INDUCED CARDIOMYOPATHY

FIELD OF THE INVENTION:

The present invention is in the field of medicine, in particular cardiology.

BACKGROUND OF THE INVENTION:

Pheochromocytomas-Paraganglioma (PPGL) are rare tumors (1) that produce and store catecholamines. The release of excessive amounts of catecholamines can lead to life- threatening cardiovascular complications. The prevalence of catecholamine-induced cardiomyopathy (CIC) was of 8-11% of patients with PPGL (2-3). Some of the mechanisms involved in CIC include the activation of beta 1 -adrenoceptors and alpha-2 adrenoreceptors. The excitation of beta 1 -adrenoceptors controls the inotropic and chronotropic activities of the heart and then myocardial oxygen demand (4-5). Alternatively, over-stimulation of the alpha-2 adrenoreceptors of the small coronary arteries and arterioles could result in non-physiologic vasoconstriction leading to myocardial ischemia. (6). Importantly, single-nucleotide polymorphisms (SNP) of the beta-1 and alpha 2c adrenergic receptors result in changed myocyte receptor function and enhanced synaptic norepinephrine release and thus could result in heart failure (7,8). In this respect, some of these polymorphisms have been associated with the risk of developing heart failure and also are associated with beta-blocker response in heart failure (9-14).

SUMMARY OF THE INVENTION:

The present invention is defined by the claims. In particular, the present invention relates to a method of determining whether a patient suffering from a pheochromocytoma is at risk of developing a catecholamine-induced cardiomyopathy.

DETAILED DESCRIPTION OF THE INVENTION:

Pheochromocytomas produce and store catecholamines. The release of excessive amounts of catecholamine can lead to life-threatening catecholamine-induced cardiomyopathy (CIC). Single-nucleotide polymorphisms of the betal and alpha-2c adrenergic receptors result in enhanced myocyte receptor function and enhanced norepinephrine release. The aim of the inventors was to test the hypothesis that such genetic variation may impact the risk of developing CIC.

Thirty-one patients, including 9 with a history of life-threatening CIC, were analyzed for alpha- 2-adrenergic receptors: ADRA2C, beta-1 and beta-2 adrenergic receptors: ADRB 1 and ADRB2 genotyping. Life-threatening CIC was defined either by a history of heart failure or cardiogenic shock associated with dilated or Takotsubo cardiomyopathy. Subjects were genotyped for five variants in three adrenergic pathway genes encoding receptors as ADRA2C (one variant: rs61767072 for del322_325), ADRB1 (2 variants: rsl801252 for Ser49Gly and rsl801253 for Arg389Gly) and ADRB2 (2 variants: rsl042713 for Argl6Gly and rsl042714 for Gln27Glu). Single-locus analysis revealed that variants in ADRA2C (alpha 2CDel322-325) and ADRB1 (rs 1801252) were more common among patients with life-threatening catecholamine-induced cardiomyopathy than among controls (allele frequency, 0.44 vs. 0.05; P<0.001 0.28 vs. 0.09 p<0.05 respectively). The lack of alpha 2CDel322-325 polymorphism has a negative predictive value of 95% for the onset of CIC.

After confirmation in a replication study, Alpha 2CDel322-325 and ADRB1 (rsl801252) through the identification of patients at low-risk of developing life threatening CIC should be of valuable help in determining a therapeutic strategy in patients with paraganglioma at high risk of surgical complication.

Accordingly, the first object of the present invention relates to a method of determining whether a patient suffering from a pheochromocytoma or a paraganglioma is at risk of developing a catecholamine-induced cardiomyopathy comprising detecting in a nucleic acid sample obtained from the patient the presence or absence of at least one genetic variant in the ADRA2C or ADRB1 gene.

As used herein, the term “pheochromocytoma” has its general meaning in the art and refers to a rare, catecholamine-secreting tumor derived from chromaffin cells. The term pheochromocytoma (in Greek, phios means dusky, chroma means color, and cytoma means tumor) refers to the color the tumor cells acquire when stained with chromium salts. When a tumor composed of the same cells as a pheochromocytoma develops outside the adrenal gland, it is referred to as a “paraganglioma”. These neuroendocrine tumors are capable of producing and releasing massive amounts of catecholamines, metanephrines, or methoxytyramine, which result in the most common symptoms, including hypertension (high blood pressure), tachycardia (fast heart rate), and diaphoresis (sweating).

In some embodiments, the patient has a bilateral pheochromocytoma.

As used herein, the term “catecholamine-induced cardiomyopathy” has its general meaning in the art and refers to the occurrence of a cardiovascular event such as heart failure or cardiogenic shock associated with dilated or Takotsubo cardiomyopathy (i.e. a sudden, transient cardiac syndrome that involves dramatic left ventricular apical akinesis and mimics acute coronary syndrome).

As used herein, the term "risk" in the context of the present invention, relates to the probability that an event will occur over a specific time period and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no- conversion. "Risk evaluation," or "evaluation of risk" in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of relapse, either in absolute or relative terms in reference to a previously measured population. The methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk of conversion. In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk. In some embodiments, the present invention may be used so as to discriminate those at risk from normal. Thus the expression "determining whether a patient is at risk of developing a catecholamine-induced cardiomyopathy” as used herein means that the patient to be analyzed by the method of the present invention is allocated either into the group of patients of a population having an elevated risk, or into a group having a reduced risk of developing a catecholamine-induced cardiomyopathy. An elevated risk as referred to in accordance with the present invention, preferably, means that the risk of developing a catecholamine-induced cardiomyopathy within a predetermined predictive window is elevated significantly (i.e. increased significantly) for a patient with respect to the average risk for a cardiovascular event or cardiac mortality in a population of patients. A reduced risk as referred to in accordance with the present invention, preferably, means that the risk of developing a catecholamine-induced cardiomyopathy within a predetermined predictive window is reduced significantly for a patient with respect to the average risk for a cardiovascular event or cardiac mortality in a population of patients. Particularly, a significant increase or reduction of a risk is an increase or reduction or a risk of a size which is considered to be significant for prognosis, particularly said increase or reduction is considered statistically significant. The terms "significant" and "statistically significant" are known by the person skilled in the art. Thus, whether an increase or reduction of a risk is significant or statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools.

As used herein, the term "nucleic acid sample” refers to any biological sample isolated from the patient liable to contain nucleic acid for the purpose of the present invention. Samples can include by way of example and not limitation, body fluids (e;g. saliva) and/or tissue extracts such as homogenates or solubilized tissue obtained from the subject.

In some embodiments, the sample is a blood sample. The term “blood sample” means any blood sample derived from the patient that contains nucleic acids. More preferably, the nucleic acid sample is a PBMC sample. The term “PBMC” or “peripheral blood mononuclear cells” or “unfractionated PBMC”, as used herein, refers to whole PBMC, i.e. to a population of white blood cells having a round nucleus, which has not been enriched for a given sub population. Typically, these cells can be extracted from whole blood using Ficoll, a hydrophilic polysaccharide that separates layers of blood, with the PBMC forming a cell ring under a layer of plasma. Additionally, PBMC can be extracted from whole blood using a hypotonic lysis which will preferentially lyse red blood cells. Such procedures are known to the expert in the art. The template nucleic acid need not be purified. Nucleic acids may be extracted from a sample by routine techniques such as those described in Diagnostic Molecular Microbiology: Principles and Applications (Persing et al. (eds), 1993, American Society for Microbiology, Washington D.C.).

As used herein, the term “ADRA2C” has its general meaning in the art and refers to the gene encoding for the adrenoceptor alpha 2C. The NCBI reference number is Gene ID: 152 and the ENSEMBL ID (i.e. the gene identifier number from the Ensembl Genome Browser database) is Ensembl:ENSG00000184160. The term is also known as ADRA2L2; ADRARL2; ADRA2RL2; or ALPHA2CAR.

As used herein, the term “ADRB1” has its general meaning in the art and refers to the gene encoding for the adrenoceptor beta 1. The NCBI reference number is Gene ID: 153 and the ENSEMBL ID (i.e. the gene identifier number from the Ensembl Genome Browser database) is Ensembl:ENSG00000043591. The term is also known as RHR; B1AR; FNSS2; ADRBIR; or BETAIAR.

As used herein, the term "genetic variant" has its general meaning in the art and denotes any of two or more alternative forms of a gene occupying the same chromosomal locus. The alteration typically consists in a substitution, an insertion, and/or a deletion, at one or more (e.g., several) positions in the gene. Genetic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term is also known as “polymorphism”. In some embodiments, the genetic variant is a single nucleotide polymorphism. As used herein, the term "single nucleotide polymorphism" or "SNP" has its general meaning in the art and refers to a single nucleotide variation in a genetic sequence that occurs at appreciable frequency in the population.

In some embodiments, the method of the present invention comprises detecting the alpha- 2CDel322-325 adrenoceptor (AR) polymorphism wherein detecting said polymorphism indicates a low risk of developing a catecholamine-induced cardiomyopathy for the patient.

As used herein, the term “alpha-2CDel322-325” refers to the polymorphism that leads to the deletion of four consecutive amino acids (residues at 322 to 325) in ADRA2C protein, and that causes a substantial loss of agonist-mediated receptor function. In some embodiments, the method of the present invention comprises detecting the rsl801252 polymorphism.

As used herein, the term “rsl801252” has its general meaning in the art and refers to the genetic variant responsible for the responsible of Ser49Gly mutation in ADRB1 protein.

Typically the presence of the minor allele “G” indicates that the subject has a low risk of developing a catecholamine-induced cardiomyopathy.

Detecting the genetic variant may be determined according to any genotyping method known in the art. Typically, common genotyping methods include, but are not limited to, TaqMan assays, molecular beacon assays, nucleic acid arrays, allele-specific primer extension, allele- specific PCR, arrayed primer extension, homogeneous primer extension assays, primer extension with detection by mass spectrometry, sequencing, multiplex primer extension sorted on genetic arrays, ligation with rolling circle amplification, homogeneous ligation, OLA, multiplex ligation reaction sorted on genetic arrays, restriction-fragment length polymorphism, single base extension-tag assays, and the Invader assay. Such methods may be used in combination with detection mechanisms such as, for example, luminescence or chemiluminescence detection, fluorescence detection, time-resolved fluorescence detection, fluorescence resonance energy transfer, fluorescence polarization, mass spectrometry, and electrical detection. Various methods for detecting polymorphisms include, but are not limited to, methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA, comparison of the electrophoretic mobility of variant and wild type nucleic acid molecules, and assaying the movement of polymorphic or wild-type fragments in polyacrylamide gels containing a gradient of denaturant using denaturing gradient gel electrophoresis. Sequence variations at specific locations can also be assessed by nuclease protection assays such as RNase and SI protection or chemical cleavage methods.

In some embodiments, genotyping is performed using the TaqMan assay, which is also known as the 5' nuclease assay. The TaqMan assay detects the accumulation of a specific amplified product during PCR. The TaqMan assay utilizes an oligonucleotide probe labeled with a fluorescent reporter dye and a quencher dye. The reporter dye is excited by irradiation at an appropriate wavelength, it transfers energy to the quencher dye in the same probe via a process called fluorescence resonance energy transfer (FRET). When attached to the probe, the excited reporter dye does not emit a signal. The proximity of the quencher dye to the reporter dye in the intact probe maintains a reduced fluorescence for the reporter. The reporter dye and quencher dye may be at the 5’ most and the 3’ most ends, respectively, or vice versa. Alternatively, the reporter dye may be at the 5 ’ or 3 ’ most end while the quencher dye is attached to an internal nucleotide, or vice versa. In yet another embodiment, both the reporter and the quencher may be attached to internal nucleotides at a distance from each other such that fluorescence of the reporter is reduced. During PCR, the 5’ nuclease activity of DNA polymerase cleaves the probe, thereby separating the reporter dye and the quencher dye and resulting in increased fluorescence of the reporter. Accumulation of PCR product is detected directly by monitoring the increase in fluorescence of the reporter dye. The DNA polymerase cleaves the probe between the reporter dye and the quencher dye only if the probe hybridizes to the target SNP-containing template which is amplified during PCR, and the probe is designed to hybridize to the target SNP site only if a particular SNP allele is present. Preferred TaqMan primer and probe sequences can readily be determined using the SNP and associated nucleic acid sequence information provided herein. A number of computer programs, such as Primer Express (Applied Biosystems, Foster City, Calif.), can be used to rapidly obtain optimal primer/probe sets. It will be apparent to one of skill in the art that such primers and probes for detecting the nucleic acids of the present invention are useful in diagnostic assays for stenosis and related pathologies, and can be readily incorporated into a kit format.

Detecting the genetic variant may also be performed by sequencing. A variety of automated sequencing procedures can be used, including sequencing by mass spectrometry. The nucleic acid sequences of the present invention enable one of ordinary skill in the art to readily design sequencing primers for such automated sequencing procedures. Commercial instrumentation, such as the Applied Biosystems 377, 3100, 3700, 3730, and 3730x1 DNA Analyzers (Foster City, Calif.), is commonly used in the art for automated sequencing. Nucleic acid sequences can also be determined by employing a high throughput mutation screening system, such as the SpectruMedix system.

Once the patient is identified as having or being at risk of having a catecholamine-induced cardiomyopathy, the physician can decide further therapeutic options. In particular, when the risk is considered as being low, it can be decided not to proceed with the surgical resection of the tumor(s) and to proceed with the follow-up of the patient. In particular, the present invention is particularly suitable for patients with bilateral pheochromocytoma in whom surgery can result in adrenal failure. The method of the present invention is also particularly suitable for patients at high surgical risk.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

Methods

Population

All patients monitored for a PPGL (except one) in the Department of Arterial Hypertension of Toulouse University were considered eligible. Among them, those for whom DNA was available and who gave their informed consent were included in the study. Informed consent for genetic testing was collected for all patients. Biological collection was included in the bio collection declared by the biological resource center of the academic hospital of Toulouse and the protocol was approved by the Ethics Committee of Sud-Ouest et Outre-Mer I et II (DC- 2015-2450). Clinical and biological characteristics were recorded. Thirty-one patients were analyzed. Life-threatening CIC was defined either by a history of heart failure or cardiogenic shock associated with dilated or Takotsubo cardiomyopathy (15).

Genetic analysis

All subjects were genotyped for five variants in three adrenergic pathway genes encoding receptors as ADRA2C (one variant: rs61767072 for del322_325), ADRBl (2 variants: rsl801252 for Ser49Gly and rsl801253 for Arg389Gly) and ADRB2 (2 variants: rsl042713 for Argl6Gly and rsl042714 for Gln27Glu). The genotyping was performed on genomic DNA from blood samples (MagNA Pure Total NA isolation kit, Roche Life Science) by sequencing PCR products spanning one or two variant positions as previously mentioned (6, 10, 13). BigDye Terminator Chemistry was used according to the manufacturer’s instructions and analyzed on an ABI 3100 sequencer (all from Applied Biosystems).

Statistical analysis

Continuous variables were recorded as means ± standard deviation and categorical variables as percentages. Patients (n=22) exempt of CIC and patients with a history of CIC (n=9) were compared. We used the Chi2 test for categorical variables and the Student or the Wilcoxon tests for continuous variables as appropriate. Cox proportional-hazards stepwise regression analysis was used to identify the independent predictors of CIC. The time to event corresponded to the age at which a person received the diagnosis of PPGL (16). Before entered in the models, continuous variable with skewness distribution (Shapiro’s test) were log transformed. A variable has to be significant at the 0.20 level before it can be entered into the model, while the variable has to be significant at the 0.05 level for it to remain in the model. Statistical analyses were performed using SAS software (version 9.4 for Windows).

Results:

The characteristics of the study population are presented in Table 1. All patients (n=31) had surgical resection of a pheochromocytoma (n=29) or a paraganglioma (n=2). All patients except one are Caucasians. 22 patients were free of CIC. Of 9 patients with a history of CIC, 5 had a Takotsubo cardiomyopathy and 4 a dilated cardiomyopathy, 5 developed heart failure and 3 had a cardiogenic shock requiring extracorporeal membrane oxygenation. There were triggering circumstances in 2/9 patients: a planned surgery and an altercation at work respectively. Age, sex, and the prevalence of syndromic disease were not significantly different between the groups. Also, at time of diagnosis, calcium channel blockers were more frequently prescribed in patients exempt of CIC. A significant increase in 24h urine metanephrine levels was detected in patients with CIC. Single-locus analysis (Table 2) revealed that 2CDel322- 325 and rsl801252 were more common among patients with CIC than among controls. No significant association was observed for the other tested genotypes: ADRB1 (rsl801253), ADRB2 (rsl800888, rsl042714, rsl042713) and the onset of CIC. Also, 89% of the patients (8/9) with CIC had alpha 2CDel322-325 polymorphism as compared with 9% (2/22) of the patients exempt from this complication. Hence, in this population, the lack of alpha 2CDel322- 325 polymorphism has a negative predictive value of 95%. Eventually, in stepwise survival analysis using Cox's regression model when urinary metanephrin (respectively urinary normetanephrin) expressed in fold the upper normal range were inserted into the model, alpha 2CDel322-325 polymorphism only predicted the onset of CIC (relative risk 13.59 95 percent confidence interval limits: 1.68-10.94; P=0.01).

Discussion:

The main finding of the study was that 2CDel322-325 and ARDB1 (rsl801252) polymorphisms were biomarkers of the risk of CIC in patients with PPGL. Some studies have previously identified such relationship between one SNP and acute events (17,18). Also, this finding is biologically plausible. Indeed, the alpha-2c receptor inhibits norepinephrine release from cardiac sympathetic nerves. In humans, a 4-amino acid deletion polymorphism is associated with decreased G-protein coupling. Then, the presence of the dysfunctional (X2cDel322-325 receptor could lead to increased norepinephrine release and consequently sympathetic overstimulation which could be a risk factor for CIC (19). In line with this hypothesis, the (2/21) 9.5 % prevalence of this deletion in patients who did not develop CIC is closed to that observed (6 %) in Caucasian population (20) and in striking contrast with the (8/9)88 % observed prevalence in patients who developed CIC. Regarding the betal -adrenergic receptor which is the receptor for norepinephrine on the cardiomyocyte, two common polymorphisms have been described (21). Of them, ARDB1 (rsl801252) is significantly associated with the onset of CIC. ARDB1 (rsl801252) causes an amino acid substitution from serine to glycine at amino acid position 49 at the intracellular C-terminus of the betal - adrenergic receptor and is associated with increased agonist-promoted receptor down- regulation in transfected cell lines (21). Thus, the presence of ARDB1 (rsl801252) would be expected to alter contractile response at the myocyte in response to norepinephrine which in turn may predispose to heart failure (21). In this respect, ARDB1 (rsl801252) has been associated with all -cause mortality in hypertensive patient (22). Also, the (4/22) 18 % prevalence of this polymorphism in patients who did not develop CIC is closed to that observed (20 %) in Caucasian population (21).

Clinical perspectives:

Genetic biomarkers able to rule out the onset of life-threatening CIC should provide help for the evaluation of the benefit/risk balance of the surgery, especially in patients with bilateral pheochromocytoma in whom surgery can result in adrenal failure even when partial adrenalectomy was performed (23), or in patients at high surgical risk. Indeed, as a consequence of bilateral adrenalectomy, patients became steroid dependent and will develop in 30% of cases either adrenal crisis or symptoms consistent with steroid over replacement (24). Also, patients with adrenal insufficiency suffer from impaired quality of life (25) and are at increased mortality risk (26,27). Furthermore, in the small proportion- about 3% (28)- of patients with secreting head or neck paraganglioma or in patients with mediastinal paraganglioma supplied by coronary arteries, surgical morbidity remains significant, In such cases, reliable biomarkers associated with a very low incidence of life-threatening CIC can help physicians better determine which patients may benefit from medical or surgical treatment. Conclusion:

We showed that alpha2c-adrenergic receptors polymorphism and b eta 1 -adrenergic receptor polymorphism predict a low risk of incidence of life-threatening CIC in patients with PPGL. These findings deserve to be confirmed and should be a useful tool in determining therapeutic strategy in patients with PPGL at high risk of surgical complication.

TABLES:

Table 1: Characteristics of the study population

Life-threatening Patients who did not develop catecholamine-induced life-threatening cardiomyopathy (n=9) catecholamine-induced cardiomyopathy (n=22)

Age (years) 47.78±14.38 50.55±12.40

Body mass index (kg/m ² ) 23.10±4.17 25.21±4.86 Urinary Normetanephrine 3596.44±3450.82 2198.24±1948.52 (pg/24h)

Urinary Metanephrine 6011.44±6687.12 2261.10±4252.33

(pg/24h)*

Tumor size (mm) 60.25±28.03 44.65±22.45

Pass score 3.29±2.63 2.65±3.21 N (%)

Male sex 3 (33.33) 8 (36.38)

Preexisting hypertension 2 (22.22) 12 (57.14) Prescribed drugs at time of diagnosis Alphablockers 0 5 (2.73)

Betablockers 1 (11.11) 8 (36.36) ACEI 1 (11.11) 4 (18.18)

ARB 0 4 (18.18)

Calcium channel blockers† 0 13 (59.09)

Diuretic 0 4 (18.18)

Type 2 diabetes 0 4 (18.18)

Antidepressant medication 1 (11.11) 2 (9.09)

Paraganglioma 0 2 (9.10)

Syndromic disorder 2 (22.22) 4 (18.18)

Neurofibromatosis 1 1 (11.11) 1 (4.55)

Multiple endocrine neoplasia 1 (11.11) type 2A

Germline mutation in 1 (4.55)

TMEM127 gene Germline mutation in 1 (4.55)

Succinate Dehydrogenase

Complex Flavoprotein

Subunit A

Germline mutation in 1 (4.55)

Succinate Dehydrogenase

Complex Flavoprotein Subunit B

Smoking status

Never 3 (33.33) 11 (50)

Former smoker 4 (44.44) 7 (31.82)

Active 2 (22.22) 4 (18.18) ACEI Angiotensin-converting enzyme inhibitor ARB Angiotensin receptor blocker *p<0.05, †p<0.01 Table 2: Genetic analysis

Alleles Allele p Genotype frequency

ADRA2C Wt/wt Wt/del Del/del P rs61767072

Life-threatening 0.44 $ 1 8 0 $ catecholamine- induced cardiomyopathy

Controls 0.05 19 2 0

ARDB2 r si 042714 Gly/Gly Cyt/Gly Cyt/Cyt

Life-threatening 0.56 2 (22.22) 6 (66.67) 1 (11.11) catecholamine- induced cardiomyopathy Controls 0.36 2 (9.10) 12 8 (38.36)

(54.55)

ADRB2 rsl042713 Arg/Arg Arg/Gly Gly/Gly

Life-threatening 0.72 4 (44.44) 5 (55.56) 0 catecholamine- induced cardiomyopathy Controls 0.68 9 (40.91) 12 1 (4.55)

(54.55)

ADRB1 rsl801252 * A/A A/G G/G

Life-threatening 0.28 4 (44.44) 5 (55.56) 0 catecholamine- induced cardiomyopathy Controls 0.09 18 4 (18.18) 0

(81.82)

ADRB1 rsl801253 C/C C/G G/G

Life-threatening 0.33 5 (55.56) 2 (22.22) 2 (22.22) catecholamine- induced cardiomyopathy Controls 0.14 16 6 (27.27) 0

(72.73)

$p<0.0001

*p<0.05

REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.