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- PATIENTS and METHODS
Regular blood transfusions from infancy until adulthood in β-thalassaemia major patients have substituted severe bone deformities with less marked skeletal lesions as osteoporosis. Osteoporosis is characterized by low bone mass and disruption of bone architecture, resulting in reduced bone strength and increased risk of fractures. Genetic factors have an important role in determining bone mineral density (BMD). We have investigated the possible association between BMD and two polymorphisms in 135 β-thalassaemic patients: (i) a substitution GΤ in a regulatory region of the COLIA1 gene encoding for the major protein of bone (type 1 collagen), and (ii) a one-base deletion in intron 4 (713–8del C) of transforming growth factor beta 1 (TGF-β1) gene. We have found a remarkable incidence (90%) of osteopenia and osteoporosis among regularly transfused patients. Bone mass was lower in men than in women (P = 0·0023), with a more prevalent osteopenia/osteoporosis of the spine in men than in women (P = 0·001). The sample was stratified on the basis of BMD expressed as Z-score, i.e. normal, osteopenic and osteoporotic patients, and genotype frequencies of each group were evaluated. TGF-β1 polymorphism failed to demonstrate a statistical difference in BMD groups. However, subjects with heterozygous or homozygous polymorphism of the COLIA1 gene showed a lower BMD than subjects without the sequence variation (P = 0·012). The differences among genotypes were still present when the BMD was analysed as adjusted Z-score and when men and women were analysed separately (P = 0·022 and 0·004 respectively), with men more severely affected. Analysis of COLIA1 polymorphism could help to identify those thalassaemic patients at risk of osteoporosis and fractures.
The new transfusion regimens and early iron-chelating therapy have improved the survival of thalassaemia major patients (Olivieri, 1999) and have substituted the marked bone abnormalities previously described (Cooley et al, 1927) with less severe skeletal lesions. In fact, osteopenia with cortical thinning, increased trabeculation of the spine and severe osteoporosis remain serious complications, even in well-transfused and iron-chelated patients (Vichinsky, 1998). Several factors may affect bone metabolism and turnover in β-thalassaemia patients, such as hormone deficiency, vitamin deficiency, iron overload and chelation therapy (Wonke, 1998; Singer & Vichinsky, 1999).
Transforming growth factor beta 1 (TGF-β1) in bone matrix has been implicated as a possible mediator of coupling between bone resorption and formation (Derynck et al, 1985; Bonewald & Mundy, 1990). Langdahl et al (1997) and Bertoldo et al (2000) found that a one-base deletion in intron 4 (713–8delC) of TGF-β1 was associated with severe osteoporosis and increased bone turnover in normal and osteoporotic women. At the same time, the genes encoding for collagen types Iα1 and Iα2 (COLIA1 and COLIA2 respectively) have also been implicated in bone mass defects, and some studies showed that COLIA1 polymorphism was associated with reduced BMD and predisposed men and women to osteoporotic fractures (Grant et al, 1996; Langdahl et al, 1998; Uitterlinden et al, 1998).
Being aware of the constant interaction between genetic and environmental factors in bone mass determination, the aim of this study was to investigate the allelic distribution for 713–8delC and COLIA1 polymorphisms in a β-thalassaemia transfusion-dependent Italian population and its relationship with bone mass.
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- PATIENTS and METHODS
The antrophometric, haematological and densitometric data are shown in Table I. The BMD of the examined subjects was expressed as the Z-score according to the WHO classification (World Health Organization, 1994). A large majority of the patients showed bone mass below one standard deviation from the mean value of the healthy subjects (Table I). Together, 121 out of 135 (90%) subjects with thalassaemia major showed osteopenia or osteoporosis of the spine and 105 out of 135 (78%) had low bone mass at the femoral neck (data not shown). Bone mass was lower in men than in women (P = 0·0023) (Table I) with a more prevalent osteopenia/osteoporosis of the spine in men than in women (χ2 = 13·4; P = 0·001) (data not shown).
Table I. Antrophometric, haematological and densitometric data in thalassaemia major patients. The data are expressed as mean (± SEM).
| ||Pooled (n = 135)||Men (n = 57)||Women (n = 78)|
|Age (years)||24·0 ± 0·4||25·4 ± 0·3||22·5 ± 0·4|
|Weight (kg)||55·8 ± 0·65||61·4 ± 0·5||51·8 ± 0·4|
|Height (cm)||159·3 ± 0·78||166 ± 0·52||154·5 ± 0·54|
|Blood (g/kg/year)||130·5 ± 0·5||129·9 ± 0·5||131·0 ± 0·6|
|Ferritin (μg/l)||1892·1 ± 1·5||1692·0 ± 1·3||2038·4 ± 1·6|
|Haemoglobin (g/dl)||9·07 ± 0·03||9·06 ± 0·03||9·08 ± 0·03|
|DFX (g/kg/year)||20·56 ± 0·17||20·97 ± 0·19||20·26 ± 0·16|
|Age started DFX (years)||5·81 ± 0·22||6·12 ± 0·29||5·88 ± 0·21|
|Years on DFX||15·9 ± 0·28||16·22 ± 0·32||15·76 ± 0·25|
|Lumbar BMD (Z-score)|| −2·19 ± 0·09|| −2·59 ± 0·16*|| −1·93 ± 0·10|
Table II reports the distribution of TGF-β1 and COLIA1 genotypes in the sample under study and in the normal reference population. Proportions among different genotypes were as expected according to the Hardy–Weinberg equilibrium. In the thalassaemic group, there were no differences in the genotype frequencies between men and women for the two polymorphisms studied (χ2 = 1·34, P = 0·246 and χ2 = 2·75, P = 0·25 respectively). Comparison between thalassaemia major and normal subjects showed no differences in the TGF-β1 and Sp1 genotype frequencies (χ2 = 1·45, P = 0·483 and χ2 = 2·21, P = 0·330 respectively).
Table II. Genotype frequencies for COLIA1 and TGF-β1 polymorphisms in thalassaemia major patients (% in brackets).
| ||Pooled (n = 135)||Men (n = 57)||Women (n = 78)||Controls (n = 112)|
| SS||74 (55)||35 (61)||39 (50)||74 (66)|
| Ss||55 (41)||20 (35)||35 (45)||34 (30)|
| ss||6 (4)||2 (4)||4 (5)||4 (4)|
| TT||112 (83)||50 (88)||62 (79)||92 (82)|
| Tt||23 (17)||7 (12)||16 (21)||19 (17)|
| tt||0||0||0||1 (1)|
COLIA1 Sp1 polymorphism and BMD
The mean Z-score values of lumbar spine BMD and clinical data divided by COLIA1 Sp1 polymorphism are reported in Table III.
Table III. Antrophometric, haematological and densitometric data according to genotypes of COLIA1 polymorphism. The data are expressed as mean (± SEM)
|Weight (kg)||55·5 ± 0·6||55·9 ± 0·3||59·5 ± 0·8||0·352|
|Height (cm)||158·8 ± 0·8||159·5 ± 0·7||164·8 ± 0·9||0·186|
|Blood (g/kg/year)||129·7 ± 0·5||131 ± 0·6||128·8 ± 0·5||0·078|
|Ferritin (μg/l)||1759 ± 1·4||2023 ± 1·5||2323 ± 0·9||0·478|
|Haemoglobin (g/dl)||9·07 ± 0·03||9·05 ± 0·03||9·1 ± 0·04||0·734|
|DFX (g/kg/year)||20·6 ± 0·2||20·2 ± 0·2||21·1 ± 0·2||0·071|
|Age started DFX (years)||5·91 ± 0·24||5·33 ± 0·25||5·7 ± 0·18||0·751|
|Years on DFX||15·7 ± 0·3||15·5 ± 0·3||16·3 ± 0·3||0·489|
|Lumbar BMD (Z-score)|| −2·06 ± 0·13|| −2·21 ± 0·15|| −3·23 ± 0·46||0·014|
|Adj lumbar BMD (Z-score)|| || || || |
| Pooled|| – 2·0 ± 0·11|| −2·19 ± 0·13|| −3·12 ± 0·26||0·012|
| Men|| – 2·20 ± 0·18|| −3·0 ± 0·24*|| −4·70 ± 0·30||0·022|
| Women|| −1·80 ± 0·13|| −1·68 ± 0·14|| −2·28 ±0·63||0·004|
The ‘Ss’ and ‘ss’ genotypes had a lower bone mass than subjects with ‘SS’ genotype (P = 0·014); the homozygous patients for ‘s’ allele had the lowest bone mass. There were no differences among genotypes in all potential confounding clinical factors. When polymorphism, spontaneous puberty, hypogonadotrophic hypogonadism and diabetes were tested together in a general linear model to verify their association with the level of bone mass, the COLIA1 polymorphism continued to maintain a significant association (P = 0·04, P = 0·610, P = 0·203 and P = 0·389 respectively). The differences among genotypes were still present when the BMD was analysed as the adjusted Z-score and when the two sexes were analysed separately (men P = 0·022 and women P = 0·004). Moreover, in male thalassaemic patients, the presence of the ‘s’ allele was associated with more severe osteoporosis of the spine than in female patients (P = 0·0006 in ‘Ss’ patients) (Table III). While spine BMD values were still lower in ‘ss’ subjects, the difference between men and women was not significant because of the small number of homozygotes. When the study population was stratified on the basis of Z-score values at the lumbar spine BMD in normal (Z-score > −1) and osteopenic/osteoporotic (Z-score < −1) groups, a different allelic distribution was found (P = 0·047) (Table IV). The ‘ss’ genotype (5%) was present only in the osteopenic/osteoporotic group.
Table IV. Genotypes frequencies of COLIA1 polymorphism in normal (vertebral BMD > −1) and osteopenic/osteoporotic patients (vertebral BMD < −1) (% in brackets).
|Normal||12 (86)||2 (14)||0|
|Osteopenic/osteoporotic||62 (51)||53 (44)||6 (5)|
TGF-β1 polymorphism and BMD
There were no differences among genotypes in all examined clinical data (data not shown). When the mean Z-score and the adjusted Z-score of lumbar spine BMD of the subjects with ‘TT’ genotype was compared with those with the ‘Tt’ genotype there was a lower bone mass in ‘Tt’ genotype, but the difference was not significant (Table V and data not shown). When men and women were analysed separately, no significant differences were found in Z-scores between the two genotypes. Moreover, in male thalassaemic patients, the presence of more severe osteoporosis of the spine than female patients was not associated with the TGF-β1 polymorphism, although ‘Tt’ men showed a trend to a lower bone mass than ‘Tt’ women (Table V). When the study population was divided by Z-score values at the lumbar spine BMD in normal and osteopenic/osteoporotic subjects, we found no differences in genotype frequencies in thalassaemic subjects with Z-score < −1 compared with thalassaemic subjects with Z-score > −1 (Table VI).
Table V. Vertebral BMD (expressed as Z-score) according to genotypes of TGF-β1 polymorphism. The data are expressed as mean (± SEM).
|Pooled|| −2·15 ± 0·10|| −2·24 ± 0·23||0·74|
|Men|| −2·61 ± 0·17|| −2·41 ± 0·46*||0·68|
|Women|| −1·80 ± 0·11|| −2·16 ± 0·23||0·10|
Table VI. Genotypes frequencies of TGF-β1 polymorphism in normal (vertebral BMD > −1) and osteopenic/osteoporotic patients (vertebral BMD < −1) (% in brackets).
|Normal||12 (86)||2 (14)|
|Osteopenic/osteoporotic||100 (83)||21 (17)|
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- PATIENTS and METHODS
A variety of skeletal disorders including rickets, scoliosis, spinal deformities, nerve compression, fractures and severe osteoporosis have been reported in thalassaemia major patients (Giardina et al, 1995; Vichinsky, 1998). In particular, osteoporosis and bone fractures seem to be present in a variable percentage of the patients from 33% to 20% (Giardina et al, 1995). Recently, Jensen et al (1998) showed a prevalence of low and severely low bone mass in 45% and 51% of thalassaemia major patients respectively. Our findings in a group of adult transfusion-dependent patients show that the bone loss is a common feature in well-treated thalassaemic patients. The prevalence of low bone mass is very high with 90% of the thalassaemic patients having a lower Z-score than controls. According to Jensen et al (1998), the prevalence of osteopenia and osteoporosis is higher in men than in women and is more prevalent in the spine than the femoral neck. These differences between two skeletal sites are as a result of different bone architecture: the lumbar spine has prevalent trabecular bone and the femoral neck has a mixture of trabecular and cortical bone.
Evidence from family and twin studies has clearly shown that genetic factors play an important role in the pathogenesis of osteoporosis. An estimated 46–80% of the total variance in adult bone mass is attributed to genetic determinants (Pocock et al, 1987; Krall & Dawson-Hughes, 1993). Recently, great interest has been generated regarding the relationship between some polymorphisms, e.g. vitamin D receptor and oestrogen receptor genes, and bone density (Morrison et al, 1994; Kobayashi et al, 1996). However, the consistency of this effect has not yet been established and controversy surrounding the relationship between these polymorphisms and BMD demands further investigation (Peacock, 1995; Han et al, 1997).
The association between 713-8delC of the TGF-β1 gene and osteoporosis has been demonstrated in different populations (Langdahl et al, 1997; Yamada et al, 1998; Bertoldo et al, 2000). Moreover, other studies have demonstrated that a novel polymorphism in the collagen type Iα1 (COLIA1) gene is over-represented in osteoporotic patients, and is associated with reduced bone density (Grant et al, 1996) and high risk of fractures (Langdahl et al, 1998; Uitterlinden et al, 1998). Recently, it has been shown (Wonke, 1998, Wonke et al, 1998) that male thalassaemia patients who are heterozygous or homozygous at the polymorphic Sp1 site have a lower BMD than females and had no improvement in spinal osteoporosis in response to treatment with bisphosphonates. In both male and female patients, we found a consistent association between Sp1 polymorphism and differences in bone density at the spine, with evidence of gene-dose effects. Furthermore, BMD values were higher in women than men in each genotype. Therefore, we suggest that this polymorphism may represent a genetic ‘background’ influencing the development of secondary osteoporosis in thalassaemia major, as demonstrated in other secondary osteoporosis conditions such as in diabetes mellitus (Hampson et al, 1998), and potentially increasing per se the high risk of vertebral fractures documented in thalassaemia major.
The association between the gene polymorphisms and bone mass in thalassaemia major needs to be considered, bearing in mind that the disease is a well-documented type of secondary osteoporosis with multiple negative influences, also exogenous, on bone metabolism that could appear to be as a result of the effect of the polymorphisms. In fact, many factors in thalassaemia major can lead to an unbalanced bone remodelling and increased bone resorption. These factors include hormonal deficiency, bone marrow expansion, iron overload and desferrioxamine toxicity (Vichinsky, 1998). In our study, the impact of antrophometric and haematological factors on lumbar bone mass values was negligible when tested in a multiple regression analysis model. Only blood and DFX cumulative amounts reached a relationship with bone mass near to the statistical significance (P = 0·078 and P = 0·071 respectively).
The molecular mechanism underlying the association between both polymorphisms and the BMD remain to be defined. A possible explanation of the different association pattern between the two polymorphisms and BMD could be that the TGF-β1 polymorphism appears to influence bone mass by mainly increasing bone turnover (Langdahl et al, 1997; Bertoldo et al, 2000). Bone turnover is also dramatically affected by thalassaemia and its treatment per se, and this may represent the cause of the lack of a strong association with bone mass in thalassaemic patients. On the contrary, COLIA1 polymorphism can affect bone mass by influencing other aspects of bone metabolism, i.e. collagen structure. It therefore appears as an independent risk factor for genetic susceptibility to osteoporosis.
This study provides strong evidence of an association between the Sp1 polymorphism of the COLIA1 gene and vertebral osteoporosis in a sample of Italian β-thalassaemia major patients, with men more severely affected. Our results raise the possibility that genotyping at the Sp1 site could be of clinical value in identifying the thalassaemic patients at risk of osteoporosis and fractures.