. Univ. of Milan. Italy; 7Dpt. of Biomedical Sci.. Univ. of Modena. Italy E-mail: [email protected] The probaJnd was a 30y Pakistani female with corneal opacities, proteinuria (2.6g/day), and abnormal lipoprotein profile (T-C 124, HDL-C 6, TG 111, Apo AI 42 mg/dl). Free cholesterol was 96%, LCAT activity was undetectable (n.v. 25 55nmol/ml/h) and LCAT mass was 4.79~tg/ml (n.v. 3.1-6.7). Renal biopsy showed foam cell accumulation in the glomeruli and interstitial tissues and thickening o f BowmaJn's capsule and o f the glomerulax capillary basement
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membrane. The direct sequence o f LCAT gene revealed that the proband was homozygous for a T>C transition in exon 2 (c.295 T>C), predicted to cause an arginine (CGG) for tryptophaaa (TGG) substitution at position 75 (at position 99 if one includes the signal peptide of 24 amino acids) of LCAT protein (p.W99R). The mutation was confirmed by the restriction fragment analysis after Msp I digestion: a 447 bp fragment encompassing exon 2, intron 2, exon 3 and intronic flaxtking regions was amplified and digested with Msp I; the digestion products (245, 154 and 4 8 b p from the wild type and 154, 136, 109 and 4 8 b p from the mutant allele) were separated on 3% agarose gel. This mutation is novel and a PSIC score of 3.995 resulted from computational analysis using the program Poly-Phen (wu~v.borh-.embl-heidelberg.de/PolvPhen/), which predicts effects of amino acid changes on protein function. On the other hand, the tryptophan at position 99 is conserved in human, baboon, rabbit, mouse, rat, chicken and c. elegaJns.
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HETEROZYGOUS MUTATION OF LCAT GENE CAUSING SEVERE HYPOALPHALIPOPROTEINEMIA IN TWO BROTHERS WITH CORONARY ARTERY DISEASE
L. Pisciotta 1 , L. Calabresi 2, R. Sturaro 3, A. Bellocchio 1 , "d Guido 1 , S. Calandra4, S. Bertolini 1 . 1Dpt. of hzte~Tml Med.. Univ. of Genoa; 2Dpt.
Ptla~vlacol. Sci.. Unic~ of Milan; 3Metabolic Centre. San Remo Hospital," t. of Biomedical Sci.. Unic~ of Modena. Ball, E-maih [email protected] We have investigated two brothers (71 and 69y) with type 2 DM, arterial hypertension, dyslipidemia and CAD. The older brother had suffered from MI at 50 and underwent C A B G at 67, the younger had angina and underwent PTCA at 66. The plasma lipid profile, before treatment, in these subjects was: T-C 323, LDL-C 234, HDL-C 23, TG 324, Apo AI 83, Apo B 173 and T-C 224, LDL-C 161, HDL-C 19, TG 220, Apo AI 90, Apo B 118, respectively. The patients were genotyped for the following allelic variants: APOE e2/e3/e4, APOA5 1131 T/C and c.56 C/G (S/W), APOC3 482 C/T, LPL D9N and N291S. Both subjects were e3e3; the older was heterozygous and the younger homozygous for the APOC3 482 T triglyceride-raising allele. The sequence o f LDLR, APOB and PCSK9 gene was negative. In view o f the low level o f HDL-C and Apo AI we sequenced the candidate genes (ABCA1, APOA1 and LCAT) and found that both patients were heterozygous for a c.1007 A > G transition in exon 6 o f LCAT gene, predicted to cause a cysteine (TGC) for tyrosine (TAC) substitution at position 312 (at position 336 if one includes the signal peptide of 24 amino acids) o f LCAT protein (p.Y336C). In these two subjects we found: CER 56.1 and 54.0nmol/ml/h (n.v. 30 30), LCAT activity 20.4 and 13.2nmol/ml/h (n.v. 25 55), LCAT mass 2.63 and 2.01 ~tg/ml (n.v. 3.1-6.7), respectively. This mutation was not previously described and seems to be pathogenic since the tyrosine at position 336 is conserved in human, baboon, rabbit, mouse, rat and chicken.
['~'] ADDITIVE EFFECT OF MUTATIONS IN LDLR AND PCSK9 GENES ON THE PHENOTYPE OF FAMILIAL HYPERCHOLESTEROLEMIA L. Pisciotta 1 , C. Priore Oliva 2, A.B. Cefalfi 3, D. Noto 3, A. Bellocchio 1 , R. Fresa 1 , A. Cantafora4, D. Patel 5, M. Averna 3, R Tarugi 2, S. Calandra2, S. Bertolini 1 . 1Dpt. of lnte~Tzal Med.. Unic~ of Genoa; 2Dpt. of Biomedical
Sci.. Unic~ of Modena; 3Dpt. of hzte~Tml Med.. Unic~ of Pale~vm; ~Nat. hzst. of Health. Rome. Italy; 5Lipoprotein Group. MRC (Tinical Sciences ('entre. London. UK E-maih [email protected] Patients homoz, or compound heteroz, for LDLR mutations or double heteroz. for LDLR and apo B (FDB) mutation have higher LDL-C, more extensive xanthomatosis (xts) and more severe premature CAD than simple heteroz. for mutations in either of these genes or for missense mutations in PCSK9 gene. It is not known whether combined mutations in LDLR and PKCS9 cause such a severe phenotype. We sequenced Apo B and PCSK9 genes in two patients with the clinical diagnosis of homoz. FH who were heteroz, for LDLR gene mutations. Proband Z.R (31 y., xts, LDL-C 13.39 mmol/L, MI, 3VCAD) was heteroz, for an LDLR mutation (p.E228K) inherited from her father (LDL-C 8.07mmol/L) and a PCSK9 mutation (p.R496W) from her mother (LDL-C 5.58mmol/L). Probaaad L.R. and her sister (48 and 62y, LDL-C 11.51 and 10.47mmol/L, xts and carotid ATS) were heteroz, for an LDLR mutation (p.Y419X) inherited from their mother (LDL-C 6.54mmol/L) and a PCSK9 mutation (p.N425S) probably from their deceased father. The LDL-C levels in double heteroz, o f these two families were respectively 56% and 44% higher than those found in simple heteroz, for the two LDLR mutations. The two PCSK9 mutations are novel and were not found in 110 controls and 80 patients with co-dominant hypercholesterolemia. These observations indicate that rare missense mutations o f PCSK9 may worsen the clinical phenotype of patients carrying LDLR mutations.
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NOVEL MUTATION OF APOA1 GENE IN A LARGE FAMILY WITH HYPOALPHALIPOPROTEIN EMIA
L. Pisciotta 1 , C. Grondona 1 , E. Pino 1 , A. Bellocchio 1 , R. Fresa 1 , V. Guido 1 , S. Calandra2, S. Bertolini 1 . 1Dpt. of hzte~Tml Med.. Univ. of Genoa; 2Dpt. of
Biomedical Sci.. Univ. of Modena. Italy E-mail: [email protected] During a survey of patients with primary hypoalphalipoproteinemia we identified a novel mutation ofAPOA1 gene. The proband was a 38 year-old male with mixed hyperlipidemia and low HDL level (T-C 276, LDL-C 185, HDL-C 28, TG 314, Apo A172, Apo B 133 mg/dl). The subject was apparently healthy, was a non-smoker, had e3e3 genotype and no mutations or pathogenic variants in LPL, APOA5, APOC3, LDLR, APOB, PCSK9, ABCA1 and LCAT gene. ECG stress test was negative, however, echographic evaluation o f the carotid arteries revealed fibrous plaques with 20 25% stenosis. Sequencing the APOA1 gene we found that the probaaad was heterozygous for a cytosine insertion in exon 3 (c.49 50 ins C). This mutation causes a frameshift of the reading frame and the predicted protein has a stretch o f 9 novel amino acids that follows Serl6 of the signal peptide and precedes a termination codon (p.Q17PfsX10). The mutation was inherited from the deceased mother, who had suffered from angina mad ischemic cerebrovascular disease, since the father, although affected by mixed hyperlipidemia, CAD and dramatic carotid atherosclerosis, was not the carrier o f the mutation. The mutation was also found in one o f the proband's sisters (41y, HDL-C 38 and Apo AI 86 mg/dl), in one o f his brothers (33y, HDL-C 22 and Apo AI 89mg/dl) and in the proband's daughter ( l l y , HDL-C 27 mad Apo A169 mg/dl). The probaaad's sister with APOA1 mutation was also a carrier o f the LPL N291S variant. The proband's son was unaffected (6y, HDL-C 62 and Apo AI 163 mg/dl).
NOVEL MUTATION OF LDLR GENE IN A PATIENT WITH [93]APSEUDOXANTHOMA ELASTICUM (PXE): IMPLICATIONS FOR THE TREATMENT L. Pisciotta 1, P. Tarugi 2, A. Bellocchio 1 , R. Fresa 1 , C. Fioravaaati1 , C. Grondona 1 , S. Calandra2, I. Ronchetti 2, S. Bertolini 1 . IDpt. of Inte~Tzal
Med.. Univ. of Genoa; 2Dpt. of Biomedical Sci.. Univ. of Modena. Italy E-mail: [email protected] PXE is characterized by lesions of the skin (yellowish papules), retina (angioid streaks, peripapillary atrophy, haemorrhages) and cardiovascular system (early atherosclerosis, vessel calcification and rupture, cardiomyopathy, fibrous thickening of endocaxdium and valves). We have studied a 60y female with PXE due to mutations in ABCC6 gene (16p13.1): c.951 C > G in exon 8, p.S317R and c.3421 C > T in exon 24, p . R l l 4 1 X . The patient had skin lesions, severe retinopathy with visual impairment and angina. In addition, she had hypercholesterolemia (HCH): T-C 473, LDL-C 384, Apo B 238 mg/dl. Coronary angiography showed a 2V-CAD with a critical stenosis in the proximal LCx. Since H C H is not a feature of PXE and it was present in the probaJnd's three sons, we sequenced the candidate genes (LDLR, APOB, PCSK9) and found that the patient was heterozygous for a novel mutation in exon 12 of LDLR (c.1721 G > A , p.R574H). Coronary revascularization in this patient was inadvisable for the risk ofhaemorrhages and blindness. On the other hand, since she had a history of adverse events with some statins her H C H had not been treated for a long period. After angiographic findings we started the treatment with ezetimibe 10 mg/day and subsequently added simvastatin, increasing step by step the daily dose from 5 to 60 mg/day in the space o f 6 months. With the last dosage we obtained a reduction o f LDL-C from 384 to 111 ± 9 mg/dl and o f CRP-HS from 9.7 to <3mg/L. The patient has become asymptomatic for angina and SPET imaging showed a substantial reduction of the perfusion defect.
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NOVEL MUTATION OF LCAT GENE IN A PAKISTANI PATIENT WITH LECITHIN:CHOLESTEROL ACYLTRANSFERASE DEFICIENCY
L. Pisciotta 1 , M Charlton-Menys 2, L. Calabresi 3, R. Fresa 1 , E. Pino 1 , E Masturzo 1 , S. Calandra4, S. Bertolini 1 . 1Dpt. ofInte~Tzal Med.. Unic~ of
Genoa. Italy; 2Dpt. of Medicine. (;~ildren's Univ. Hospitals. Manchester; UIw 3Dpt. of Pha~vzacol. Sci.. Univ. of Milan. Italy; 7Dpt. of Biomedical Sci.. Univ. of Modena. Italy E-mail: [email protected] The probaJnd was a 30y Pakistani female with corneal opacities, proteinuria (2.6g/day), and abnormal lipoprotein profile (T-C 124, HDL-C 6, TG 111, Apo AI 42 mg/dl). Free cholesterol was 96%, LCAT activity was undetectable (n.v. 25 55nmol/ml/h) and LCAT mass was 4.79~tg/ml (n.v. 3.1-6.7). Renal biopsy showed foam cell accumulation in the glomeruli and interstitial tissues and thickening o f BowmaJn's capsule and o f the glomerulax capillary basement
$21
membrane. The direct sequence o f LCAT gene revealed that the proband was homozygous for a T>C transition in exon 2 (c.295 T>C), predicted to cause an arginine (CGG) for tryptophaaa (TGG) substitution at position 75 (at position 99 if one includes the signal peptide of 24 amino acids) of LCAT protein (p.W99R). The mutation was confirmed by the restriction fragment analysis after Msp I digestion: a 447 bp fragment encompassing exon 2, intron 2, exon 3 and intronic flaxtking regions was amplified and digested with Msp I; the digestion products (245, 154 and 4 8 b p from the wild type and 154, 136, 109 and 4 8 b p from the mutant allele) were separated on 3% agarose gel. This mutation is novel and a PSIC score of 3.995 resulted from computational analysis using the program Poly-Phen (wu~v.borh-.embl-heidelberg.de/PolvPhen/), which predicts effects of amino acid changes on protein function. On the other hand, the tryptophan at position 99 is conserved in human, baboon, rabbit, mouse, rat, chicken and c. elegaJns.
[•]
HETEROZYGOUS MUTATION OF LCAT GENE CAUSING SEVERE HYPOALPHALIPOPROTEINEMIA IN TWO BROTHERS WITH CORONARY ARTERY DISEASE
L. Pisciotta 1 , L. Calabresi 2, R. Sturaro 3, A. Bellocchio 1 , "d Guido 1 , S. Calandra4, S. Bertolini 1 . 1Dpt. of hzte~Tml Med.. Univ. of Genoa; 2Dpt.
Ptla~vlacol. Sci.. Unic~ of Milan; 3Metabolic Centre. San Remo Hospital," t. of Biomedical Sci.. Unic~ of Modena. Ball, E-maih [email protected] We have investigated two brothers (71 and 69y) with type 2 DM, arterial hypertension, dyslipidemia and CAD. The older brother had suffered from MI at 50 and underwent C A B G at 67, the younger had angina and underwent PTCA at 66. The plasma lipid profile, before treatment, in these subjects was: T-C 323, LDL-C 234, HDL-C 23, TG 324, Apo AI 83, Apo B 173 and T-C 224, LDL-C 161, HDL-C 19, TG 220, Apo AI 90, Apo B 118, respectively. The patients were genotyped for the following allelic variants: APOE e2/e3/e4, APOA5 1131 T/C and c.56 C/G (S/W), APOC3 482 C/T, LPL D9N and N291S. Both subjects were e3e3; the older was heterozygous and the younger homozygous for the APOC3 482 T triglyceride-raising allele. The sequence o f LDLR, APOB and PCSK9 gene was negative. In view o f the low level o f HDL-C and Apo AI we sequenced the candidate genes (ABCA1, APOA1 and LCAT) and found that both patients were heterozygous for a c.1007 A > G transition in exon 6 o f LCAT gene, predicted to cause a cysteine (TGC) for tyrosine (TAC) substitution at position 312 (at position 336 if one includes the signal peptide of 24 amino acids) o f LCAT protein (p.Y336C). In these two subjects we found: CER 56.1 and 54.0nmol/ml/h (n.v. 30 30), LCAT activity 20.4 and 13.2nmol/ml/h (n.v. 25 55), LCAT mass 2.63 and 2.01 ~tg/ml (n.v. 3.1-6.7), respectively. This mutation was not previously described and seems to be pathogenic since the tyrosine at position 336 is conserved in human, baboon, rabbit, mouse, rat and chicken.
['~'] ADDITIVE EFFECT OF MUTATIONS IN LDLR AND PCSK9 GENES ON THE PHENOTYPE OF FAMILIAL HYPERCHOLESTEROLEMIA L. Pisciotta 1 , C. Priore Oliva 2, A.B. Cefalfi 3, D. Noto 3, A. Bellocchio 1 , R. Fresa 1 , A. Cantafora4, D. Patel 5, M. Averna 3, R Tarugi 2, S. Calandra2, S. Bertolini 1 . 1Dpt. of lnte~Tzal Med.. Unic~ of Genoa; 2Dpt. of Biomedical
Sci.. Unic~ of Modena; 3Dpt. of hzte~Tml Med.. Unic~ of Pale~vm; ~Nat. hzst. of Health. Rome. Italy; 5Lipoprotein Group. MRC (Tinical Sciences ('entre. London. UK E-maih [email protected] Patients homoz, or compound heteroz, for LDLR mutations or double heteroz. for LDLR and apo B (FDB) mutation have higher LDL-C, more extensive xanthomatosis (xts) and more severe premature CAD than simple heteroz. for mutations in either of these genes or for missense mutations in PCSK9 gene. It is not known whether combined mutations in LDLR and PKCS9 cause such a severe phenotype. We sequenced Apo B and PCSK9 genes in two patients with the clinical diagnosis of homoz. FH who were heteroz, for LDLR gene mutations. Proband Z.R (31 y., xts, LDL-C 13.39 mmol/L, MI, 3VCAD) was heteroz, for an LDLR mutation (p.E228K) inherited from her father (LDL-C 8.07mmol/L) and a PCSK9 mutation (p.R496W) from her mother (LDL-C 5.58mmol/L). Probaaad L.R. and her sister (48 and 62y, LDL-C 11.51 and 10.47mmol/L, xts and carotid ATS) were heteroz, for an LDLR mutation (p.Y419X) inherited from their mother (LDL-C 6.54mmol/L) and a PCSK9 mutation (p.N425S) probably from their deceased father. The LDL-C levels in double heteroz, o f these two families were respectively 56% and 44% higher than those found in simple heteroz, for the two LDLR mutations. The two PCSK9 mutations are novel and were not found in 110 controls and 80 patients with co-dominant hypercholesterolemia. These observations indicate that rare missense mutations o f PCSK9 may worsen the clinical phenotype of patients carrying LDLR mutations.