|
 
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| ORIGINAL ARTICLE |
|
| Year : 2021 | Volume
: 5
| Issue : 2 | Page : 49-55 |
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Prevalence of H63D and C282Y mutations in hereditary hemochromatosis (HFE) gene in Tripoli region of Libya
Laila Mohamed Elghawi1, Kaltoom Hassan Mahanna2, Abdulla M Bashein3
1 Department of Genetic Engineering, School of Engineering and Applied Sciences, Libyan Academy, Tripoli, Libya
2 Faculty of Medicine, Gharyan University, Gharyan, Libya
3 Department of Biochemistry, Faculty of Medicine, University of Tripoli; Reference Laboratory, National Center for Disease Control, Tripoli, Libya
| Date of Submission | 03-May-2021 |
| Date of Acceptance | 06-Jun-2021 |
| Date of Web Publication | 23-Jul-2021 |
Correspondence Address:
Dr. Abdulla M Bashein
Department of Biochemistry, Faculty of Medicine, University of Tripoli, Tripoli
Libya
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/ljms.ljms_27_21
Background and Aims: Hereditary hemochromatosis (HH) is an autosomal recessive disorder, characterized by increased intestinal absorption of iron. Excessive amount of iron accumulates in the liver, pancreas, and heart, etc., and eventually leading to organ failure due to iron toxicity and death if untreated. The most common causes of HH are the C282Y and H63D mutations in HFE gene. This study aimed to identify the prevalence of H63D and C282Y alleles among the Libyan population in Tripoli region and to compare the results with other published data. Materials and Methods: This study included 300 randomly selected unrelated Libyan male blood donors, aged between 18 and 50 years. In-house hydrolysis probe real-time polymerase chain reaction and high-resolution melting analysis protocols were developed and employed as screening tools for H63D and C282Y genotyping, respectively, and direct DNA sequencing was used to confirm the results. Results: Seven subjects (2.33%) were detected as homozygous H63D mutation and 72 (24%) were detected as heterozygous, and only one subject was detected as a heterozygous C282Y mutant (0.33%) and no homozygous C282Y mutation was detected. Conclusion: In Libyans residing in Tripoli region, the allele frequency of C282Y was very rare and allele frequency of H63D was common.
Keywords: C187G, G845A, high-resolution melting, rs1799945, rs1800562
How to cite this article:
Elghawi LM, Mahanna KH, Bashein AM. Prevalence of H63D and C282Y mutations in hereditary hemochromatosis (HFE) gene in Tripoli region of Libya. Libyan J Med Sci 2021;5:49-55 |
How to cite this URL:
Elghawi LM, Mahanna KH, Bashein AM. Prevalence of H63D and C282Y mutations in hereditary hemochromatosis (HFE) gene in Tripoli region of Libya. Libyan J Med Sci [serial online] 2021 [cited 2023 Mar 31];5:49-55. Available from: https://www.ljmsonline.com/text.asp?2021/5/2/49/322203 |
| Introduction | |  |
Hereditary hemochromatosis (HH) is an autosomal recessive disorder, which is characterized by increase of iron absorption from the diet[1] that results in increased iron levels in circulation and toxic accumulation of iron in the body's organs such as liver, pancreas, gonads, and heart, leading to organ failure and death if untreated.[2],[3],[4],[5] The liver is the organ most affected by hemochromatosis because of its relatively large blood flow.[6] In the past decades, several genes were identified as being central to the maintenance of iron homeostasis. One such gene is HFE, a major histocompatibility class I-like gene that, when mutated, may cause HH.[7] It is reported that the most common genetic causes of HH disease are the C282Yand H63D mutations in the HFE gene.[8]
HFE gene encodes a membrane protein (HFE protein) that co-localizes and interacts at the cell surface through its α1 and α2 domains with the major protein responsible for cellular iron uptake, namely the transferrin receptor 1 (TFR1). This association reduces TFR1 affinity for the circulating iron-transporter transferrin, thereby limiting iron uptake and thus directly implicating HFE in the modulation of cellular iron levels.[9],[10],[11],[12]
C282Y mutation is a single-base change that results in the transition of G-to-A at nucleotide position 845 (c. G845A; rs1800562) of the HFE gene within exon 4, in the codon of amino acid 282, which substitutes a tyrosine for a cysteine (Cys282Tyr). Loss of this cysteine disrupts the disulfide bond needed for formation of the α3 domain leading to misfolding of the mutant HFE protein, lack of association with chaperone β2-microglobulin, and failure to transport to and presentation on the cell surface,[13] preventing the association of HFE with TFR1 at cell surface, thereby increasing iron uptake.[14] H63D is a single-base change that results in transversion of C-to-G at nucleotide position 187 (c. C187G, rs1799945) of the HFE gene within exon 2, in the codon of amino acid 63, which substitutes an aspartic acid for histidine (His63Asp).[14] However, H63D does not have a direct effect on the interaction between HFE and TFR. Since it is located in the α1 domain of the HFE protein where the histidine participates in a salt bridge with a residue in the HFE α1 domain and thereby causes a local change in the tertiary structure of the HFE protein, this disruption may lead to a local rearrangement of the α1 domain and possibly interfere with either the correct function,[10],[14] and/or stability of the HFE protein.[15]
The prevalence of these two missense mutations around the world varies from one population to another. While C282Y is the common mutation in the Caucasian populations, ranging from 1.07% in Greece to 9.9% in North Ireland,[16],[17],[18],[19],[20] it is very rare or absent in Africans, Asians, and aboriginal Australian populations.[19],[21] Several studies suggested either a “Celtic” or a “Viking” origin of the C282Y mutation about 2000 years ago and spread since then.[19],[21],[22],[23] Almost certainly, the rare prevalence of C282Y mutation in non-Caucasian occurrences results in admixture.[19] However, H63D mutation has a global distribution. The H63D mutation has been thought to be older, while the C282Y mutation has emerged relatively recently.[24] In Libya, we were not able to identify any previous studies dealing with the frequency of these mutations among Libyans in Tripoli region. Therefore, this study aimed to investigate the prevalence of H63D and C282Y mutations of HFE gene among Libyans residing in Tripoli region and to compare the results with those of other populations worldwide.
| Materials and Methods | | |
Study population and samples
Three hundred unrelated blood donors were recruited randomly to participate in this study at Al-Jala Maternity Hospital in Tripoli. All of the participating individuals were males of Libyan origin residing in Tripoli region, aged between 18 and 50 years (mean ± standard deviation = 31.38 ± 6.78 years). Four milliliter of venous blood samples was collected in EDTA tubes from each of the participants and transported to the laboratory of the National Center for Disease Control in Tripoli for DNA isolation and subsequent molecular genetic studies. Written informed consent was obtained from all of the participants to use their blood for the research project. The study was approved by the Scientific Ethics Committee of the Libyan Academy.
Genomic DNA extraction
Genomic DNA from all blood samples was extracted from 200 μl of EDTA anti-coagulated whole blood samples using QIAamp DNA Blood Mini Kit (QIAGEN, Valencia, USA) according to the manufacturer's instructions. Genomic DNA was eluted in 200 μl (AE) elution buffer and stored at −20°C until use.
Primers and probes design
The occurrence of HFE H63D and C282Y mutations in study subjects was explored by in-house hydrolysis probe real-time polymerase chain reaction for H63D and high-resolution melting (HRM) analysis protocols for C282Y genotyping and direct DNA sequencing was used to confirm the results.
The GenBank: NG_008720.2 sequence was used to design the primers and probes for the H63D and C282Y mutations detection. The primers and probes were designed using Oligo Analyzer 1.5 software (Gene Link™) (Kuopio, Finland) and bought from Metabion (Metabion, Martinsried, Germany). The sequences of the primers and probes used for H63D mutation detection, using hydrolysis probe real-time polymerase chain reaction (PCR), were as follows: H63DF 5'-TTGGGCTACGTGGATGACC-3' (Tm = 59.5°C) as a forward primer[25] and H63DR 5'-TCTGGCTTGAAATTCTACTGGAAA-3' (Tm = 61.1°C) as a reverse primer.[25] The allele-specific probes were designed during this study, these include H63DW5'-FAM-CGTGTTCTATGATCATGAGAGTC-BHQ-1-3' (Tm = 61.1°C) as a wild type probe and H63DM 5'-HEX-CGTGTTCTATGATGATGAGAGTC-BHQ-1-3' (Tm = 61.16°C) as a mutant probe. The H63D primers would produce a 100 bp amplicon. The sequences of the primers used for C282Y mutation screening, using HRM, were as follows: C282YF 5'-GAACCTAAAGACGTATTGCCCAA-3' (Tm = 61.1°C) as a forward primer (designed during the current study) and C282YR 5'-AGATCACAATGAGGGGCTGATC-3' (Tm = 62.1°C) as a reverse primer.[25] The C282Y primers would produce a 136 bp amplicon. The primers used for direct sequencing were previously published.[26] The sequences of the primers used for H63D mutation detection using direct DNA sequencing were as follows: H63DF 5'-ACATGGTTAAGGCCTGTTGC-3' (Tm = 58.4°C) as a forward primer and H63DR 5'-ATGTGATCCCACCCTTTCAG-3' (Tm = 58.4°C) as a reverse primer. The H63D primers would produce a 245 bp amplicon. The sequences of the primers used for C282Y mutation to confirm the results, using direct DNA sequencing, were as follows: C282YF 5'-TACCCCCAGAACATCACCAT-3' (Tm = 58.4°C) as a forward primer and C282YR 5'-CCTGGCTCTCATCAGTCACA-3' (Tm = 60.5°C) as a reverse primer. The C282Y primers would produce a 225 bp amplicon. The accuracy of the designed primers and probes sequences was verified by comparison with the GenBank database using Basic Local Alignment Search Tool (http://www.ncbi.nlm.nih.gov/BLAST/).
Genotyping of H63D using allelic discrimination real-time polymerase chain reaction
The allelic discrimination PCR was carried out using Rotor-Gene Q real-time rotary analyzer (QIAGEN GmbH). Reaction mix of 25 μl contained 12.5 μl of 2x QIAGEN Multiplex PCR Master Mix that is made up of HotStarTaq® DNA Polymerase, Multiplex PCR Buffer*, and dNTP Mix from QIAGEN GmbH, 0.75 μl of 10 pmol/μl of each primer and 1 μl of 10 pmol/μl of each probe, and 2 μl (5–50 ng) genomic DNA. The thermal profile for H63D mutation detection contained one cycle of Taq polymerase activation at 95°C for 15 min followed by forty cycles of DNA denaturation at 94°C for 30 s, primers and probes annealing at 57°C for 90 s, and extension at 72°C for 60 s. FAM and HEX fluorescent signals were collected at the end of extension step.
Genotyping of C282Y mutation using high resolution melting analysis
The HRM analysis was carried out using the Rotor-Gene Q real-time rotary analyzer (QIAGEN GmbH), using a ready-to-use Type-it HRM PCR Kit (QIAGEN). PCR amplification was carried out using a reaction mix of 25 μl contained 12.5 μl 2X Type-it HRM PCR Master mix (HotStarTaq Plus DNA Polymerase, Type-it HRM PCR Buffer [with EvaGreen dye], Q-Solution, dNTPs mix, and RNase-Free Water), 2 μl (5–50 ng) genomic DNA, 9 μl de-ionized water, and 0.75 μl of 10 pmol/μl to give a final concentration of 0.3 pmol of each primer.
The thermal profile consisted of one cycle of an initial denaturation step (Taq polymerase activation) at 95°C for 4 min followed by 40 cycles of denaturation step at 95°C for 30 s, annealing step at 58°C for 30 s, and extension step at 72°C for 20 s. Signal acquisition was performed at the extension step. In the HRM phase, melting curves were generated by continuous fluorescence signal acquisition using the Green channel from 80°C to 90°C, with each step raised by 0.05°C per second. Melting curves were produced by the decrease in fluorescence with the rise in the temperature; and accordingly, nucleotide variation results in different curve patterns.
Genotyping of H63D and C282Y mutations using direct DNA sequencing
Allelic discrimination and HRM results were confirmed by Sanger sequencing of the region of interest. PCR was performed using TC-412 Thermocycler (Techne, Duxford, Cambridge) by using a mi-Red Load Hot Taq Mix kit from metabion (Germany). Reaction mix of 25 μl was prepared that contained 5 μl of 5X Red Load Taq Mix (5X Red Load Hot Taq Mix is an optimized ready to use solution containing Hot Taq Polymerase: 0.05 u/μl, dNTPs [dATP, dCTP, dGTP, dTTP] [200 μM], reaction buffer with KCl and MgCl2 [2 mM], red dye, gel loading buffer and stabilizers) from metabion (Martinsried-Germany), 0.75 μl of 10pmol/μl of each primer, 16.5 μl de-ionized water, and 2 μl (5–50 ng) genomic DNA. The thermal profile for both (H63D and C282Y) polymorphism contained one cycle of initial denaturation at 94°C for 4 min followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 57°C and 58°C (H63D and C282Y), respectively, for 30 s, and extension at 72°C for 30 s, final extension 72°C for 2 min.
The PCR product was purified by the QIAquick PCR purification kit (Qiagen, Chatsworth, CA) according to the manufacturer's instructions. Then, the sequencing reaction was performed on a TC-412 Thermocycler (Techne, Duxford, Cambridge) by using Big Dye Terminator v3.1 Cycle Sequencing Kit (PE Applied Biosystems, Foster City, CA).
Forward and reverse DNA sequencing reactions were assembled separately by mixing the following: 4 μl BDT v3.1 reaction mix, 4 μl 5X sequencing buffer, 1 μl 3.2pmol/μl primer, 1 μl purified PCR product, and 10 μl de-ionized water. The mixture was mixed by flicking and the cycle sequencing was performed on the 20 μl sequencing reaction in a TC-412 thermocycler (Techne, Duxford, Cambridge) under an initial denaturation at 96°C for 1 min followed by 25 cycles of denaturation at 96°C for 10 s, annealing at 57°C and 58°C for (H63D and C282Y), respectively, for 5 s, and extension at 60°C for 4 min. The cycle sequencing product was purified by ethanol/EDTA precipitation, the precipitate was resuspended in 20 μl Hi Di formamide, denatured at 950C for 2 min, pipetted into a 96-well plate, and analyzed with an ABI PRISM® 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) of the Department of Biochemistry of the University of Tripoli. The resultant sequences were edited and the database analysis was done using BLAST (http://www. ncbi. nlm. nih. gov/BLAST).
| Results | | |
Genotyping of H63D mutation using allelic discrimination polymerase chain reaction
H63D genotyping was performed using standard allelic discrimination analysis with two probes labeled with FAM and HEX fluorescent for WT and mutant genotypes, respectively. Four PCR amplification patterns are expected. The WT samples (C187C genotype) would be identified by the detection of FAM fluorescence at the green channel only, which is attributed to degradation of the WT probe, the mutant samples (G187G genotype) would be identified by the detection of the HEX fluorescence at yellow channel only, which is attributed to degradation of the mutant probe, and the heterozygous samples (C187G genotype) would be identified by the detection of both dyes FAM and HEX, and in the nontemplate control (NTC) to which no DNA was added, there would be no fluorescence detected. [Figure 1] shows that there is a clear segregation of different genotypes in the scatter plot produced. It shows the result of allelic discrimination analysis of 15 samples as WT or C187C genotype (top left), three samples as heterozygous or C187G genotype (top right), two samples as homozygous mutant or G187G genotype (bottom right), and NTC (bottom left). | Figure 1: Scatter plot: Testing of the HFE gene H63D mutation with hydrolysis probe allele discrimination assays with automatic allele calling; FAM fluorescence indicates the degradation of the wild-type H63D probe (Y axes), is plotted against the HEX fluorescence, which indicates the degradation of the mutant H63D probe (X axes) using the baseline-corrected fluorescence (dR)
Click here to view |
Genotyping of C282Y mutation using high resolution melting analysis
[Figure 2] shows the normalized melting curve of HRM of C282Y (G845A) mutation. This figure shows that the HRM protocol was clearly able to reveal differences in the melting curve shape that correlated to the different genotypes and hence differentiating between the different genotypes, namely wild-type (G845G) and heterozygous mutant (G845A) by producing two different melting profiles. No homozygous mutant cases (A845A) were detected. | Figure 2: High-resolution melting analysis. Normalized melt curves for C282Y mutation. The melting curves show two different patterns, for wild type (845GG), and heterozygous (845GA). Differentiating different genotypes of C282Y mutation. It shows that the heterozygous is first to melt, followed by the wild type
Click here to view |
Genotyping of H63D and C282Y mutations using direct DNA sequencing
The results obtained using allelic discrimination and HRM analysis were confirmed by direct DNA using Big Dye Terminator v3.1 Cycle. [Figure 3] shows examples of the different H63D genotypes obtained. It compares the wild-type (C187C), heterozygous (C187G), and homozygous mutant (G187G) genotypes and [Figure 4] shows examples of the different C282Y genotypes obtained. It compares the homozygous wild-type (G845G) and heterozygous mutant (G845A) genotypes. | Figure 3: Sequencing results using Big Dye Terminator v3.1 Cycle Sequencing. Comparing the wild-type C187C (a), heterozygous C187G (b) and homozygous G187G (c) genotypes of H63D mutation. The position of the mutated nucleotide is marked by an arrow
Click here to view |
 | Figure 4: Sequencing result using Big Dye Terminator v3.1 Cycle Sequencing. Comparing the wild-type G845G (a) and heterozygous G845A (b) genotypes of C282Y mutation. The position of the mutated nucleotide is marked by an arrow
Click here to view |
Frequencies of H63D and C282Y mutations in the study population
Genotype frequencies were estimated in 300 subjects of the male Libyan population. A total of 600 chromosomes were analyzed for the presence of H63D and C282Y mutations in HFE gene.
Regarding H63D, 7 subjects (2.33%) were detected as mutant homozygous and 72 subjects (24%) were detected as a heterozygous mutant, while for C282Y, only 1 (0.33%) subject was detected as a heterozygous mutant [Table 1]. No subjects were detected as compound heterozygous. | Table 1: Genotype frequency of H63D and C282Y mutations in hemochromatosis gene in the study population
Click here to view |
The allele frequency of H63D (C187G) for C allele was 514 (85.67%) and for G was 86 (14.33%), and the allele frequency of C282Y (G845A) for G allele was 599 (99.83%) and for A was 1 (0.17%).
| Discussion | | |
HFE H63D (C187G) and C282Y (G845A) mutation (s) play an important role in the development of HH. To the best of our knowledge, no previous studies investigated the prevalence of HFE H63D (C187G) and C282Y (G845A) mutation (s) in the Libyan population residing in Tripoli region.
We used protocols based on allelic discrimination and HRM analysis to screen for the study mutations and the results were confirmed using direct DNA sequencing. HRM analysis is a simple and efficient molecular diagnostic tool, inexpensive in comparison with the probe based methods, closed tube format that is homogenous, accurate, and rapid[27],[28], and being a closed tube, reduces the possibility of PCR carry over and hence contamination and it does not need post PCR gel analysis and mutagenic reagents such as ethidium bromide for visualization of fragments. Data analysis is performed automatically using special software.
We tried to use HRM for the screening of both mutations, but it did not clearly differentiate between H63D genotypes because the change is C187G and Tm shift of C to G is very small. Hence, we designed primers and probes to detect the mutation using allelic discrimination technique. The developed protocols proved to be reliable for genotyping.
Our results showed that the prevalence of C282Y (G845A) mutation in our subjects was very uncommon. It is slightly greater than that reported in a previous study from the Libyan population living in Benghazi region[29] and less than that reported in Tunisia,[30] while it is close to that seen in Syria.[31] However, its frequency is very low compared with European populations such as North Ireland,[32] Norway,[19] Sweden,[33] North Italy,[34] and North Spain[35] and American United States population,[36] while it is indicated to be high compared with South African, Asian, Australian,[19] Saudi Arabian,[19] Jordanian,[37], and Algerian Mzab[24] populations.
Our results as well as other reports from Africa and the Middle East concluded that C282Y (G845A) is rare, suggesting the Celtic origin of this mutation[19],[21],[22],[23],[24],[29],[31],[33],[37],[38]. It has been suggested that the HFE C282Y mutation occurred in mainland Europe before about 4000 BC.[39]
Our results show a relatively high prevalence of H63D mutation among the study subjects, which is less than the highest European frequency found in North Spain[35] and slightly lower than that of Libyans living in Benghazi region,[29] South Spain,[40] Tunisia,[38] and North Ireland.[32] It is close to that seen in South Italy,[41] Greece,[19] United States,[36] and North Italy.[34] Furthermore, it is noted to be slightly high compared with those reported from Sweden,[33] Syria,[31] Jordan,[37] and Norway.[19] However, it is evidently high compared with the Algerian Mzab population[24] and Saudi Arabia population,[19] while it is noted to be very high compared with people living in Kenya, Nigeria, China, Vanuatuans, and Mexico.[19]
Evidently, the allele frequency of H63D mutation is found in the frequency of about 8.5%–17% in North Africa and the Middle East populations.[19],[24],[29],[31],[38] These observations agree with the fact that H63D mutation is not restricted to European populations.[19],[24],[29],[31],[38]
In the past, in addition to aboriginal Amazigh (Berber), Libya was inhabited by Phoenicians, Greeks, Romans, Arabs, Ottomans, Jews, and Africans.[42] These different ethnicities are distributed across a country of approximately 1.7 million square kilometers with different ratios, reflecting the distribution of genetic diversity. We suggest that the presence of C282Y mutation in our population is due to historical admixture with the European population. However, our result may suggest that the Europeans left a very limited effect on the Libyan population genetic diversity.
Roth et al. in 1997 assumed that the H63D mutation is found around the world and the restriction of the C282Y mutation to European populations may suggest that the H63D preceded C282Y.[24]
| Conclusion | | |
For the first time, the current study determined the prevalence of H63D (C187G) and C282Y (G845A) mutations in Libyans residing in Tripoli region. Among Libyan population residing in Tripoli region, the overall allele frequency of H63D was common (14.33%), and the allele frequency of C282Y was very rare (0.17%).
Acknowledgments
This work was supported in part by the Libyan Authority for Research, Science and Technology, the Department of Biochemistry University of Tripoli, and the National Center for Disease Control in Tripoli-Libya. The authors would like to thank all the participants.
Financial support and sponsorship
This work was supported by the Libyan Authority for Research, Science and Technology, the Department of Biochemistry University of Tripoli, and the National Center for Disease Control in Tripoli-Libya.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1]
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