SNPs known to modulate HbF levels and response to hydroxyurea therapy.
Abstract
Sickle cell disease (SCD) consists of inherited monogenic hemoglobin disorders affecting over three million people worldwide. Efforts to establish precision medicine based on the discovery of genetic polymorphisms associated with disease severity are ongoing to inform strategies for novel drug design. Numerous gene mutations have been associated with the clinical complications of SCD such as frequency of pain episodes, acute chest syndrome, and stroke among others. However, these discoveries have not produced additional treatment options. To date, Hydroxyurea remains the only Food and Drug Administration-approved agent for treating adults with SCD; recently it was demonstrated to be safe and effective in children. The main action of Hydroxyurea is the induction of fetal hemoglobin, a potent modifier of SCD clinical severity. Three inherited gene loci including XmnI-HBG2, HBS1L-MYB and BCL11A have been linked to HBG expression, however the greatest progress has been made to develop BCL11A as a therapeutic target. With the expanded availability of next generation sequencing, there exist opportunities to discover additional genetic modifiers of SCD. The progress made over the last two decades to define markers of disease severity and the implications for achieving precision medicine to treat the complications of SCD will be discussed.
Keywords
- fetal hemoglobin
- single nucleotide polymorphism
- drug discovery
- genome-wide association studies
1. Introduction
Sickle cell anemia is caused by an A to T point mutation in the sixth codon of the β-globin (
About one in 500 African-American and one in 36,000 Hispanic-American children are born with SCD disease [2], which is diagnosed at birth by newborn screening in the United States. The carrier state or sickle cell trait is detected in 1:13 African Americans and 1:100 Hispanic Americans [3] with an estimated 2.5 million Americans with sickle cell trait [4]. Worldwide about 3.2 million people have SCD and 43 million have sickle cell trait [5] with 80% occurring in sub-Saharan Africa mainly as a protective mechanism against malaria. Moreover, the
Hemoglobin is a tetrameric protein, composed of two α-like and two β-like globin polypeptide chains, which transports oxygen to the body tissues. During human development, two switches in the type of hemoglobin synthesized occur, a process known as hemoglobin switching [1]. The first switch at 6–8 weeks of development involves ε-globin gene silencing and activation of the
2. Clinical manifestations of sickle cell disease
Over the last 30 years, survival in people living with SCD has improved significantly due to decreased death rates during infancy. However, morbidity remains high due to central nervous system and pulmonary complications during childhood and end-organ damage in adults [9, 10]. The average life expectancy of people with SCD is 50 years in the United States [11]. Individuals with SCD experience a chronic hemolytic anemia caused by HbS polymerization under deoxygenated conditions, which [12] produces RBC membrane damage and a shortened life span of 14–21 days. As a result, HbSS patients have an average hemoglobin level of 6–8 g/dL with an elevated reticulocyte count and plasma lactate dehydrogenase level [13]. Furthermore, the damaged membrane leads to inflexible and dehydrated sickled RBCs and abnormal adhesion to the vascular endothelium producing the vasculopathy observed in persons with SCD [13].
The most common pathophysiology of SCD is vaso-occlusive (VOC) events produced by tissue ischemia leading to pain and acute or chronic injury to the spleen, brain, lungs, kidneys, and bones [13]. Individuals with a severe SCD sub-phenotype have more frequent VOC events, a higher white blood cell count, a lower HbF level, and increased blood vessel flow resistance under deoxygenation conditions [14–16]. The most common clinical manifestation of SCD is acute painful episodes which occur mainly in the extremities, but can involve the abdomen, back, and chest [17, 18].
As HbF falls below protective levels at around 6–12 months of age, dactylitis involving pain and swelling of the hands and feet is an early manifestation of SCD and is a risk factor for diseased severity [19]. Splenic sequestration occurs in 30% of children between the ages of 6 months to 3 years, which can cause severe life-threatening anemia and death if not treated promptly. Over time, repeated episodes of VOC in the spleen lead to infarction and a markedly increased risk for infection due to encapsulated bacteria such as
Other types of VOC events include acute chest syndrome [22, 23], silent and acute cerebral infarcts [24, 25], and osteonecrosis of the femoral head. Episodes of acute chest syndrome can be caused by pulmonary VOC, infection, and/or fat emboli from bone marrow infarcts [22]. Long-term damage in the lungs can precede pulmonary hypertension [26] in older children and adults with SCD causing high morbidity and mortality. By adolescents, 50% of individuals with SCD suffer silent cerebral infarcts [27] and 10% of children over the age of 2 experience overt strokes requiring chronic transfusions [28, 29]. The process of VOC can affect any organ system producing a wide variety of complications in SCD involving the heart, liver, gall bladder, kidney, and skin [30].
3. Treatment of vaso-occlusive complications
Blood transfusions are the mainstay of therapy for individuals suffering from acute and chronic complications of SCD. Red blood cell transfusions improve the oxygen-carrying capacity and prevent sickling by decreasing the HbS level to <30% of total hemoglobin [31–33]. Transfusions are also used for the acute exacerbation of anemia associated with splenic sequestration and aplastic crisis caused by Parvo B19 virus infection [34]. The most common symptom in persons with SCD is acute and chronic pain due to tissue ischemia, which is correlated with long-term survival [35]. Therefore, early aggressive treatment of pain episodes to prevent complications is the standard of care [36]. Recent research has provided insights into mechanisms of pain related to tissue injury (nociceptive), nerve injury (neuropathic), or unknown causes (idiopathic). Effective pain treatment is most often achieved using opioid narcotics combined with nonsteroidal anti-inflammatory drug.
To address the long-term effects of repeated pain episodes, extensive research has been conducted to develop drugs that induce HbF, which inhibits HbS polymerization [37] to improve the clinical symptoms of SCD. Based on findings in the Multicenter Study of Hydroxyurea [38], this agent is the only Food and Drug Administration-approved drug for the treatment of adults with SCD [39]. Subsequent studies in children including BABY HUG demonstrated that hydroxyurea (HU) is an effective HbF inducer and can be used safely in the first year of life [40]. Unfortunately, HU has a 30% nonresponse rate in adults, causes bone marrow suppression, and has detrimental effects on fertility [38, 41]. Therefore, the development of novel therapeutic agents based on inherited mutations that alter the expression of the
4. Genetic modifiers of sickle cell disease severity
While homozygosity for the βS-globin gene mutation (
4.1. Vaso-occlusive pain
SCD patients experience a wide variety of clinical pain ranging from acute mild/severe to persistent chronic pain. The underlying mechanisms of differences in pain rates are complex and likely involve a number of genetic polymorphisms in several biological systems. Studies have been conducted that provide insights into SNPs associated with the frequency and severity of pain in SCD. Jhun et al. [42] identified mutations in the dopamine D3 receptor (Ser9Gly heterozygotes) associated with a lower acute pain rate. The most commonly used opioid medications including codeine and hydrocodone require cytochrome P450 2D6 (CYP2D6) for drug activation, which can impact the efficacy of these agents. The CYP2D6 gene is highly polymorphic, with variant alleles that result in decreased, absent, or ultra-rapid metabolism [43]. Altered CYP2D6 enzymatic activity in CYP2D6*17 (reduced activity), CYP2D6*5 (gene deletion), and CYP2D6*4 (absent function) is correlated with the analgesic response to codeine and hydrocodone. Therefore, genotyping the CYP2D6 gene is a reasonable approach for developing personalized medicine for the treatment of pain in persons with SCD. Moreover, missense or frame-shift mutations in CYP2C9 decrease or abolish enzymatic activity, respectively, which impairs opioid activation [44, 45]. Likewise, an SNP in the promoter of the gene encoding the enzyme uridine 5′-diphospho (UDP)-glucuronosyltransferase 2B7 (−840G/A) responsible for morphine glucuronidation in the liver is associated with lower morphine metabolites in sickle cell patients suggesting that higher doses of morphine may be required to achieve adequate pain control [46].
4.2. Acute chest syndrome/pulmonary hypertension
Acute chest syndrome continues to contribute to significant morbidity and mortality in children and adults with SCD [47]; therefore, the discovery of genetic modifiers of this complication has the potential for high impact and the design of precision medicine. Redha et al. [48] investigated the association of the vascular endothelial growth factor A (VEGFA) 583C/T mutation with acute chest rates in children with SCD. The presence of the 583T/T genotype was associated with increased serum VEGF levels while the VEGFA 583C/T caused reduced VEGF serum levels.
The rate of RBC hemolysis and release of free heme in the circulation are associated with clinical severity of SCD. Heme oxygenase-1 (
Over the last decade, numerous studies have been conducted to define risk factors associated with pulmonary artery hypertension [50, 51], which defines a severe sub-phenotype of SCD leading to premature death. SNPs in genes involved in the regulation of endothelial function, which alter the synthesis of the endothelium-derived vasodilators nitric oxide and prostacyclin, have been implicated [52]. An extended screen of 297 SNPs in 49 candidate genes [53] identified mutations in the transforming growth factor (TGF) superfamily including the activin A type II-like 1 receptor (ACVRL1), bone morphogenetic protein (BMP) receptor 2, bone morphogenetic protein 6, and the β-1 adrenergic receptor (ADRB1) associated with pulmonary artery hypertension. A multiple regression model using age and hemoglobin as covariates demonstrated that SNPs in ACVRL1, BMP6, and ADRB1 independently contribute to pulmonary hypertension risk. These findings offer promise for identifying patients at risk for this complication and developing novel therapeutic targets for SCD.
A recent study by Al-Habboubi et al. [54] examined the association between VEGF secretion and VOC rates among 210 individuals with SCD. Mutations in VEGFA including rs2010963 heterozygous and rs833068 and rs3025020 homozygous states were associated with increased pain rates. Moreover, Yousry et al. [55] observed that the homozygous mutant eNOS 786T/T was significantly associated with a high risk of acute chest syndrome. By contrast, the wild-type eNOS 4a/4b genotype was protective against VOC and pulmonary hypertension while the homozygous haplotype (C, 4a) was significantly associated with the risk of VOC pain, acute chest syndrome, and pulmonary hypertension. Thus, eNOS SNPs may be useful as a genetic marker of prognostic value in SCD to predict a severe disease sub-phenotype.
4.3. Cerebral vascular disease
SCD is the most common cause of ischemic stroke occurring in 10% of children under 15 years of age; by contrast, hemorrhagic strokes are observed more commonly in adults over 30 years of age [56]. Genetic polymorphisms in multiple genes have been implicated in childhood stroke risk. For example, a mutation in vascular adhesion molecule-1 (
Ischemic stroke is common in children with SCD producing high morbidity and mortality. A meta-analysis by Sarecka-Hujar et al. [62] demonstrated the association of SNP 677C/T in the methylenetetrahydrofolate reductase gene with the risk of stroke. Abnormalities in the coagulation pathway have been implicated in the pathogenesis of cerebral bleeding. For example, protein Z, a vitamin K-dependent glycoprotein structurally related to the vitamin K-dependent coagulation factors, is devoid of catalytic activity and inhibits the generation of thrombin. Mahdi et al. [63] identified three SNPs in the protein Z gene promoter (rs3024718, rs3024719, and rs3024731) and one intronic SNP rs3024735 associated with stroke risk suggesting that reduced protein Z levels produced a procoagulant state and increased risk for thrombotic diseases including ischemic stroke. These studies provide evidence for genetic markers that can be used to assess stroke risk in SCD and targeted for therapeutic intervention.
4.4. Osteonecrosis
Repeated episodes of bone infarction caused by vaso-occlusive events precede osteonecrosis of the head of the femur and humerus, a disabling complication of SCD [64, 65]. The discovery of SNPs in genes involved in bone morphogenesis, metabolism, and vascular disease will identify individuals at high risk for osteonecrosis. Previously, 233 SNPs in seven genes including
4.5. Priapism
Thirty percent of males with SCD experience the potentially devastating complication of priapism associated with a clinically severe disease sub-phenotype. Proteins involved in neuro-regulatory and adrenergic pathways, nitric oxide biology, and ion channels have been implicated in the pathophysiology of priapism [68–71]. More recently, clinical studies have identified genetic markers of priapism that produce erectile dysfunction and determine the ability to respond to phosphodiesterase inhibitors. Nolan et al. [72] identified SNPs in the
4.6. Nephropathy
Sickle nephropathy is a serious complication of SCD that can lead to renal failure and is rapidly becoming a major cause of death in adults. In view of the high medical burden and poor health outcome of end-stage renal disease, genetic markers of nephropathy risk are desirable. Youssry et al. [73] identified soluble FMS-like tyrosine kinase-1, a member of the vascular endothelial growth factor receptor family, as a biomarker for sickle nephropathy. In addition, Ashley-Koch et al. [53] demonstrated that the myosin, heavy chain 9, non-muscle (
4.7. Leg ulcers
Cutaneous leg ulcers occur more often in adult sickle cell patients with low baseline hemoglobin levels and increased hemolysis rates indicated by high lactate dehydrogenase, bilirubin, and reticulocyte levels. The V34L G/T SNP (rs5985) in the factor XIII gene (F13A1) has been associated with leg ulcers [74]. Other studies have implicated factor V Leiden [75], the fibroblast growth factor receptor [76], and the HLA-B3525 antigen [77] in the pathogenesis of leg ulcers. A larger study involving 243 sickle cell patients [78] examined SNPS in 60 candidate genes that have a putative role in the pathophysiology of SCD. The association of SNPs in
5. Genetic modifiers of fetal hemoglobin
5.1. HBB locus haplotypes
Inherited genetic mutations that modulate
5.2. Genome-wide association studies (GWAS)
The normal switch from HbF to HbA synthesis occurs during the first year of life reaching adult levels of HbF <1% by 12 months of age. A group of disorders known as hereditary persistence of HbF expression is caused by inherited deletions in the
5.3. Xmn1-HBG2
In 1985, the C/T SNP at nucleotide −158 of the
5.4. HBS1L-MYB (HMIP) region
Early studies conducted in a family of Asian Indian origin using segregation analysis demonstrated a modifier of
5.5. BCL11A
After the completion of the Human Genome Project and the development of genome-wide techniques, GWAS became the preferred approach to identify inherited genetic modifiers of disease phenotypes. The first GWAS to identify HbF modifiers utilized a selected genotyping study design, targeting 179 individuals with contrasting extremes of F-cell numbers [89]. The
5.6. Mechanism of regulating HBG expression
Many decades of research have revealed that two types of mechanisms play a major role in modifying HbF levels: (1) direct transactivation of the
SNP | Gene | Phenotype | Reference |
---|---|---|---|
rs1186868 | Baseline HbF | Uda et al. [90] | |
rs766432 | Baseline HbF | Sedgewick et al. [92] | |
rs4671393 | Baseline HbF | Lettre et al. [94] | |
rs7557939 | Baseline HbF | Lettre et al. [94] | |
rs7482144 | Baseline HbF | Lettre et al. [94] | |
rs10128556 | Baseline HbF | Galarneau et al. [110] | |
|
Baseline HbF | Sebastiani et al. [91] | |
rs5024042 | Baseline HbF | Solovieff et al. [93] | |
rs4895441 | Baseline HbF | Lettre et al. [94] | |
rs9494145 | Baseline HbF | Bae et al. [95] | |
rs9399137 | Baseline HbF | Creary et al. [107] | |
rs28384513 | Baseline HbF | Galarneau et al. [110] | |
rs12103880 | Baseline F-cells | Bhatnagar et al. [109] | |
|
ALOX5AP | HbF induced by HU | Sebastiani et al. [91] |
|
HbF induced by HU | Sebastiani et al. [91] | |
rs17599586 | HbF induced by HU | Ware et al. [108] | |
rs2295644 | HbF induced by HU | Ware et al. [108] | |
rs10483802 | HbF induced by HU | Ma et al. [105] | |
rs2182008 | HbF induced by HU | Ma et al. [105] | |
rs10494225 | HbF induced by HU | Ma et al. [105] | |
rs7130110 | HbF induced by HU | Sebastiani et al. [91] | |
rs7977109 | HbF induced by HU | Ma et al. [105] | |
HbF induced by HU | Ma et al. [105] | ||
rs4282891 | HbF induced by HU | Kumkhaek et al. [111] | |
rs2310991 | HbF induced by HU | Kumkhaek et al. [111] |
The mechanism by which the HMIP region silences
5.7. Genetic modifiers of response to hydroxyurea therapy
Data from the Multicenter Hydroxyurea Study [38] suggest that not all persons with SCD respond to HU treatment with increased HbF expression. Therefore, genetic markers to predict response to HU would support the development of precision medicine by limiting unnecessary exposure to a chemotherapy drug that causes bone marrow suppression and decreased fertility [41]. Although limited, studies have identified genetic modifiers of HbF response to HU. For example, SNPs in the
5.8. MicroRNA-mediated control of HBG gene expression
Recent studies have focused on posttranscriptional mechanisms of
Other miRNAs have been implicated in
6. Precision medicine for sickle cell disease
Completion of the Human Genome Project greatly improved efforts to develop gene-based treatment strategies for β-hemoglobinopathies. Early efforts to identify genetic modifiers of clinical severity and sub-phenotypes of disease severity in SCD consisted of candidate gene studies. Insights were gleamed into risk factors for acute VOC pain events such as SNPs in the dopamine D3 receptor [42]. Expanded investigations to understand the wide range of opioid dose required by individual sickle cell patients led to the characterization of mutations in the CYP2D6 gene required for opioid activation and classification of slow, intermediate, and rapid metabolizers [43]. However, additional studies with larger sample sizes and/or direct DNA sequencing are required to develop gene markers of disease severity for the development of precision medicine to inform clinical decision making.
A great urgency exists to identify genetic factors associated with risk for acute chest syndrome, the leading cause of morbidity and mortality in children and adults with SCD. Mutations in
Based on the absence of clinical symptoms in infants and the amelioration of symptoms in persons with hereditary persistence of HbF, the most effective strategy to modulate disease severity in persons with SCD is
Additional genetic studies that utilize high-throughput DNA (whole genome and exome) and RNA/miRNA (RNA-seq) sequencing will increase our knowledge of mechanisms involved in
Funding source
National Heart Lung and Blood Institute, National Institutes of Health to BSP (R01HL069234).
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