This paper describes the genetic etiology of sporadic amyotrophic
lateral sclerosis in a single population. Polymerase chain reaction-restriction
fragment length polymorphism and DNA sample sequencing of 3 common HFE
gene variants (C282Y and H63D and S65C) were performed on 10 randomly selected
samples of H63D gene variant (124 patients with sporadic amyotrophic lateral
sclerosis) and 10 wild types of H63D samples (210 controls). The C282Y
and S65C gene variant were absent.
There
were 24 cases (7.18%) with H63D heterozygous variants, including 16 cases (13%)
in the sporadic amyotrophic lateral sclerosis group and 8 cases (4%) in the
healthy control group. The polymorphism frequency of the H63D gene variant in the
sporadic amyotrophic lateral sclerosis group was significantly different than
that in the control group (p
Amyotrophic lateral sclerosis (ALS) is an idiopathic and fatal neurodegenerative disease of the human motor system. Like other neurodegenerative diseases, about 10% of ALS is classified as familial diseases, while the remaining 90% are considered sporadic because they appear to occur randomly throughout the community (Rowland and Shneider, 2001). Further study on the genetic etiology of ALS provides basic insights into the cellular mechanism of neuronal degeneration, and also contributes to the design and testing of disease modeling and targeted therapy. Therefore, it is not surprising that a large number of resources have been used to search for pathogenic variants. Sporadic ALS is suspected to be associated with genetic susceptibility to environmental risk factors (Talbott et al., 2016).
SOD1 is the first gene found to cause ALS (Rosen, 1993). With the rapid development of technology, gene discovery has been dramatically accelerated. Some new ALS genes have also been found, including TARDBP (Sreedharan et al., 2008) , profilin 1 (PFN1) gene (Wu et al., 2012), C9ORF72 (Renton et al., 2011) , valosin-containing protein (VCP) gene (Johnson et al., 2010), UBQLN2 (Deng et al., 2011), and other genes.
The existence of the SOD1 variant leads to the theory of the role
of oxidative stress in the pathogenesis of the disease. One of the leading causes
of oxidative stress is the disorder of cellular iron homeostasis. Improper cell
iron management and oxidative stress are associated with many neurodegenerative
diseases. One of the mechanisms by which cells cannot regulate iron status
properly is through variants in the HFE gene (Pietrangelo, 2004). It
was found that H63D variant cells had the phenotype of promoting glutamate
toxicity (Mitchell et al., 2011), which was one of the causes of ALS. The p.
His 63 Asp polymorphism of the
HFE gene (c.187C
In this paper, we determine the relationship between the C282Y (p.Cys 282 Tyr, A at rs1800562 instead of G), H63D (p. His 63 Asp, G at rs1799945 instead of C), and S65C (p.Ser65Cys, T at i3002468 instead of A) variants in HFE gene (chromosome 6p21.3)and the risk of sporadic ALS (SALS) in a single population.
polymorphism | primer | restriction enzyme | PCR products | base change | digestion product | ||
mutations | |||||||
wild | homozygous | heterozygous | |||||
C282Y | 5’-TGGCAAGGGTAAACAGATCC-3’ (forword) | Rsa I | 387 bp | G |
247 bp | 247 bp | 247 bp |
140 bp | 111 bp | 140 bp | |||||
5’-CTCAGGCACTCCTCTCAACC-3’ (reverse) | 29 bp | 111 bp | |||||
29 bp | |||||||
S65C | 5’-ACATGGTTAAGGCCTGTTGC-3’ (forword) | Hinf I | 208 bp | A |
147 bp | 208 bp | 208 bp |
61 bp | 147 bp | ||||||
5’-GCCACATCTGGCTTGAAATT-3’(reverse) | 61 bp | ||||||
H63D | 5’-ACATGGTTAAGGCCTGTTGC-3’ (forword) | Mbo I | 208 bp | C |
138 bp | 208 bp | 208 bp |
5’-GCCACATCTGGCTTGAAATT-3’ (reverse) | 70 bp | 138 bp | |||||
70 bp |
Variable | Patients n = 124 | Controls n = 210 | p -Value |
Male, n | 69 | 133 | 0.165 |
Female, n | 55 | 77 | |
Mean age (SD), y | 50.00 (10.09) | 45.02 (10.64) | 0.66 |
Spinal onset, n | 103 | ||
Bulibar onset, n | 21 | ||
Symptom duration 36m | 98 | ||
Symptom duration > 36m | 26 | ||
Age at disease onset ≤ 45y | 48 | ||
Age at disease onset > 45y | 76 | ||
Note a: p -value for Chi-square test; b: p -value for t -test. |
One hundred twenty-four patients (69 males and 55 females) with SALS were recruited from the Second Hospital of Hebei Medical University, the Peking University Third Hospital, the West China Hospital, Sichuan University, and the Third Hospital of Hebei Medical University. Patients met the El Escorial criteria for definite, probable, or possible ALS (de et al., 2008). Two hundred ten healthy people matched by age, gender, and nationality were included as the control group. All patients and controls were of Han ethnicity. The ethics committee approved the research plan of the second hospital of Hebei Medical University. All individuals gave written informed consent.
Venous blood (5 ml) was collected from patients and healthy subjects,
anticoagulated with sodium citrate, and stored in a refrigerator at 4
SPSS 21 version software was used for statistical processing.
Gene | Genotype/Allele | Patients (n) n = 124 | Controls (n) n = 210 |
HFE H63D | Wild type (CC) | 108 | 202 |
Heterozygous (CG) | 16 | 8 | |
Homozygous (GG) | 0 | 0 | |
p Value | 0.597 | 1.00 |
Gene | Gene variant/Allele | Patients, n = 124 No (%) | Controls,n = 210 No (%) | p Value* |
HFE H63D | Wild type (CC) | 108 (87.10) | 202 (96.19) | 0.002 |
Heterozygous (CG) | 16 (12.90) | 8 (3.81) | ||
Homozygous (GG) | 0 (0.00) | 0 (0.00) | ||
C | 232 (93.55) | 412 (98.10) | 0.003 | |
G | 16 (7.45) | 8 (1.90) | ||
Note *: p -Value for Chi-square test; a: p -Value for the CC genotype relative to CG genotype of H63D. |
The baseline characteristic was shown in Table 2. There was no
statistical difference in age and gender between the control group and the
patient group (p
The polymorphism frequency of the H63D variant in the SALS group
was significantly different from that in the control group (p
We found that the H63D variant was associated with SALS, which is consistent with previous studies (Wang et al., 2004). Our results are consistent with findings in different populations (Eum et al., 2015; He et al., 2011; Sutedja et al., 2007). Compared with the finding of He et al. (2011), we used different amplification primers and different fragment lengths. We used the course of the disease as the stratification factor for statistical analysis. The patient group in this study had different data sources, and the conclusion was complementary to He et al. (2011), which further expands the population data. The results of Li et al. (2014) and Van Rheenen et al. (2013) showed the opposite conclusions and confirmed that the H63D variant was not associated with ALS risk. The difference in the genetic background in ethnicity of the population could explain the difference of experimental results.
SALS was reported to be associated with the H63D variant in Dutch
(Eum et al., 2015), and the H63D variant was associated with a higher age of
onset. The mean age of onset in Chinese ALS patients was 49.8 to 54.3 years old
in hospital-based studies (Chen et al., 2015; Liu et al., 2014, 2018, 2019; Rosenbohm et al., 2018; Wei et al., 2015), indicating
that the onset age of ALS patients in China is earlier than that in other
countries. The average age of the experimental subjects was 50.00
The H63D variant in the HFE gene is associated with iron homeostasis disorder and oxidative stress and plays a vital role in the pathogenesis of ALS. Although the relationship between the HFE gene and ALS is still controversial, there is strong evidence in animal experiments. In animal models, the presence of H67D variant of the HFE gene (homologous to H63D in humans) disrupts iron homeostasis in the brain. It is associated with increased oxidative stress in the brain (Nandar et al., 2013) and severe disruption of cholesterol metabolism (Ali-Rahmani et al., 2014). Changes in iron homeostasis and increased oxidative stress were also observed at the cellular level (Lee et al., 2007), and increase glutamate release (Mitchell et al., 2011) and endoplasmic reticulum pressure (Liu et al., 2011). Each of the above mechanisms is considered to be the promoter of ALS pathogenesis (Rothstein, 2009). Therefore, the data suggests that H63D-HFE is a genetic modifier of ALS risk.
Nandar et al. (2014) established a double transgenic mouse line carrying the H67D variant of the HFE gene (homologous with human H63D) and SOD1 (G93A) variant. They found that the H67D variant of the HFE gene can double the survival time and course of disease of transgenic mice. The underlying mechanism is that the H63D variant of the HFE gene changes the pathophysiology of ALS through oxidative stress, gliosis, and cell dysfunction (Nandar et al., 2014). The C282Y variant, rather than the H63D variant, has a more significant impact on serum iron concentration and liver deposition (Adams et al., 2005). In the present work, C282Y and S65C variants were not present in both patients and controls. The frequency of the CG genotype in the SALS group was significantly higher than that in the control group, and the difference was still significant at the allele level. However, the sample size of this study is too small for subsample analysis. More samples are needed for future research.
The results suggest that the H63D variant plays a role in the development of SALS. Still, we need to be cautious in extrapolating different geographical regions, as further independent HFE variant association studies are needed.
The ethics committee approved the research plan of the second hospital of Hebei Medical University. All individuals gave written informed consent.
The authors thank the patients and clinicians at The Second Hospital of Hebei Medical University, Peking University Third Hospital, West China Hospital, Sichuan University, and The Third Hospital of Hebei Medical University, who helped to collect and organize data. This study was supported by the Hebei Province Medical Science Research Key Project (08079).
The authors promise that there is no conflict of interest in this study.