Down-regulation of Long Non-coding RNA H19 and ADCK4 gene in Children with Nephrotic Syndrome

doi:10.21203/rs.3.rs-4883076/v1

AIM OF THE WORK: The objective of this study is to examine the correlation between the long non-coding RNA H19 and ADCK4 genes and the susceptibility to nephrotic syndrome in children from Egypt.

METHODS: A cross-sectional research was conducted on 50 cases with nephrotic syndrome and 50 controls of apparently healthy children with no past or family history of renal or chronic diseases. Each participant signed their informed consent form. The study excluded malignancy, chronic infectious disorders, drug-induced membranous glomerulonephritis (MGN) or lupus nephritis.

RESULTS: There was a significant divergence among cases and control regarding RQ (ADCK4) (p-value < 0.001) and RQ (H-19) (p-value < 0.001). Nevertheless, no notable distinction was observed between the two forms of nephrotic syndrome in terms of RQ (ADCK4) (p-value =0.935) and RQ (H-19) (p-value 0.525).

CONCLUSION:In nephrotic syndrome, the RNA H19 and ADCK4 genes are downregulated, with no notable distinction observed between the steroid-sensitive and steroid-resistant patient groups.

RNA H19 gene

ADCK4 gene

Nephrotic Syndrome

Long non-coding RNA H19 is downregulated in children with NS.
The ADCK4 gene is downregulated in children with nephrotic NS.
In children with nephrotic syndrome, whether it is SSNS or SRNS, the levels of both lncRNA H19 and ADCK4 are decreased.
H19 knockdown decreased ADCK4 expression in human primary renal podocytes, but H19 overexpression increased it.
Electrolyte imbalance may occur in some patients with NS.

Nephrotic syndrome (NS) is a widespread kind of renal illness observed in youngsters (Safaei & Maleknejad, 2009). It is the outward sign of glomerular diseases connected with severe (nephrotic range) proteinuria. Nephrotic range proteinuria is defined as proteinuria > 40mg/m2/hr or a ratio of urine protein to creatinine that is more than 2. The three main medical manifestations of nephrotic syndrome are oedema, hyperlipidemia (cholesterol > 200 mg/dL), and hypoalbuminemia (≤ 2.5 g/dL), which are caused by significant protein losses in the urine(Pais P. & Avner ED., 2011).

While most children with NS develop SSNS, it is essential to note that 10–20% of children and 40% of adults with NS do not respond well to glucocorticoid treatment and other immunosuppressive therapy. This lack of response can result in the progression of end-stage kidney disease (ESKD) (Ashraf et al., 2013a; Pokrajac et al., 2018). Most instances of steroid-sensitive nephrotic syndrome (SSNS) can be distinguished by widespread podocyte foot-process effacement on electron microscopy and normal glomeruli on light microscopy. On the other hand, cases of steroid-resistant nephrotic syndrome (SRNS) typically manifest with either diffuse mesangial sclerosis (DMS) or focal segmental glomerulosclerosis (FSGS) (Machuca et al., 2009).

DMS is characterised by mesangial matrix growth along with hypertrophy and minor cobblestone hyperplasia of podocytes, whereas FSGS also includes effacement of the foot process without glomerular immune complex deposits. DMS and FSGS are the outcome of a more serious and prolonged injury process that eventually causes glomerular scarring and increased loss(Ravaglia et al., 2022).

There is still no known cause of NS. Highly developed podocytes are crucial for preserving the renal glomerular filtration barrier. A podocyte’s malfunction, such as abnormal apoptosis or decreased migratory ability, can potentially dismantle the barrier and result in glomerulosclerosis and proteinuria (Barutta et al., 2022). The aarF domain, including kinase 4 (ADCK4), is extensively expressed in glomerular podocytes, and NS, which is resistant to steroids, is markedly encouraged by mutations in ADCK4 (Xu et al., 2020a).

Non-coding RNAs (lncRNAs), which are regulatory RNAs that have not been translated, play a crucial role in controlling several biological processes. These processes include the cell cycle, differentiation, genomic and chromosomal alterations, transcriptional activation and interference, and nuclear transcription (Liu et al., 2022; Statello et al., 2021).

Because lncRNAs are involved in every stage of the gene regulatory procedure, deregulation of these transcripts may occur in several pathophysiological conditions, including cancer and disorders involving genomic imprinting (Fenoglio et al., 2013). The pathogenesis of several kidney disorders has been linked to lncRNAs in recent years as it has become more widely acknowledged (Moreno et al., 2021a).

H19 knockdown decreased ADCK4 expression in human primary kidney podocytes, but H19 overexpression increased it. Additional research indicated that H19 may control ADCK4’s promoter activity (Xu et al., 2020a).

This study assessed the levels of lncRNA H19 and ADCK4 in the blood of 50 healthy and 50 children with NS. We observed a decrease in the expression of both long non-coding RNA H19 and ADCK4 in children with nephrotic syndrome (NS) who had either steroid-sensitive nephrotic syndrome (SSNS) or steroid-resistant nephrotic syndrome (SRNS).

2.1. Study group:

This cross-sectional study was conducted between January and October of 2023 through a collaboration between the Medical Biochemistry and Molecular Biology Department and the Paediatric Department of Menoufia University.

Fifty children were diagnosed with nephrotic syndrome based on specific criteria: The International Study of Kidney Disease in Children (ISKDC) has provided a concise and academic definition of Nephrotic Syndrome (NS). According to ISKDC, NS is characterised by significant proteinuria (exceeding 40 mg/m2/h, or 1000 mg/m2/24 h), resulting in low levels of albumin (less than 2.5 g/dL), oedema, and elevated lipid level (“The Primary Nephrotic Syndrome in Children. Identification of Patients with Minimal Change Nephrotic Syndrome from Initial Response to Prednisone,” 1981a). Thirty-three of them were steroid sensitive, and the samples were collected during relapse; the other 17 patients were steroid-resistant, based on the following definitions SSNS: complete remission following four weeks of normal dosage prednisone or prednisolone(“The Primary Nephrotic Syndrome in Children. Identification of Patients with Minimal Change Nephrotic Syndrome from Initial Response to Prednisone,” 1981b). Relapse: recurrence of nephrotic range proteinuria. SRNS: absence of total remission after four weeks of treatment with a daily dose of prednisone or prednisolone (Rovin et al., 2021). Fifty controls of healthy children with no past or family history of renal or chronic diseases.

This study was approved by the Research Ethics Committee of Menoufia University (IRP NO: 10/2023 BIO 5) and conforms to the research guidelines implemented at the Pediatric Department at Menoufia University. Before each patient was enrolled in the trial, their parents gave informed consent.

The study enrolled participants aged 1 to 18 years old who had a steroid-sensitive type. Samples were collected during relapse. The study did not include any cancer cases, chronic infectious diseases (such as hepatitis B and C viruses), drug-induced membranous glomerulonephritis (MGN), or lupus nephritis.

Each participant underwent a comprehensive heritage-taking and meticulous clinical examination.

2.2. Blood sample:

Before administering any medication, the blood samples were obtained by venipuncture in an entirely aseptic setting. (details attached in supplementary file)

Every subject in this research underwent laboratory testing, which included standard colourimetric techniques utilising kits provided by Spinreact (Spain) to determine serum levels of triacylglycerol (TG) and total cholesterol (TC).

The supernatant serum was separated from the clot and stored at -80°C until the serum urea levels were analysed using an enzymatic colourimetric method (Sena, 2006) an enzymatic colourimetric approach to measure serum creatinine (Bowers & & Wong, 1980), SGOT and SGPT (Scherwin, 2003). (supplementary file)

Albumin was measured by using a modified Bromocresol green colourimetric method (#ALB100250) (Point & Albumin. B., n.d.). (Supplemental file)

Utilising the QIAamp RNA Blood MiniKit (Qiagen, USA), total RNA was isolated from blood. (Extraction details supplemental file)

RNAH19 mRNA levels were measured using the following primers from (Midland, Texas):

Long non-coding Forward primer Template: AGCGGGTCTGTTTCTTTACTT.

Reverse primer Template: AGCTGGGTAGC ACCATTTC.ADCK4.

Gene (also known as COQ8B).

Forward primer Template: TCCGATTCATGCAGACTGAC.

Reverse primer Template: CTGTGAACTCTGTCCCAAACT.

Primers for human glyceraldehyde-3-phosphate dehydrogenase GAPDH

Forward primer Template: CAGGGCTGCTTTTAACTCTGGTAA.

Reverse primer Template: GGGTGGAATCATATTGGAACATGT.

A 20 µL mixture was prepared by adding five µL of the cDNA to 10 µL of 2x SYBR® Low ROX master mix, one µL of each primer, and four µL of RNase-free water. The reaction was conducted in 45 cycles, with denaturation at 94ºC for 30 seconds, annealing at 55ºC for 30 seconds, and extension at 72ºC for 30 seconds. The data was analysed using Version 2.0.1 of the Applied Biosystems 7500 software. The mRNA levels were evaluated using the relative quantification (RQ) technique, which utilises the ΔΔCt method to measure the specified gene concerning a control and an endogenous reference gene (GAPDH).

2.3. Statistical analysis of the data

Version 20.0 of the IBM SPSS software program fed and analysed data into the computer (Armonk, NY: IBM Corp).

The study population was divided into two groups.

Group I (patients)

fifty children with NS, ages 1.5 to 15 years old, with 26 males and 24 females. (Table 1)

Group II (controls)

fifty normal children with no history of renal diseases, their ages ranged from 1.5 to 14 years old, with 30 males and 20 females. (Table 1)

The clinical types of nephrotic patients were as follows: steroid-sensitive nephrotic syndrome in 33 patients (66%) and steroid-resistant nephrotic syndrome in 17 patients (34%). (Fig. 1)

Table (2)

Figure (1)

Patients exhibited significantly lower serum albumin levels than the control group (p-value = 0.002). Conversely, patients displayed significantly higher levels of total protein in urine (p-value < 0.001), as well as elevated serum cholesterol and triglycerides (p-value < 0.001) in comparison to the control group. (Table 2)

The serum creatinine levels showed no significant difference between the cases and controls (p-value = 0.819). However, the BUN levels were significantly higher in the cases than the controls (p-value < 0.001). (Table 2)

The cases had significantly lower serum sodium levels than the control group (p-value < 0.001). Conversely, patients had significantly higher serum potassium levels than the control group (p-value < 0.001). Additionally, patients had significantly lower serum total calcium levels compared to controls (p-value < 0.001). (Table 2)

Liver enzymes (ALT and AST) were insignificantly different between cases and controls (p-values = 0.677 and 0.178, respectively). (Table 2)

Table (2)

In the study, the findings presented in Table 3 and Fig. 2 indicate that the RQ (ADCK4) and RQ (H-19) values were notably lower in patients compared to controls. The statistical analysis revealed a significant difference between the two groups, with p-values of less than 0.001 for both variables. Nevertheless, there was no notable distinction observed between the two forms of nephrotic syndrome in terms of RQ (ADCK4) (p-value = 0.935) and RQ (H-19) (p-value = 0.525). (Figs. 3 and 4)

Table (3)

Figure (2)

Figure (3)

Figure (4)

Idiopathic nephrotic syndrome (INS) is more prevalent in certain age groups, racial groups, and geographic regions. INS is detected globally. According to estimates, the annual incidence of steroid-sensitive nephrotic syndrome (SSNS) in children under 16 years in the USA and Europe is 1–3 per 100,000, with a cumulative frequency of 16 per 100,000 children (Niaudet & Boyer, 2016a; Verma & Patil, 2024).

It is widely recognised that there is a higher prevalence of males in children, with a male-to-female ratio as high as 3.8:1. In contrast, adolescents of both genders are affected similarly (Mahamat Abderraman et al., 2023; Niaudet & Boyer, 2016a). This study found no significant differences regarding demographic data between cases and controls. The patients comprised 26 males (52%) and 24 females (48%), indicating a male predominance.

Most children diagnosed with nephrotic syndrome (NS) will go on to develop steroid-sensitive nephrotic syndrome (SSNS). Nevertheless, a significant portion of adults (40%) and children (10–20%) with NS do not experience sustained remission even after undergoing treatment with glucocorticoid medication and other immunosuppressive therapies (Ashraf et al., 2013a; Trautmann et al., 2023). In the present study, the clinical types of NS were classified as SSNS in 33 patients (66%) and steroid-resistant nephrotic syndrome (SRNS) in 17 patients (34%), with a predominance of the SSNS type.

The study found that patients had lower serum albumin levels than controls, while patients had higher total urine protein levels than controls. In addition, the levels of serum cholesterol and triglycerides were significantly higher in the patients compared to the controls. The reason for this can be attributed to the majority of patients having steroid-sensitive nephrotic syndrome (SSNS) and are currently going through a relapse, which indicates an ongoing state of active disease. The serum protein levels in patients with NS are notably lower in most cases, with 80% below 50 g/L and 40% below 40 g/L. Albumin concentrations often decrease to below 20 g/L and occasionally below ten g/L (Niaudet & Boyer, 2016a).

A higher LDL/HDL cholesterol ratio occurs when total and LDL cholesterol levels increase, while HDL cholesterol, specifically HDL2, stays low or stable. (Vaziri, 2003). Patients with severe hypoalbuminaemia had higher levels of VLDL and triglycerides (Niaudet & Boyer, 2016a).

In our study, the serum creatinine levels showed no significant difference between the cases and controls. However, the blood urea nitrogen (BUN) levels were significantly higher in the cases compared to the controls. One way to understand this is by looking at blood urea nitrogen levels and serum creatinine levels. In patients with NS, these levels are usually normal or slightly elevated, indicating a slight decrease in glomerular filtration rate (GFR), as described by Niaudet and Boyer (Niaudet & Boyer, 2016a). Patients with active proteinuria frequently experience a slight decline in renal function because of decreased glomerular permeability, which improves when the patient enters remission (Marques et al., 2022; Menon, 2019).

The study found that cases had lower serum sodium levels compared to controls. Additionally, patients had higher serum potassium levels and lower serum total calcium levels compared to controls. This could be explained by the low sodium levels being linked to hypovolaemia-induced dilution due to improper renal water retention and inappropriate release of antidiuretic hormones. The slight drop in plasma sodium content is frequently a hyperlipidaemia-related artefact (Niaudet & Boyer, 2016a). Oliguric patients may have elevated serum potassium levels. Due to hypoproteinaemia, serum calcium levels are consistently low. The usual range of ionised calcium can be affected by normal but inappropriate amounts of calcitriol and urine loss of 25-hydroxyvitamin D3 [27, 28].

The study revealed a significant decrease in RQ (ADCK4) and RQ (H-19) levels among patients compared to controls. There is a growing recognition of the involvement of lncRNAs in the pathophysiology of various kidney disorders. LncRNAs regulate genes and pathways associated with acute kidney injury (AKI) pathophysiology. They serve as potential therapeutic targets and reliable indicators for assessing renal damage [29].

LncRNA taurine-upregulated 1 (Tug1) was implicated in diabetic nephropathy (DN), according to a publication by Li et al. and Moreno et al. [29, 30]. The activity of lupus nephritis (LN) patients was correlated with heightened expression of the pro-apoptotic lncRNA-p21 in their monocytes and urine cells [31].

Expression of lncRNA LOC105375913 was increased in patients with focal segmental glomerulosclerosis (FSGS). LOC105375913 suppressed miR-27b, promoting the overexpression of Snail and tubulointerstitial fibrosis, according to additional research conducted in tubular cells and study animals. This lncRNA was activated through the C3a/p38/XBP-1s pathway [29].

Patients diagnosed with IgA-negative mesangial proliferative glomerulonephritis exhibit differential expression of numerous lncRNAs, potentially playing a role in the pathogenesis of the condition. A recent study utilised microarray analysis to identify over 250 differentially expressed lncRNAs and mRNAs in monocytes from patients with IgA nephropathy and healthy individuals. These genes were found to regulate the innate immune response [32,33] primarily. A case study published in 2020 by Zhai et al. [32] discovered that the ADCK4 gene caused their patient’s isolated proteinuria.

The incidence of ADCK4-associated FSGS was 7.5% (n = 4) in children aged five and older with multidrug-resistant FSGS, according to research done in 2017 by Park E et al. on 53 children with NS and FSGS histopathology. They concluded that when older children with steroid-resistant FSGS present, ADCK4 mutations should be considered [33].

In this study, we evaluated the demonstration of RQ (ADCK4) and RQ (H-19) in patients with nephrotic syndrome, which is either steroid-resistant or steroid-sensitive. Our study revealed that there was downregulation of both RNA H19 and ADCK4 genes in all cases of nephrotic syndrome and no significant difference between the steroid-sensitive group and steroid-resistant group of patients. Xu J. et al. [34] observed a positive correlation between the expression of H19 and ADCK4 in children with NS based on their study of 30 children with nephrotic syndrome and 30 healthy control children. It was also discovered that H19 might control the expression of ADCK4 in human primary renal podocytes. H19 overexpression increased the expression of ADCK4 in human primary renal podocytes, but H19 knockdown decreased it.

The present study sets itself apart from previous research by especially concentrating on the down-regulation of long non-coding RNA H19 and the ADCK4 gene in paediatric patients diagnosed with nephrotic syndrome (NS). Although prior studies have investigated the genetic and molecular basis of NS, our research stands out due to its comprehensive analysis of the relationship between H19 and ADCK4 in both steroid-sensitive (SSNS) and steroid-resistant (SRNS) variants of the disease [35–37]. In contrast to previous investigations, our findings demonstrate that the down-regulation of these markers is consistent in both SSNS and SRNS patient groups. In addition, our research reveals a new understanding of the mechanism, showing that reducing H19 leads to a direct decrease in ADCK4 expression in renal podocytes. This suggests a regulatory connection that has not been previously recorded. These findings not only enhance our understanding of the underlying mechanisms of NS but also suggest possible biomarkers that might be used for diagnosing the condition and monitoring its therapy.

The main limitation of our study was the lack of histopathological data on patients, which might have affected the significance of the data. Another limitation was that only one paper studied the same point to compare the results.

We concluded that downregulation of both RNA H19 and ADCK4 genes occurs in all cases of nephrotic syndrome. There is no significant difference between the sensitive group and the steroid-resistant group of patients.

ADCK4;	Aarf Domain Containing Kinase 4
Na;	Sodium
K;	Potassium
Ca;	Calcium
BUN;	Blood Urea Nitrogen
DMS;	Mesangial Sclerosis
ESKD;	End-Stage Kidney Disease
FSGS;	Focal Segmental Glomerulosclerosis
IBR;	Institutional Review Board
ISKDC;	International Study Of Kidney Disease In Children
LncRNAs;	Long Non-coding RNAs
MGN;	Membranous Glomerulonephritis
NS;	Nephrotic Syndrome
SSNS;	Steroid-Sensitive Nephrotic Syndrome
Tug1;	taurine-upregulated 1

ACKNOWLEDGEMENTS

The authors thank the parents of the children for their active participation in our study.

Author Contributions

MF, SS, AME, AME, ABZ, and SMM: idea, design, and data interpretation. MF, SS and SMM participant enrolment and data collection. MF, SS and SMM: manuscript writing SS, SMM, AME, AME, ABZ: statistical analysis. MF, SS, AME, AME, ABZ and SMM: manuscript revision. All authors contributed to the article and approved the submitted version.

Funding

The authors confirmed that no funds had been received from any organisation and that the research had been done independently.

Competing Interests

The authors declare no competing interests.

Ethical Approval

The Institutional Review Board (IRB) of the Menoufia Faculty of Medicine approved the study (ID number: 10/2023 BIO 5). Research work was performed following the Declaration of Helsinki.

Informed Consent

Informed consent was obtained from all the legally authorised representatives of participants included in the study.

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Zhai, S., Zhang, L., Sun, B., Zhang, Y., & Ma, Q. (2020). Early-onset COQ8B (ADCK4) glomerulopathy in a child with isolated proteinuria: a case report and literature review. BMC Nephrology, 21(1), 406. https://doi.org/10.1186/s12882-020-02038-7

Table (1): Demographic data of the studied groups.

Demographic data	Patients (n = 50)		Controls (n = 50)		Test of Sig.	P
	No.	%	No.	%
Sex
Male	26	52.0	30	60.0	χ²= 0.649	0.420
Female	24	48.0	20	40.0
Age (years)
Min. – Max.	1.50 – 15.0		1.50 – 14.0		U= 1082.0	0.246
Median (IQR)	8.0 (4.50 – 12.0)		6.50 (4.0 – 10.0)

IQR: Inter quartile range, x²: Chi square test, U: Mann Whitney test

p: p value for comparing between the two studied groups.

Table (2): Comparison between the two studied groups according to the laboratory data

Laboratory data	Patients (n = 50)	Controls (n = 50)	Test of sig.	p
ALT (U/L)
Min. – Max.	20.0 – 35.0	22.0 – 35.0	U= 1190.0	0.677
Mean ± SD.	28.36 ± 3.88	27.98 ± 3.61	U= 1190.0	0.677
AST (U/L)
Min. – Max.	25.0 – 73.0	20.0 – 46.0	U= 1055.0	0.178
Mean ± SD.	38.22 ± 13.69	33.18 ± 8.11	U= 1055.0	0.178
Serum albumin (g/dl)
Min. – Max.	1.17 – 3.99	3.50 – 4.90	U= 813.50^*	0.002^*
Mean ± SD.	2.43 ± 0.71	4.15 ± 0.43	U= 813.50^*	0.002^*
Serum Creatinine (mg/dl)
Min. – Max.	0.30 – 1.40	0.20 – 9.0	t= 0.230	0.819
Mean ± SD.	0.70 ± 0.23	0.74 ± 1.21	t= 0.230	0.819
BUN (mg/dl)
Min. – Max.	19.0 – 48.0	8.0 – 27.0	U= 92.000^*	<0.001^*
Mean ± SD.	33.68 ± 8.78	15.66 ± 5.27	U= 92.000^*	<0.001^*
Total protein in urine (mg/day)
Min. – Max.	400.0 – 6200.0	34.0 – 78.0	U= 0.000^*	<0.001^*
Median (IQR)	1850.0 (950.0 – 2500.0)	56.0 (38.0 – 69.0)	U= 0.000^*	<0.001^*
Na (mmol/ L)
Min. – Max.	125.4 – 144.0	136.0 – 144.0	t= 7.357^*	<0.001^*
Mean ± SD.	134.2 ± 4.44	139.6 ± 2.78	t= 7.357^*	<0.001^*
K (mmol/ L)
Min. – Max.	4.30 – 5.40	3.80 – 4.20	t= 24.156^*	<0.001^*
Mean ± SD.	4.93 ± 0.22	4.0 ± 0.16	t= 24.156^*	<0.001^*
Ca (mg/dl)
Min. – Max.	4.90 – 10.10	8.50 – 10.0	t= 11.136^*	<0.001^*
Mean ± SD.	7.19 ± 1.30	9.31 ± 0.33	t= 11.136^*	<0.001^*
Cholesterol (mg/dl)
Min. – Max.	159.0 – 364.0	80.0 – 160.0	t= 24.029^*	<0.001^*
Mean ± SD.	277.86 ± 43.33	119.33 ± 17.29	t= 24.029^*	<0.001^*
Triglyceride (mg/dl)
Min. – Max.	140.0 – 298.40	50.0 – 102.0	t= 19.926^*	<0.001^*
Mean ± SD.	214.94 ± 46.92	78.0 ± 12.64	t= 19.926^*	<0.001^*

IQR: Inter quartile range, SD: Standard deviation

t: Student t-test, U: Mann Whitney test

p: p-value for comparing between the two studied groups

*: Statistically significant at p ≤ 0.05

Table (3): Comparison between the two studied groups regarding RQ (ADCK4) and RQ (H-19)

	Patients (n = 50)	Controls (n = 50)	U	p
RQ (ADCK4)
Min. – Max.	0.01 – 1.86	0.33 – 1.50	206.50^*	<0.001^*
Median (IQR)	0.26 (0.18 – 0.45)	1.0 (0.90 – 1.20)	206.50^*	<0.001^*
RQ (H-19)
Min. – Max.	0.0 – 1.67	0.66 – 1.20	82.0^*	<0.001^*
Median (IQR)	0.44 (0.32 – 0.56)	0.99 (0.90 – 1.0)	82.0^*	<0.001^*

IQR: Inter quartile range, U: Mann Whitney test

p: p value for comparing between the two studied groups

*: Statistically significant at p ≤ 0.05

No competing interests reported.

supplementaryfiles.docx

Down-regulation of Long Non-coding RNA H19 and ADCK4 gene in Children with Nephrotic Syndrome

Status:

Version 1

Abstract

Figures

IMPACT

Introduction

Patient and methods

2.1. Study group:

2.2. Blood sample:

2.3. Statistical analysis of the data

Results

Discussion

Limitation of the Study

Conclusion

Abbreviations

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1