Molecular characterization and frequency of transforming growth factor beta 1 gene polymorphism and its relation to bone complications in Egyptian patients with β-thalassemia

Somaya M Elgawhary; Manal N Mohammed; Heba M Ahmed; Ahmed M Elamir; Hossam M Abdelaziz

doi:10.4103/ejh.ejh_13_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 47 | Issue : 3 | Page : 174-180

Molecular characterization and frequency of transforming growth factor beta 1 gene polymorphism and its relation to bone complications in Egyptian patients with β-thalassemia

Somaya M Elgawhary, Manal N Mohammed, Heba M Ahmed, Ahmed M Elamir, Hossam M Abdelaziz
Department of Clinical Pathology, Faculty of Medicine, Fayoum, Egypt

Date of Submission	22-Mar-2022
Date of Acceptance	04-Apr-2022
Date of Web Publication	03-Jan-2023

Correspondence Address:
Hossam M Abdelaziz
Department of Clinical Pathology, Faculty of Medicine, Fayoum 63514
Egypt

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/ejh.ejh_13_22

Abstract

Objective The objective of this study was to determine the frequency of transforming growth factor beta 1 (TGFβ1) C-509T gene polymorphism and its relation to bone complications in patients with β-thalassemia major in Egypt. Background Osteoporosis is the most prevalent bone complication in patients with β-thalassemia major despite regular blood transfusions and iron chelation therapy. It is characterized by low bone mineral density (BMD) resulting in reduced bone strength and increased risk of fractures. Genetic factors play an important role in the determination of BMD. The TGFβ1 gene, which encodes TGFβ1, is a strong candidate for susceptibility to osteoporosis, and several studies have reported associations between BMD and different polymorphisms of TGFβ1, although these studies have yielded conflicting results. Study design and methods Single nucleotide polymorphism in the TGFβ1 gene promoter (C-509T) was investigated in 100 regularly treated Egyptian children with β-thalassemia major by PCR/RFLP genotyping. BMD was measured by dual-energy radiograph absorptiometry and expressed as Z score. Results The frequency of TGFβ1 gene polymorphism C-509T genotypes in all studied patients was 6% for homozygous CC, 85% for heterozygous CT, and 9% for homozygous TT. C allele frequency was 48.5%, whereas T allele frequency was 51.5%. BMD Z score was significantly higher in TT genotype compared with CC genotype, with P value less than 0.05. Patients were grouped on the basis of BMD Z score: 51 (51%) patients with BMD deficit (Z score <−1) and 49 (49%) with normal BMD (Z score ≥−1). TGFβ1 gene polymorphism C-509T genotypes were distributed differently between the two groups; the TT genotype frequency was lower in patients with BMD deficit (P<0.05). Conclusion TGFB1 gene polymorphism C-509T is associated with BMD and genetic susceptibility to osteoporosis and may play a role in the pathogenesis and modification of bone complication in β-thalassemia major. BMD deficit is common in Egyptian children with β-thalassemia major. Analysis of this polymorphism at an early age could help in identification of thalassemic children at risk of osteoporosis and early management. However, large-scale studies are required to confirm these findings.

Keywords: bone mineral density, osteoporosis, polymorphism, transforming growth factor beta 1 gene, β-thalassemia major

How to cite this article:
Elgawhary SM, Mohammed MN, Ahmed HM, Elamir AM, Abdelaziz HM. Molecular characterization and frequency of transforming growth factor beta 1 gene polymorphism and its relation to bone complications in Egyptian patients with β-thalassemia. Egypt J Haematol 2022;47:174-80

How to cite this URL:
Elgawhary SM, Mohammed MN, Ahmed HM, Elamir AM, Abdelaziz HM. Molecular characterization and frequency of transforming growth factor beta 1 gene polymorphism and its relation to bone complications in Egyptian patients with β-thalassemia. Egypt J Haematol [serial online] 2022 [cited 2023 Mar 30];47:174-80. Available from: http://www.ehj.eg.net/text.asp?2022/47/3/174/366858

Introduction

β-thalassemia syndromes are one of the most common autosomal recessive hereditary disorders worldwide, with high prevalence in the populations of Mediterranean, Middle East, Central Asia, Indian subcontinent, and far east regions [1].

The clinical variability in the β-thalassemia syndromes has swayed researchers toward identifying genetic modifiers of severity for these disorders. Such genetic modifiers could potentially lead to the development of more specific and effective therapies. Genetic modifiers exert their effect on three levels: primary, secondary, and tertiary [2].

Primary modifiers refer to the type of alterations affecting the β-globin gene [1]. Secondary modifiers include variations in genes affecting α/β-globin chain equilibrium [3]. However, the tertiary modifiers are gene variations affecting the phenotype regarding the complications caused by β-thalassemia syndromes such as hyperbilirubinemia, thrombophilia, and bone disease [4].

Osteoporosis is the most prevalent bone complication in patients with β-thalassemia major. It is characterized by low bone mineral density (BMD) and deterioration of bone tissue micro-architecture with increased fracture risk [5]. Several factors have been implicated in playing an important role in the pathogenesis of osteoporosis, and several candidate gene polymorphisms have been found to regulate this process [6].

Polymorphisms of several genes, known to influence BMD, including collagen type I alpha 1, vitamin D receptor, and transforming growth factor beta 1 (TGFβ1), have been previously investigated among patients with thalassemia. However, the results were controversial [7].

Patients and methods

A total of 100 patients with β-thalassemia major attending Fayoum University Hospitals for regular blood transfusion and chelation therapy were enrolled in our study. Approval by the ethical committee of Faculty of Medicine, Fayoum University, was obtained before commencement of the study. Moreover, informed written consent was obtained from legal guardians of all participants participating in this study.

Inclusion criteria were as follows:

(1) Patients with β-thalassemia major compliant with regular blood transfusion and chelation therapy.

(2) Patients under 18 years of age.

(3) Both sexes.

Exclusion criteria were as follows:

(1) Patients with hemolytic anemia other than β-thalassemia major.

(2) Age over 18 years.

(3) Patients with any associated diseases known to affect BMD.

All patients were subjected to the following:

(1) Complete history taking and clinical examination.

(2) BMD measurement by dual-energy radiograph absorptiometry (DXA) scan of the lumbar spine (L1–L4) using GE Lunar DPX Duo Bone Densitometer (GE Medical Systems GmbH, Freiburg, Germany, Headquarters: GE Medical Systems-LUNAR Madison, WI, USA). The BMD results were converted to age-specific and sex-specific Z scores based on the normative reference data for BMD in Egyptian children.

(3) Routine laboratory investigations: complete blood picture and reticulocyte count; hemoglobin electrophoresis; and serum calcium, phosphorus, alkaline phosphatase (ALP), and ferritin.

(4) Detection of TGFβ1 C-509T gene polymorphism by PCR-RFLP:
- (a) Extraction of genomic DNA: total genomic DNA was extracted from peripheral blood leukocytes using the GeneJET Blood Genomic DNA purification kit (Thermo Scientific; Catalog No.: K0782).
- (b) Determination of extracted DNA quantity and quality: the concentration and purity of DNA was determined by NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA).
- (c) Amplification of genomic DNA by PCR:

Reagents:

(i) COSMO PCR RED Master Mix: (Willofort, UK, Catalog No: WF10203001). The mix contains the COSMO DNA polymerase enzyme, as well as all the necessary components required for a successful PCR. Components of the COSMO PCR Master Mix include COSMO Taq DNA polymerase, MgCl2, dNTPs, and storage buffer.

(ii) Primers (Willofort, UK):
- 1. Forward primer (F): 5’-AGTAAATGTATGGGGTCGCAG-3’
- 2. Reverse primer (R): 5’-GGTGTCAGTGGGAGGAGGG-3’

(d) Detection of PCR amplification products using gel electrophoresis and ultraviolet light trans-illumination: the samples were then run in parallel on 3% agarose gel using gel electrophoresis and visualized on ultraviolet transilluminator (SYNGENE GVM 20 X) to detect the presence of the amplified 153 bp DNA band.

(e) Digestion of the amplified DNA by the specific restriction enzyme: reagents: Bsu 36I restriction enzyme supplied with its fast digest buffer (New England Biolabs; Catalog No. R0524S, Hitchin, UK). Bsu 36I is a restriction endonuclease that recognizes the sequence: 5’… C C^↓T N A G G…3’ / 3’… G G A N T_↑C C…5’

(5) Detection and interpretation of TGFβ1 C-509T gene polymorphism: the amplified digested products were separated on 3% agarose gel electrophoresis, stained with ethidium bromide and visualized under ultraviolet light. The results were documented using a digital camera.
- (a) C allele: two bands at 117 and 36 bp were detected.
- (b) T allele: one band at 153 bp was detected.
- (c) Homozygous genotype CC: two bands 117 and 36 bp were detected.
- (d) Heterozygous genotype CT: three bands at 117, 36, and 153 bp were detected.
- (e) Homozygous genotype TT: one band at 153 bp was detected.

Statistical analysis

Data were analyzed using the Statistical Package of Social Science (SPSS) software, version 22 in Windows 7 (SPSS Inc., Chicago, Illinois, USA). The Kolmogorov–Smirnov test was used to verify the normality of distribution of variables.

For quantitative parametric data, independent Student t test was used to compare measures of two independent groups of quantitative data, and one-way analysis of varaince test was used in comparing more than two independent groups of quantitative data.

For quantitative nonparametric data, Mann–Whitney test was used in comparing two independent groups, and Kruskal–Wallis test was used in comparing more than two independent groups.

For qualitative data, χ² test was used to compare two of more than two qualitative groups.

The level P value less than 0.05 was considered the cutoff value for significance.

Results

A total of 100 patients with β-thalassemia major regularly attending Fayoum University Hospitals for blood transfusion and iron chelation therapy were enrolled in the present study. The patients’ age ranged from 2 to 13 years, with a mean±SD of 7.9 ± 3.4 years. There were 52 (52%) males and 48 (48%) females.

Total calcium level ranged from 7.9 to 10.6 mg/dl, with a mean±SD of 9.5 ± 0.52 mg/dl. Ionized calcium level ranged from 3.9 to 5.2 mg/dl, with a mean±SD of 4.7 ± 0.36 mg/dl. Phosphorus level ranged from 3.9 to 6.5 mg/dl, with a mean±SD of 4.9 ± 0.4 mg/dl. The ALP level ranged from 113 to 396 U/l, with a mean±SD of 204.4 ± 66.3 U/l. The ferritin level ranged from 719 to 2500 ng/ml with a mean±SD of 1506.3 ± 482.6 U/l. Lumbar BMD Z score ranged from −7.1 to 2.3, with mean±SD Z score of −1.33 ± 1.8.

Results of PCR-RFLP analysis of TGFβ1 C-509T gene polymorphism in the whole patient group showed that homozygous CC (wild allele) was found in six (6%) patients, heterozygous CT was found in 85 (85%), and homozygous TT was found in nine (9%) patients. Regarding allele frequency, wild C allele frequency was 48.5% (97/200), whereas T allele frequency was 51.5% (103/200).

There was no statistically significant difference (P>0.05) between different TGFβ1 C-509T polymorphism genotypes regarding age and sex distribution ([Table 1]).

Table 1: The comparative analysis between different transforming growth factor beta 1 C-509T polymorphism genotypes regarding laboratory and densitometry data

Click here to view

There was a statistically significant difference (P<0.05) between different genotypes regarding phosphorus and ALP with lower mean levels among cases with TT genotype compared with CT and CC genotypes. There was no statistically significant difference (P>0.05) between different genotypes regarding other laboratory data. Mean BMD Z score was significantly higher in TT genotype compared with CC genotype (P<0.05) ([Table 2]).

Table 2: The comparison between different C allele groups of transforming growth factor beta 1 C-509T polymorphism among β-thalassemia cases regarding age, laboratory, and densitometry data

Click here to view

There was a statistically significant lower mean of ionized calcium and higher mean of phosphorus and ALP, with P value less than 0.05, among cases that had the C allele of TGFβ1 C-509T polymorphism (CC+CT genotypes) compared with cases with no C allele (TT genotype). On the contrary, there was no statistically significant difference (P>0.05) between the two groups regarding other laboratory data and BMD Z score ([Table 3]).

Table 3: The comparison between different T allele groups of transforming growth factor beta 1 C-509T polymorphism among β-thalassemia cases regarding age, laboratory, and densitometry data

Click here to view

There was no statistically significant difference (P>0.05) between patients having the T allele (TT+CT genotypes) and patients with no T allele (CC genotype) regarding age, laboratory data, and BMD Z score ([Table 4]).

Table 4: The comparison of genotype distribution and allele frequency between patients with bone mineral density deficit (Z score <−1) and patients with normal bone mineral density (Z score ≥−1)

Click here to view

The CC genotype frequency was higher in the patient group with BMD deficit, whereas TT genotype frequency was higher in the patient group with normal BMD (P<0.05). There was no significant difference regarding heterozygous CT genotype frequency between the two groups. The C allele frequency was significantly higher and T allele frequency was significantly lower in the group of patients with BMD deficit compared with patients with normal BMD (P<0.05).

Discussion

β-thalassemia major causes hemolytic anemia, poor growth, and skeletal abnormalities during infancy. Affected children require regular lifelong blood transfusions. Cooley et al. [8], described the first patients with anemia, splenomegaly, and cranial and facial bone enlargement. These bone changes were due to the marked expansion of the bone marrow, secondary to anemia and ineffective erythropoiesis.

Osteoporosis represents an important cause of morbidity in β-thalassemia major, and its pathogenesis has not been completely clarified. Genetic factors play an important role in the pathogenesis of osteoporosis, and several candidate gene polymorphisms have been implicated in the regulation of this process [9].

TGFβ1 gene was one of the first suggested candidate genes to contribute to genetic risk for osteoporosis for several reasons; TGFβ1 is expressed by osteoblast cells and present in high concentrations in bone tissue. It is involved in the control of bone resorption and formation [10].

The aim of this study was to determine the frequency of TGFβ1 C-509T gene polymorphism and its relation to bone complications in patients with β-thalassemia major in Egypt.

The present study included 100 children with β-thalassemia major under regular blood transfusion and chelation therapy. They were selected among patients attending Fayoum University Hospitals. All patients were subjected to history taking, routine laboratory investigations, and determination of BMD by DXA. Genotyping was conducted for TGFB1 polymorphism C-509T using PCR/RFLP analysis.

DXA is currently the most reliable and widely used method for measuring BMD, and this technique assesses bone mass classically at the lumbar spine and proximal femur [11].

Of 100 patients with β-thalassemia major, 51 (51%) included in this study had BMD deficit, whereas 49/100 (49%) had normal BMD. Bone mineral status has been extensively investigated in adult thalassemics but less in thalassemic children [12].

Ishaq et al. [13] studied the frequency of low BMD in patients with thalassemia in Pakistan. A total of 150 children with thalassemia major with age range from 3 to 12 years old were included in their study, and low BMD was recorded in 73 (49%) children with beta-thalassemia major. Similar results were reported by another study in the city of Tehran, Iran, which investigated the prevalence of low bone mass in patients with the diagnosis of beta-thalassemia major (n=203), aged 10–20 years, and the potential risk factors for osteoporosis in this specific patient population. The study showed that the prevalence of low BMD was 50.7% with no difference in male and female patients [14].

Higher frequency of BMD deficit was reported by Nawar et al. [15], who assessed BMD in 50 Egyptian patients with β-thalassemia major (age 5–18 years) and reported that nine (18%) patients had normal BMD (Z score >−1), whereas 41 (82%) patients had low BMD.

The difference in BMD deficit frequency could be explained by the relationship between BMD Z scores and chronological age. A study by Merchant et al. [16] showed a statistical significance (P=0.046) between chronological age and BMD scores with 62.5% of cases having abnormal Z scores between ages 10 and 15 years and 92.3% cases with abnormal Z scores after 15 years.

In contrast to this study and other studies, Christoforidis and colleagues reported that BMD Z scores were within normal among 35 patients with β-thalassemia major studied, age 5–20 years. None of the patients were in the osteoporotic range (Z score <−2.5), whereas only one (5.26%) out of the 19 girls and six (37.5%) out of the 16 boys were in the osteopenic range (−1>Z score >−2.5). They concluded that their study was the first in the literature that shows normal BMD values in children with homozygous β-thalassemia, regularly transfused, and adequately chelated [17].

The gene encoding TGFβ1 is a strong functional candidate for genetic susceptibility to osteoporosis. Several polymorphisms have been identified in TGFB1, and previous work has suggested that allelic variants of TGFβ1 may regulate BMD and susceptibility to osteoporotic fracture [18].

To our knowledge, this is the first study to investigate the frequency of TGFβ1 C-509T polymorphism and its relation to BMD and osteoporosis risk in Egyptian children with β-thalassemia major. The frequency of TGFβ1 C-509T polymorphism genotypes in this study was 6% (6/100) homozygous CC genotype, 85% (85/100) heterozygous CT, and 9% (9/100) homozygous TT. Regarding allele frequency, wild C allele frequency was 48.5%, whereas T allele frequency was 51.5%.

Previously, the frequency of TGFβ1 gene promoter polymorphism C-509T has been investigated in association with changes in BMD and in association with many other diseases, although with different and contradictory results. In a study by Riyanti [19], the C-509T polymorphism was studied in 66 patients with thalassemia major in Indonesia. Homozygous CC (wild allele) was found in one (1.53%) patient, heterozygous CT was found in 42 (63.63%), and homozygous TT was found in 23 (34.84%) patients.

The possible association of a C-509T polymorphism in the promoter region of the TGFβ1 gene with BMD and genetic susceptibility to osteoporosis was investigated in 625 postmenopausal Japanese women by Yamada et al. [20]; the frequencies of the CC, CT, and TT genotypes of the C-509T polymorphism in the study population were 24, 49, and 27%, respectively.

In a large-scale study by McGuigan and colleagues, the relationship between common polymorphisms of TGFβ1 and several osteoporosis-related phenotypes including BMD measured by DXA at the lumbar spine and femoral neck was investigated in 5119 women (45–54 years of age). Regarding C-509T polymorphism, the CC genotype frequency was 51.8%, CT genotype was 39.9%, and TT genotype was 8.3% [18].

The inconsistent results of TGFβ1 C-509T polymorphism frequency between different studies reflect the often-limited small sample sizes and the variation of ethnicity, age, sex, and specific disease of the populations studied.

Regarding association of C-509T polymorphism in the promoter region of the TGFβ1 gene with BMD and genetic susceptibility to osteoporosis, BMD Z score in this study was significantly higher in TT genotype compared with CC genotype, with P value less than 0.05.

In addition, C-509T genotypes were distributed differently among patients with BMD deficit and patients with normal BMD. The TT genotype was less common in individuals with BMD deficit (P<0.05). Previous studies on the effect of C-509T polymorphism on BMD have found contradictory results; the T-allele was associated with reduced BMD in postmenopausal Japanese women [20], whereas the same allele, was associated with higher BMD in Danish men and women [21] and higher heel ultrasound stiffness index in young Canadian women [22].

The discrepancy of results between different studies investigating the association of TGFβ1 C-509T polymorphism with BMD could be due to the variation in ethnicity, age, sex, and associated clinical diagnosis of the populations studied.

Conclusion

Despite regular blood transfusions and iron chelation, decreased BMD with risk of osteoporosis remains a serious complication in patients with β-thalassemia major even at young age. A total of 51 (51%) studied children with β-thalassemia major had BMD deficit. Most patients included in this study were heterozygous for the TGFβ1 gene polymorphism C-509T (85%) followed by homozygous TT (9%) and homozygous CC (6%).

BMD Z score was significantly higher in TT genotype compared with CC genotype, with P value less than 0.05. TGFβ gene polymorphism C-509T genotypes were distributed differently among patients with BMD deficit and patients with normal BMD; TT genotype was less common in individuals with BMD deficit (P<0.05).

TGFβ1 gene polymorphism C-509T was associated with BMD and genetic susceptibility to osteoporosis in the studied population and may play a role in the pathogenesis and modification of bone complications in β-thalassemia major.

However, further studies with a larger cohort are needed to confirm the findings of this study.

Acknowledgements

Authorship contributions: Somaya M. Elgawhary: project administration, software, and validation. Manal N. Mohammed: conceptualization, visualization, and supervision. Ahmed M. Elamir: methodology, data curation, and formal analysis. Heba M. Ahmed: investigation, resources, and writing – original draft. Hossam M. Abdelaziz: investigation, resources, writing, review, and editing.

Financial support and sponsorship

Nil.

Conflicts of interest

There is no conflict of interest.

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