- Email: [email protected]
A novel mechanism for thalassaemia intermedia
RESEARCH LETTERS
of nodal-positive tumours, progression was limited mainly to class A. Finally, we saw progression of nodal-negative tumours only in class A. Taken together, our cluster analysis identifies breast-cancer patients with a high risk of recurrences, and is a step towards the establishment of an individual risk-profile system. Future directions should combine these molecular methods with the standard tumour classification system to obtain improved patient-tailored therapies. Contributors A Ahr, M Kaufmann, T Karn and U Holtrich had the original idea and designed the overall study. The molecular analyses were developed and done by U Holtrich and T Karn. Tumour biopsies were collected by C Solbach and K Strebhardt. T Seiters and A Ahr handled the patient data and analysed the association between patient classification and survival data. A Ahr, M Kaufmann, T Karn and U Holtrich analysed the results and wrote the manuscipt.
Conflict of interest statement None declared
Acknowlegments We thank Silke Deckert and Katherina Kourtis for technical assistance. This work was supported by a grant from the Deutsche Krebshilfe (10-1478-Ka2). The sponsors had no role in the study design; data collection, analysis or interpretation; or in the writing of the paper. The Genbank accession numbers of the 41 marker genes are as follows: AJ131693, U66879, U43746, L33930, L11315, U24166, X52541, M11730, N54493, AA029434, R85813, T89015, AL050276, X03635, AB006589, AF123659, X59932, M60974, X75208, U07695, M35410, M65062, M62402, NM_002417, D26512, AF076622, X17620, U41745, NM_005030, X51730, X78817, L25081, U33920, M97935, U47686, L12350, X74764, M33294, NM_005204, X51602, and AF041259. 1
2
3 4 5
Golub TR, Slonim DK, Tamayo P, et al. Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science 1999; 286: 531–37. Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 2000; 403: 503–11. Perou CM, Sorlie T, Eisen MB, et al. Molecular portraits of human breast tumours. Nature 2000; 406: 747–52. Hedenfalk I, Duggan D, Chen Y, et al. Gene-expression profiles in hereditary breast cancer. N Engl J Med 2001; 344: 539–48. Ahr A, Holtrich U, Solbach C, et al. Molecular classification of breast cancer patients by gene expression profiling. J Pathol 2001; 195: 312–20.
Department of Obstetrics and Gynecology, J W Goethe University, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany (A Ahr MD, T Karn PhD, C Solbach MD, T Seiter MD, K Strebhardt PhD, U Holtrich PhD, Prof M Kaufmann MD)
transfusion requirements. The phenotype is highly heterogeneous, ranging in severity from severe anaemia with hepatosplenomegaly and thalassaemia-like bone modifications to moderate microcytic hypochromic anaemia. In terms of genotype, thalassaemia intermedia is also heterogeneous, and has been shown to arise in four distinct ways:1 inheritance of mild -thalassaemia alleles; part rescue of -thalassaemia major by hereditary persistence of fetal haemoglobin; exacerbation of the heterozygous state by coinheritance of an extra ␣-globin gene, which worsens imbalance of the globin chains; or inheritance of a dominant -thalassaemia mutation. We report a novel mechanism by which severity of the heterozygous state is increased leading to thalassaemia intermedia. The propositus was first examined at age 10 years for anaemia, hepatosplenomegaly, and growth failure. He had a typical thalassaemic face, and skull radiographs showed a typical hair-on-end appearance. Analysis of his haemoglobin showed HbA 71%, HbF 25%, and HbA2 4%, and in-vitro globin chain synthesis gave an ␣/ globin-chain ratio of 3·9. Concentration of ferritin was 367 g/L and of bilirubin, 60 mol/L. The individual is now 18 years old and has never had a blood transfusion. His haemoglobin concentration has remained between 70 and 80 g/L. The patient’s father had a -thalassaemic trait, with HbA2 5·1% and microcytosis, whereas his mother had a normal red-cell index, haemoglobin electrophoretic profile, and ␣/ globin-chain ratio. Results of molecular studies showed that the patient’s father has the Mediterranean -thalassaemia nonsense mutation at codon 39 (C→T), which the patient inherited, whereas his mother does not have any common -thalassaemia mutations. We screened the maternally inherited allele of the propositus for a rare or de novo mutation by cloning and sequencing a PCR fragment spanning the entire maternallyderived  gene (from –240 to +1665 nucleotide relative to the cap site). We did not identify a mutation. With PCR, we also showed that neither the 3·7 kb deletion nor the anti3·7 kb triplication was present at the ␣ gene locus. After digestion of the patient’s leucocyte DNA with the restriction enzyme Mae I to detect the -thalassaemia mutation at codon 39, we noted that the band
Correspondence to: Dr André Ahr (e-mail: [email protected])
A novel mechanism for thalassaemia intermedia C Badens, M G Mattei, A M Imbert, C Lapouméroulie, N Martini, G Michel, D Lena-Russo Thalassaemia intermedia is a moderate form of thalassaemia resulting from various genetic defects. We report an undescribed mechanism leading to this condition: a somatic deletion of the -globin gene in the haemopoietic lineage of a heterozygous -thalassaemic patient. We did molecular studies and haemoglobin analysis of the patient and his parents. We found that the deletion gives rise to a mosaic of cells with either one or no functional -globin gene and it extends to a region of frequent loss of heterozygosity called LOH11A, which is located close to the -globin locus. Thus, loss of heterozygosity can be a cause of non-malignant genetic disease.
Lancet 2002; 359: 132–33
Thalassaemia intermedia is a clinical term used to describe patients who have mild thalassaemia with no regular
132
Figure 1: Restriction enzyme digestion and FISH analysis
PCR fragments of (a) exon 2 of the -globin gene digested by Mae I and (b) IVS 2 of the ␥-globin gene digested by Hind III. Lane 1=father; lane 2=mother, lane 3=propositus. (c) FISH on metaphases from lymphocytes of the propositus with a probe specific for the -globin gene extending from 2 kb upstream of the ␦-globin gene to the 3⬘ end of the -globin gene
THE LANCET • Vol 359 • January 12, 2002 • www.thelancet.com
For personal use. Only reproduce with permission from The Lancet Publishing Group.
RESEARCH LETTERS
report. C Lapouméroulie did Southern blot analyses, revised the report, and approved the final version. Prof G Michel obtained data and revised the report. D Lena-Russo did laboratory analyses, revised the report, and approved th final version. N Martini did laboratory analyses and analysed data.
Conflict of interest statement None declared. 1
Figure 2: Digestion of exon 2 of the -globin gene by Mae I
2
Lanes 1, 2, 3, 4=hemizygous colonies; lane 5=heterozygous colony.
corresponding to the maternal allele was strikingly faint relative to the one derived from the paternal allele (figure 1). With informative restrictive fragment length polymorphisms elsewhere in the -globin locus, we consistently recorded a maternal to paternal allele ratio of about one to three (figure 1). By contrast, this ratio was normal in the patient’s hair root and cheek epithelium cells (data not shown), suggesting that the maternal allele might be deleted in a percentage of the patient’s blood cells. Such mosaicism was looked for by fluorescent in-situ hybridisation (FISH) done on metaphases from lymphocytes with a probe specific for the -globin gene. Results of FISH showed the presence of one signal in 56% of the cells (figure 1). To further confirm this mosaicism in the haemopoietic lineage, we did PCR on erythroid burst-forming units obtained from the patient’s peripheral blood cells after culture in methylcellulose. Of 26 individual colonies, six were heterozygous for the mutation at codon 39 and 20 were hemizygous (figure 2). The -globin complex is located in 11p15.5 close to a region of frequent loss of heterozygosity, termed LOH11A.2 This region is associated with various cancers—such as stomach, lung, and breast—and is believed to contain a gene with tumour suppressor function.2,3 Deletion resulting in loss of heterozygosity has been shown to involve the -globin locus.4 With the polymorphic marker D11S988, we showed that the patient’s deletion extends to the LOH11A tumour suppressor region (data not shown). This deletion could potentially confer a proliferative advantage to hemizygous cells, and account for deleted cells being more numerous than non-deleted cells. Thus, we report a patient who is constitutionally heterozygous for a -thalassaemia mutation inherited from his father. During development, a second hit—deletion of the normal maternal allele in one of the early precursors of the haemopoietic lineage—led to somatic reduction, hemizygosity, and mosaicism of blood cells that have either one or no functional -globin gene. Somatic deletions giving rise to loss of heterozygosity have been discovered and studied in the context of carcinogenesis. In our report, loss of heterozygosity was not associated with malignancy, but allows phenotypic expression of the recessive -thalassaemia mutation at the intact -globin locus. This mechanism could account for some sporadic cases of thalassaemia intermedia in which only one -thalassaemia mutation is apparent.1,5 Our patient is of interest with respect to potential genereplacement therapies, since expression of a single functional -globin gene in only a proportion of red blood cells is sufficient to produce a moderate form of thalassaemia, which does not need major medical intervention. Contributors C Badens did laboratory analyses, had the original idea for and co-ordinated the study, analysed and interpreted data, and revised the report. M G Mattei did FISH analyses, revised the report, and approved the final version. A M Imbert did laboratory analyses and revised the
THE LANCET • Vol 359 • January 12, 2002 • www.thelancet.com
3 4
5
Galanello R, Cao A. Relationship between genotype and phenotype: thalassemia intermedia. Ann N Y Acad Sci 1998; 850: 325–33. O’Briant KC, Bepler G. Delineation of the centromeric and telomeric chromosome segment 11p15,5 lung cancer suppressor regions LOH11A and LOH11B. Genes Chromosomes Cancer 1997; 18: 111–14. Rodriguez E, Sreekantaiah C, Chaganti RS. Genetic changes in epithelial solid neoplasia. Cancer Res 1994; 54: 3398–406. Bepler G, O’Briant KC, Kim YC, Schreiber G, Pitterle DM. A 1,4 Mb high-resolution physical map and contig of chromosome segment 11p15.5 and genes in the LOH11A metastasis suppressor region. Genomics 1999; 55: 164–75. Rees DC, Luo LY, Thein SL, Singh BM, Wickramasinghe S. Nontransfusional iron overload in thalassemia: association with hereditary hemochromatosis. Blood 1997; 90: 3234–36.
CERGM-Laboratoire des Hémoglobines, Marseille, France (C Badens PhD, N Martini, D Lena-Russo PhD); INSERM U 491, Marseille (M G Mattei PhD); Centre de Thérapie Cellulaire et Génique, Institut Paoli-Calmettes, Marseille (A M Imbert PhD); Hématologie Pédiatrique, CHU de la Timone, Marseille (Prof G Michel MD); and INSERM U 458, Paris (C Lapouméroulie PhD) Correspondence to: Dr C Badens, CERGM-Hémoglobines, Faculté de Médecine, 27, Bd Jean Moulin, 13385 Marseille cedex 5, France (e-mail: [email protected])
Association between hepatitis C virus seropositivity, carotid-artery plaque, and intima-media thickening Nobukazu Ishizaka, Yuko Ishizaka, Eiko Takahashi, Ei-ichi Tooda, Hideki Hashimoto, Ryozo Nagai, Minoru Yamakado We investigated the relation between positivity for hepatitis C virus (HCV) and carotid-artery plaque and carotid intimamedia thickening by analysing cross-sectional data of individuals undergoing a general health screening test. Of 4784 individuals enrolled, 104 (2·2%) were seropositive for HCV. After adjustment for confounding risk factors, HCV seropositivity was found to be associated with an increased risk of carotid-artery plaque (odds ratio 1·92 [95% CI 1·56–2·38], p=0·002) and carotid intima-media thickening (2·85 [2·28–3·57], p<0·0001). These findings suggest a possible role for chronic hepatitis C in the pathogenesis of carotid arterial remodelling.
Lancet 2002; 359: 133–35
The results of several published studies suggest a link between infection with micro-organisms such as Chlamydia pneumoniae,1 cytomegalovirus, herpes simplex virus, and hepatitis A virus,2 and an increased risk of cardiovascular and cerebrovascular diseases. The finding that infection of animals with micro-organisms such as C pneumoniae3 accelerates the development of atherosclerosis also suggests a possible role for these pathogens in atherogenesis. However, the atherogenic effects of certain infective agents remain controversial. Introduction of a transgene of bacterial  galactosidase into mouse cardiovascular cells accelerated cholesterol-induced atherosclerosis,4 and
133
For personal use. Only reproduce with permission from The Lancet Publishing Group.
of nodal-positive tumours, progression was limited mainly to class A. Finally, we saw progression of nodal-negative tumours only in class A. Taken together, our cluster analysis identifies breast-cancer patients with a high risk of recurrences, and is a step towards the establishment of an individual risk-profile system. Future directions should combine these molecular methods with the standard tumour classification system to obtain improved patient-tailored therapies. Contributors A Ahr, M Kaufmann, T Karn and U Holtrich had the original idea and designed the overall study. The molecular analyses were developed and done by U Holtrich and T Karn. Tumour biopsies were collected by C Solbach and K Strebhardt. T Seiters and A Ahr handled the patient data and analysed the association between patient classification and survival data. A Ahr, M Kaufmann, T Karn and U Holtrich analysed the results and wrote the manuscipt.
Conflict of interest statement None declared
Acknowlegments We thank Silke Deckert and Katherina Kourtis for technical assistance. This work was supported by a grant from the Deutsche Krebshilfe (10-1478-Ka2). The sponsors had no role in the study design; data collection, analysis or interpretation; or in the writing of the paper. The Genbank accession numbers of the 41 marker genes are as follows: AJ131693, U66879, U43746, L33930, L11315, U24166, X52541, M11730, N54493, AA029434, R85813, T89015, AL050276, X03635, AB006589, AF123659, X59932, M60974, X75208, U07695, M35410, M65062, M62402, NM_002417, D26512, AF076622, X17620, U41745, NM_005030, X51730, X78817, L25081, U33920, M97935, U47686, L12350, X74764, M33294, NM_005204, X51602, and AF041259. 1
2
3 4 5
Golub TR, Slonim DK, Tamayo P, et al. Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science 1999; 286: 531–37. Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 2000; 403: 503–11. Perou CM, Sorlie T, Eisen MB, et al. Molecular portraits of human breast tumours. Nature 2000; 406: 747–52. Hedenfalk I, Duggan D, Chen Y, et al. Gene-expression profiles in hereditary breast cancer. N Engl J Med 2001; 344: 539–48. Ahr A, Holtrich U, Solbach C, et al. Molecular classification of breast cancer patients by gene expression profiling. J Pathol 2001; 195: 312–20.
Department of Obstetrics and Gynecology, J W Goethe University, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany (A Ahr MD, T Karn PhD, C Solbach MD, T Seiter MD, K Strebhardt PhD, U Holtrich PhD, Prof M Kaufmann MD)
transfusion requirements. The phenotype is highly heterogeneous, ranging in severity from severe anaemia with hepatosplenomegaly and thalassaemia-like bone modifications to moderate microcytic hypochromic anaemia. In terms of genotype, thalassaemia intermedia is also heterogeneous, and has been shown to arise in four distinct ways:1 inheritance of mild -thalassaemia alleles; part rescue of -thalassaemia major by hereditary persistence of fetal haemoglobin; exacerbation of the heterozygous state by coinheritance of an extra ␣-globin gene, which worsens imbalance of the globin chains; or inheritance of a dominant -thalassaemia mutation. We report a novel mechanism by which severity of the heterozygous state is increased leading to thalassaemia intermedia. The propositus was first examined at age 10 years for anaemia, hepatosplenomegaly, and growth failure. He had a typical thalassaemic face, and skull radiographs showed a typical hair-on-end appearance. Analysis of his haemoglobin showed HbA 71%, HbF 25%, and HbA2 4%, and in-vitro globin chain synthesis gave an ␣/ globin-chain ratio of 3·9. Concentration of ferritin was 367 g/L and of bilirubin, 60 mol/L. The individual is now 18 years old and has never had a blood transfusion. His haemoglobin concentration has remained between 70 and 80 g/L. The patient’s father had a -thalassaemic trait, with HbA2 5·1% and microcytosis, whereas his mother had a normal red-cell index, haemoglobin electrophoretic profile, and ␣/ globin-chain ratio. Results of molecular studies showed that the patient’s father has the Mediterranean -thalassaemia nonsense mutation at codon 39 (C→T), which the patient inherited, whereas his mother does not have any common -thalassaemia mutations. We screened the maternally inherited allele of the propositus for a rare or de novo mutation by cloning and sequencing a PCR fragment spanning the entire maternallyderived  gene (from –240 to +1665 nucleotide relative to the cap site). We did not identify a mutation. With PCR, we also showed that neither the 3·7 kb deletion nor the anti3·7 kb triplication was present at the ␣ gene locus. After digestion of the patient’s leucocyte DNA with the restriction enzyme Mae I to detect the -thalassaemia mutation at codon 39, we noted that the band
Correspondence to: Dr André Ahr (e-mail: [email protected])
A novel mechanism for thalassaemia intermedia C Badens, M G Mattei, A M Imbert, C Lapouméroulie, N Martini, G Michel, D Lena-Russo Thalassaemia intermedia is a moderate form of thalassaemia resulting from various genetic defects. We report an undescribed mechanism leading to this condition: a somatic deletion of the -globin gene in the haemopoietic lineage of a heterozygous -thalassaemic patient. We did molecular studies and haemoglobin analysis of the patient and his parents. We found that the deletion gives rise to a mosaic of cells with either one or no functional -globin gene and it extends to a region of frequent loss of heterozygosity called LOH11A, which is located close to the -globin locus. Thus, loss of heterozygosity can be a cause of non-malignant genetic disease.
Lancet 2002; 359: 132–33
Thalassaemia intermedia is a clinical term used to describe patients who have mild thalassaemia with no regular
132
Figure 1: Restriction enzyme digestion and FISH analysis
PCR fragments of (a) exon 2 of the -globin gene digested by Mae I and (b) IVS 2 of the ␥-globin gene digested by Hind III. Lane 1=father; lane 2=mother, lane 3=propositus. (c) FISH on metaphases from lymphocytes of the propositus with a probe specific for the -globin gene extending from 2 kb upstream of the ␦-globin gene to the 3⬘ end of the -globin gene
THE LANCET • Vol 359 • January 12, 2002 • www.thelancet.com
For personal use. Only reproduce with permission from The Lancet Publishing Group.
RESEARCH LETTERS
report. C Lapouméroulie did Southern blot analyses, revised the report, and approved the final version. Prof G Michel obtained data and revised the report. D Lena-Russo did laboratory analyses, revised the report, and approved th final version. N Martini did laboratory analyses and analysed data.
Conflict of interest statement None declared. 1
Figure 2: Digestion of exon 2 of the -globin gene by Mae I
2
Lanes 1, 2, 3, 4=hemizygous colonies; lane 5=heterozygous colony.
corresponding to the maternal allele was strikingly faint relative to the one derived from the paternal allele (figure 1). With informative restrictive fragment length polymorphisms elsewhere in the -globin locus, we consistently recorded a maternal to paternal allele ratio of about one to three (figure 1). By contrast, this ratio was normal in the patient’s hair root and cheek epithelium cells (data not shown), suggesting that the maternal allele might be deleted in a percentage of the patient’s blood cells. Such mosaicism was looked for by fluorescent in-situ hybridisation (FISH) done on metaphases from lymphocytes with a probe specific for the -globin gene. Results of FISH showed the presence of one signal in 56% of the cells (figure 1). To further confirm this mosaicism in the haemopoietic lineage, we did PCR on erythroid burst-forming units obtained from the patient’s peripheral blood cells after culture in methylcellulose. Of 26 individual colonies, six were heterozygous for the mutation at codon 39 and 20 were hemizygous (figure 2). The -globin complex is located in 11p15.5 close to a region of frequent loss of heterozygosity, termed LOH11A.2 This region is associated with various cancers—such as stomach, lung, and breast—and is believed to contain a gene with tumour suppressor function.2,3 Deletion resulting in loss of heterozygosity has been shown to involve the -globin locus.4 With the polymorphic marker D11S988, we showed that the patient’s deletion extends to the LOH11A tumour suppressor region (data not shown). This deletion could potentially confer a proliferative advantage to hemizygous cells, and account for deleted cells being more numerous than non-deleted cells. Thus, we report a patient who is constitutionally heterozygous for a -thalassaemia mutation inherited from his father. During development, a second hit—deletion of the normal maternal allele in one of the early precursors of the haemopoietic lineage—led to somatic reduction, hemizygosity, and mosaicism of blood cells that have either one or no functional -globin gene. Somatic deletions giving rise to loss of heterozygosity have been discovered and studied in the context of carcinogenesis. In our report, loss of heterozygosity was not associated with malignancy, but allows phenotypic expression of the recessive -thalassaemia mutation at the intact -globin locus. This mechanism could account for some sporadic cases of thalassaemia intermedia in which only one -thalassaemia mutation is apparent.1,5 Our patient is of interest with respect to potential genereplacement therapies, since expression of a single functional -globin gene in only a proportion of red blood cells is sufficient to produce a moderate form of thalassaemia, which does not need major medical intervention. Contributors C Badens did laboratory analyses, had the original idea for and co-ordinated the study, analysed and interpreted data, and revised the report. M G Mattei did FISH analyses, revised the report, and approved the final version. A M Imbert did laboratory analyses and revised the
THE LANCET • Vol 359 • January 12, 2002 • www.thelancet.com
3 4
5
Galanello R, Cao A. Relationship between genotype and phenotype: thalassemia intermedia. Ann N Y Acad Sci 1998; 850: 325–33. O’Briant KC, Bepler G. Delineation of the centromeric and telomeric chromosome segment 11p15,5 lung cancer suppressor regions LOH11A and LOH11B. Genes Chromosomes Cancer 1997; 18: 111–14. Rodriguez E, Sreekantaiah C, Chaganti RS. Genetic changes in epithelial solid neoplasia. Cancer Res 1994; 54: 3398–406. Bepler G, O’Briant KC, Kim YC, Schreiber G, Pitterle DM. A 1,4 Mb high-resolution physical map and contig of chromosome segment 11p15.5 and genes in the LOH11A metastasis suppressor region. Genomics 1999; 55: 164–75. Rees DC, Luo LY, Thein SL, Singh BM, Wickramasinghe S. Nontransfusional iron overload in thalassemia: association with hereditary hemochromatosis. Blood 1997; 90: 3234–36.
CERGM-Laboratoire des Hémoglobines, Marseille, France (C Badens PhD, N Martini, D Lena-Russo PhD); INSERM U 491, Marseille (M G Mattei PhD); Centre de Thérapie Cellulaire et Génique, Institut Paoli-Calmettes, Marseille (A M Imbert PhD); Hématologie Pédiatrique, CHU de la Timone, Marseille (Prof G Michel MD); and INSERM U 458, Paris (C Lapouméroulie PhD) Correspondence to: Dr C Badens, CERGM-Hémoglobines, Faculté de Médecine, 27, Bd Jean Moulin, 13385 Marseille cedex 5, France (e-mail: [email protected])
Association between hepatitis C virus seropositivity, carotid-artery plaque, and intima-media thickening Nobukazu Ishizaka, Yuko Ishizaka, Eiko Takahashi, Ei-ichi Tooda, Hideki Hashimoto, Ryozo Nagai, Minoru Yamakado We investigated the relation between positivity for hepatitis C virus (HCV) and carotid-artery plaque and carotid intimamedia thickening by analysing cross-sectional data of individuals undergoing a general health screening test. Of 4784 individuals enrolled, 104 (2·2%) were seropositive for HCV. After adjustment for confounding risk factors, HCV seropositivity was found to be associated with an increased risk of carotid-artery plaque (odds ratio 1·92 [95% CI 1·56–2·38], p=0·002) and carotid intima-media thickening (2·85 [2·28–3·57], p<0·0001). These findings suggest a possible role for chronic hepatitis C in the pathogenesis of carotid arterial remodelling.
Lancet 2002; 359: 133–35
The results of several published studies suggest a link between infection with micro-organisms such as Chlamydia pneumoniae,1 cytomegalovirus, herpes simplex virus, and hepatitis A virus,2 and an increased risk of cardiovascular and cerebrovascular diseases. The finding that infection of animals with micro-organisms such as C pneumoniae3 accelerates the development of atherosclerosis also suggests a possible role for these pathogens in atherogenesis. However, the atherogenic effects of certain infective agents remain controversial. Introduction of a transgene of bacterial  galactosidase into mouse cardiovascular cells accelerated cholesterol-induced atherosclerosis,4 and
133
For personal use. Only reproduce with permission from The Lancet Publishing Group.