WO2024223402A1

WO2024223402A1 - Biallelic gene mutations for the diagnosis of neonatal systemic hypertension

Info

Publication number: WO2024223402A1
Application number: PCT/EP2024/060551
Authority: WO
Inventors: Judith Melki; Yline CAPRI
Original assignee: Assistance Publique Hopitaux de Paris APHP; Institut National de la Sante et de la Recherche Medicale INSERM; Universite Paris Saclay
Current assignee: Assistance Publique Hopitaux de Paris APHP; Institut National de la Sante et de la Recherche Medicale INSERM; Universite Paris Saclay
Priority date: 2023-04-24
Filing date: 2024-04-18
Publication date: 2024-10-31
Anticipated expiration: 2025-10-24

Abstract

The present invention relates to a method and kit for identifying a subject having or at risk of having or developing an isolated neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock, comprising determining, in a sample obtained from said subject, the presence or absence of bi-allelic nucleotide variants (NV) located in NPR1 gene, said NV predicted to be pathogenic and being associated with NPR1 loss of function. The present inventors have established a statistical link between specific predicted pathogenic variants located in the NPR1 gene and neonatal systemic hypertension (NSH) in a cohort of NSH disease families. More precisely, the present inventors have established a link between specific predicted biallelic pathogenic variants contained in NPR1 in patients with NSH and/or cardiogenic shock (in multiplex and consanguineous families) associated sometime with increased Nuchal Translucency (NT). Another object of the invention relates to a method for treating neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock.

Description

BIALLELIC GENE MUTATIONS FOR THE DIAGNOSIS OF NEONATAL SYSTEMIC HYPERTENSION

FIELD OF THE INVENTION:

The invention is in the field of neonatal systemic hypertension diagnosis and therapy. In particular, the invention relates to the identification of human NPR1, a new gene in which biallelic loss of function variants are responsible for neonatal isolated systemic hypertension (NSH).

BACKGROUND OF THE INVENTION:

Incidence of neonatal systemic hypertension (NSH) is around l%b There are many causes of NSH including either acquired or congenital causes². The most common renovascular abnormality associated with systemic hypertension in neonates is thrombus formation secondary to umbilical artery catheter placement. Congenital causes of NSH are usually associated with other organ involvement with autosomal dominant or recessive inheritances. Fibromuscular dysplasia resulting in renal arterial stenosis is an important cause of renovascular NSH as well as compression of the renal arteries by tumors. Non-renal intra-abdominal tumors, including neuroblastoma or pheochromocytoma can cause NSH either due to direct compression on renal vessels and/or ureters or due to production of vasoactive substances such as catecholamines. The common cause of NSH is congenital renal abnormalities. Both autosomal dominant and recessive polycystic kidney diseases, tuberous sclerosis and Wilms tumor may present with NSH. Bronchopulmonary dysplasia (BPD) associated NSH is reported with an incidence of 43% of infants with BPD². Coarctation of the aorta may be detected in the neonate, and has been frequently implicated as a cause of NSH. Disorders of the endocrine system, including congenital adrenal hyperplasia, hyperthyroidism, hyperaldosteronism, and Williams-Beuren syndrome may also present with NSH. Diagnostic approaches to systemic hypertension in neonates include careful clinical examination and cardiac, artery, pulmonary, kidney and endocrine investigations. However, isolated NSH remains of unknown origin in many cases.

Therefore, there is a need for identifying genes that provide a more accurate diagnosis/prognosis and more tailored management of isolated neonatal systemic hypertension.

In order to gain insight into the underlying cause of these diseases, inventors took advantage of the added value of whole exome sequencing followed by variant filtering and Sanger sequencing for validation and familial segregation of selected variant in a large multiplex consanguineous family and confirmed their results in another multiplex family.

Here, mutations in new disease gene (NPR1) were identified by the inventors and for the first time it is demonstrate that biallelic loss of function variants of NPR1 are responsible for isolated neonatal onset systemic hypertension in humans, which represents a new recessive autosomal genetic cause of infantile systemic hypertension or cardiogenic shock.

Consequently, the inventors describe identification of a new NSH-causing gene, present in isolated NSH families associated sometime with increased Nuchal Translucency (NT). Screening of this gene should improve the clinical outcome for young members of these families by allowing early detection and appropriate clinical management of NSH from its earliest stages (even at embryo or foetal stage) and genetic counselling.

SUMMARY OF THE INVENTION:

A first object of the invention is a method of identifying a subject having or at risk of having or developing a neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock, comprising determining, in a sample obtained from said subject, the presence or absence of bi-allelic nucleotide variant (NV) located in NPR1 gene, said NV leading to NPR1 loss of function.

In a specific embodiment the bi-allelic nucleotide variants (NV) located in NPR1 gene leads to a marked reduction of NPR1 function either by marked reduction of RNA levels through frameshift, stop gain or spliced variants or marked loss of function of the NPR1 protein as the consequence of missense variants.

In a specific embodiment the bi-allelic nucleotide variants (NV) located in NPR1 gene a homozygous stop gain or frameshift or missense variant.

In a preferred embodiment, the NV is selected from the group consisting of NPR1 gene (NM_000906.4:c.ll59C>T [p.Arg387Ter]) and (NM_000906.4:c.l75del [p.Val59TrpfsTer8]) and

- the presence of biallelic variants (NM_000906.4:c.l l59C>T [p.Arg387Ter]) and/or (NM_000906.4:c.l75del [p.Val59TrpfsTer8]) indicates an increased risk of having or developing a neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock.

A second object of the invention is a kit for identifying whether a subject has or is at risk of having or developing a neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock, comprising: - at least a means for detecting bi-allelic NV located in NPR1 gene associated with NPR1 loss of function and

- instructions for use.

A third object of the invention is a nuclease for use in treating a neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock and/or preventing progression of a neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock in a patient, wherein the presence of nucleotide variants (NV) in NPR1 gene, said NV leading to NPR1 loss of function , in a sample previously obtained from said patient, have been detected by a method of the invention previously described.

Another object of the invention relates to a method for treating neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock with an antihypertensive drug in a subject wherein the presence or absence of bi-allelic nucleotide variants (NV) located in NPR1 gene, said NV leading to NPR1 loss of function, obtained from a sample of said subject, have been detected, by one of the methods of the invention.

DETAILED DESCRIPTION OF THE INVENTION:

Definitions:

Throughout the specification, several terms are employed and are defined in the following paragraphs.

The term “neonatal hypertension (NSH)” is a medical condition which is defined as systolic blood pressure (BP) values persistently above the 95th percentile or BP values over the 99th percentile depending on gestational age. The incidence of neonatal hypertension in the NICU ranges from 0.2% to 3% and most commonly affects term and preterm infants in the intensive care setting. Neonatal Systemic Hypertension characterized (NHS) as an early onset isolated systemic hypertension is a rare condition of unknown genetic origin. Renovascular, renal parenchymal diseases or aortic coarctation are the most common causes of secondary systemic hypertension in younger children and neonates.

Incidence of neonatal systemic hypertension (NSH) is around 1%^J. There are many causes of NSH including either acquired or congenital causes². The most common renovascular abnormality associated with systemic hypertension in neonates is thrombus formation secondary to umbilical artery catheter placement. Congenital causes of NSH are usually associated with other organ involvement with autosomal dominant or recessive inheritances. Fibromuscular dysplasia resulting in renal arterial stenosis is an important cause of renovascular NSH as well as compression of the renal arteries by tumors. Non-renal intra-abdominal tumors, including neuroblastoma or pheochromocytoma can cause NSH either due to direct compression on renal vessels and/or ureters or due to production of vasoactive substances such as catecholamines. The common cause of NSH is congenital renal abnormalities. Both autosomal dominant and recessive polycystic kidney diseases, tuberous sclerosis and Wilms tumor may present with NSH. Bronchopulmonary dysplasia (BPD) associated NSH is reported with an incidence of 43% of infants with BPD². Coarctation of the aorta may be detected in the neonate, and has been frequently implicated as a cause of NSH. Disorders of the endocrine system, including congenital adrenal hyperplasia, hyperthyroidism, hyperaldosteronism, and Williams-Beuren syndrome may also present with NSH. Diagnostic approaches to systemic hypertension in neonates include careful clinical examination and cardiac, artery, pulmonary, kidney and endocrine investigations.

According to the method of the invention “neonatal systemic hypertension” or “NSH” means “neonatal hypertension”.

Accordingly, the method of the present invention encompasses diagnostic method for neonatal systemic hypertension either isolated or associated with cardiogenic shock.

A “cardiogenic shock” is defined as a disease of the cardiovascular system, involving an acute physiological condition caused by the inability of the heart to pump sufficient blood for the needs of the body. Cardiogenic shock remains a highly lethal condition. Conventional therapy including revascularization and mechanical circulatory support aims to improve cardiac output and oxygen delivery, but increasing basic and clinical observations indicate wider circulatory and cellular abnormalities, particularly at the advanced stages of shock. Progressive cardiogenic shock is associated with microcirculatory and cellular abnormalities. Cardiogenic shock is initially characterized by a failure to maintain global oxygen delivery; however, progressive cardiogenic shock is associated with the release of pro - inflammatory cytokines, derangement of the regulation of regional blood flow, microcirculatory abnormalities, and cellular dysoxia. These abnormalities are analogous to septic shock and may not be reversed by increase in oxygen delivery, even to supranormal levels. Earlier mechanical circulatory support in cardiogenic shock may limit the development of microcirculatory and cellular abnormalities (see LIM HS Clin Cardiol. 2016 Aug; 39(8): 477-483.).

NPR1 gene

The term “NPR1” also known as “Natriuretic peptide receptor A/guanylate cyclase A” also knows as ANPa; NPRA; ANPRA; GUC2A; GUCY2A, is an atrionatriuretic peptide receptor A. In humans it is encoded by the NPR1 gene (NM_000906 NP_000897). NPR1 is a membrane-bound guanylate cyclase that serves as the receptor for both atrial and brain natriuretic peptides (ANP and BNP, respectively). NPR1 is localized in the kidney where it results in natriuresis upon binding to natriuretic peptides. However, it is found in even greater quantity in the lungs and adipocytes. Guanylate cyclases, catalyzing the production of cGMP from GTP, are classified as soluble and membrane forms (Garbers and Lowe, 1994). The membrane guanylyl cyclases, often termed guanylyl cyclases A through F, form a family of cell-surface receptors with a similar topographic structure: an extracellular ligand-binding domain, a single membrane-spanning domain, and an intracellular region that contains a protein kinase-like domain and a cyclase catalytic domain. GC-A and GC-B function as receptors for natriuretic peptides; they are also referred to as atrial natriuretic peptide receptor A (NPR1) and type B (NPR2; MIM 108961). Also see NPR3 (MIM 108962), which encodes a protein with only the ligand-binding transmembrane and 37-amino acid cytoplasmic domains. NPR1 is a membrane-bound guanylate cyclase that serves as the receptor for both atrial and brain natriuretic peptides (ANP (MIM 108780) and BNP (MIM 600295), respectively). The whole sequence of human NPR1 gene is referenced as Gene ID: 4881.

"Risk" in the context of the present invention, relates to the probability that an event will occur over a specific time period, as in the conversion to neonatal systemic hypertension (NSH), 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. Alternative continuous measures which may be assessed in the context of the present invention include time to Neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock disease conversion and therapeutic Neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock disease conversion risk reduction ratios.

"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, i.e., from a normal condition to a NSH condition or at risk of developing cardiogenic shock disease; a Neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock disease. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of Neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock diseases, such as cellular population determination in peripheral tissues, in serum or other fluid (i.e.), 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 to Neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock diseases, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk for a Neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock diseases. In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk for Neonatal systemic hypertension (NSH) and/orNSH associated cardiogenic shock diseases. In other embodiments, the present invention may be used so as to help to discriminate those having Neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock diseases from normal.

"Clinical parameters or indicia" encompasses all non-sample or non-analyte biomarkers of subject health status or other characteristics, such as, without limitation, age (Age), geographical origin (Origin), gender (Sex), family history (FamHX), height (HT), weight (WT), waist (Waist) as well as others such as clinical cardinal signs of NSH disease (like systolic blood pressure (BP) with criteria described above), cardiogenic shock, heart failure or increased 1^st trimester nuchal translucency during the pregnancy.

A "sample" in the context of the present invention is a biological sample isolated from a subject and can include, by way of example and not limitation, bodily fluids and/or tissue extracts such as homogenates or solubilized tissue obtained from a subject. Tissue extracts are obtained routinely from tissue biopsy and autopsy material. Bodily fluids useful in the present invention include blood, saliva or in case of fetus, amniotic fluid or chorionic villi or any other bodily secretion or derivative thereof. In a preferred embodiment, the sample to be tested is saliva or blood. As used herein "blood" includes whole blood, circulating epithelial cells, constituents, or any derivative of blood cells.

In a preferred embodiment the sample is a blood sample or amniotic fluid or chorionic villi. According to the invention, NPR1 mutations are genomic variants and are detected by using any type of body cells. In a preferred embodiment the cell is a blood cell, fetal cells from amniotic fluid or chorionic villi cell.

According to the invention, the sample comprises NPR1 nucleic acid, wherein NPR1 nucleic acid is genomic DNA.

A "subject" in the context of the present invention is preferably a human, and more preferably a new-born or neonate human (before 1 year old) or even a foetus.

The term "Allele" has the meaning which is commonly known in the art, that is, a copy of a gene inherited from a parent (one member of a pair) and that is located at a specific position on a specific chromosome which, when translated results in functional or dysfunctional (including non-existent) gene products. Bi-allelic refers to the 2 copies of the same gene, one copy inherited from the father and the other one from the mother.

The term "mutation" or " variant" means a sequence variation of a gene. Allelic variants can be found in the exons, introns, untranslated regions of the gene, or in the sequences that control expression of the gene. Complete gene sequencing often identifies numerous allelic variants (sometimes hundreds) for a given gene. The significance of allelic variants is often unclear until further study of the genotype (including frequency in the general population, prediction of pathogenicity of variants based on several and specific softwares) and corresponding phenotype occurs in a sufficiently large population.

The term " nucleotide variant" or "NV" refers to a type of DNA variation of a base pair or insertions/deletions. There are millions of NVs in the human genome. Most commonly, these variations are found in coding sequences of genes, non-coding regions of genes, or in intergenic regions between genes. When NVs occur within a gene or in a regulatory region near a gene, they may play a more direct role in disease by affecting the gene’s function.

The NV pertaining to the invention are known (known sequences are publicly available from the data base http://www.ncbi.nlm.nih.gov/SNP/). The mutations studied are described here after:

Diagnostic method:

By whole exome sequencing or Sanger sequencing in undiagnosed isolated NSH patients or isolated increased nuchal translucency belonging to 21 multiplex and/or consanguineous, the inventors have found predicted pathogenic variants in a new gene (NPR1). More precisely, the present inventors have identified specific variants contained in the coding sequence of the NPR1 gene, and associated with neonatal systemic hypertension (NSH) in the corresponding patients.

As disclosed in the examples herein, the inventors have screened DNA blood or tissue samples of a well characterized cohort of families with NSH or isolated increased nuchal translucency to assess the genomic effects of single nucleotide variants (SNVs) at different loci. Evidence that NSH is caused by biallelic mutations in NPR1 gene (associated with loss of functions) in two unrelated multiplex families have been provided.

More precisely, the inventors have now identified specific bi-allelic variants located in NPR1 gene, wherein the biallelic variants selected from the group consisting of (NM_000906.4:c.l l59C>T [p.Arg387Ter]) and (NM_000906.4:c.l75del [p.Val59TrpfsTer8], was not only associated with NSH but cause NSH.

Indeed, the inventors show for the first time that biallelic loss of function variants of NPR1 are responsible for isolated neonatal onset systemic hypertension in humans, which represents a new autosomal recessive genetic cause of infantile systemic hypertension or cardiogenic shock. NPR1 gene analysis should be therefore investigated in infants with early onset systemic hypertension with or without cardiogenic shock of unknown origin or isolated increased nuchal translucency. Indeed, in a multiplex family with 4 affected children, increased 1^st trimester nuchal translucency during the pregnancy was observed in 3 of them.

A first object of the invention is a method of identifying a subject having or at risk of having or developing a Neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock disease, comprising determining, in a sample obtained from said subject, the presence or absence of bi-allelic nucleotide variants (NV) located in NPR1 gene, said NV leading to (or being associated with) NPR1 loss of function.

In a specific embodiment the bi-allelic nucleotide variants (SNV) located in NPR1 gene leads to a marked reduction of NPR1 function either by marked reduction of RNA levels through frameshift, stop gain or spliced variants or marked loss of function of the NPR1 protein as the consequence of missense variants.

In a specific embodiment the bi-allelic nucleotide variant (NV) located in NPR1 gene is a homozygous stop gain variant or frameshift variant.

In particular embodiment the bi-allelic NV is selected from the group consisting of NM_000906.4:c.l 159OT [p.Arg387Ter]) and (NM_000906.4:c.l75del [p.Val59TrpfsTer8 ]) and wherein :

- the presence of the allele (NM_000906.4:c.l l59C>T [p.Arg387Ter]) and/or (NM_000906.4:c.l75del [p.Val59TrpfsTer8]) indicates an increased risk of having or developing a neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock.

According to the invention, the presence of the bi-allelic stop variants of NPR1 : NM_000906.4:c.l 159OT indicates a high risk of having or developing Neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock disease. As shown in the examples, homozygous variant NPR1: NM_000906.4:c.l l59C>T was detected in family 1 so significantly increases the risk of NSH compared with controls.

According to the invention, the presence of an homozygous variant of NPR1 : NM_000906.4:c.l75del indicates an high risk of having or developing Neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock disease. As shown in the examples, the homozygous variant ofNPRl : NM_000906.4:c.l75del was detected in family 2 so significantly increases the risk of NSH compared with controls.

In one embodiment of the invention, the method of identifying a subject having or at risk of having or developing a NSH disease and/or NSH associated cardiogenic shock disease, comprising determining the presence or absence of a bi-allelic variants located in NPR1 gene in a blood, amniotic fluid, chorionic villi or tissue sample obtained from said subject.

In another embodiment, said subject may also be one that is asymptomatic for the neonatal systemic hypertension (NSH). As used herein, an “asymptomatic” subject refers to a subject that does not exhibit NSH symptoms (high BP). In asymptomatic case, the monitoring of the patient is recommended.

In another embodiment of the invention, said subject may be one that is at risk of having or developing a Neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock disease, as defined by clinical indicia such as for example: age, gender, clinical marker (see criteria mentioned above), family history of Neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock disease or isolated increased 1^st trimester nuchal translucency during pregnancy.

As shown by the inventors, the identification of a novel gene in this study should now lead to a molecular diagnosis in most of isolated early onset NSH cases.

According to the invention, the determination of the presence or absence of said predicted pathogenic biallelic NVs of NPR1 may be determined by DNA sequencing, PCR analysis or any genotyping method known in the art. Examples of such methods include, but are not limited to, chemical assays such as allele specific hybridation, primer extension, allele specific oligonucleotide ligation, sequencing, enzymatic cleavage, flap endonuclease discrimination; and detection methods such as fluorescence, chemiluminescence, and mass spectrometry.

For example, the presence or absence of said variant may be detected in a DNA sample, preferably after amplification. F or instance, the isolated DNA may be subj ected to amplification by polymerase chain reaction (PCR), using specific oligonucleotide primers that are specific for the NV or that enable amplification of a region flanking the NV or primers able to amplify then sequence each NPR1 exon. According to a first alternative, conditions for primer annealing may be chosen to ensure specific amplification; so that the appearance of an amplification product be a diagnostic of the presence of the SNV according to the invention. Otherwise, DNA may be amplified, after which a mutated site may be detected in the amplified sequence by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art.

Actually numerous strategies for genotype analysis are available (Cooper et al., 1991; Grompe, 1993). Briefly, the nucleic acid molecule may be tested for the presence or absence of a restriction site. When a base polymorphism creates or abolishes the recognition site of a restriction enzyme, this allows a simple direct PCR genotype of the polymorphism. Further strategies include, but are not limited to, direct sequencing, restriction fragment length polymorphism (RFLP) analysis; hybridization with allele-specific oligonucleotides (ASO) that are short synthetic probes which hybridize only to a perfectly matched sequence under suitably stringent hybridization conditions; allele-specific PCR; PCR using mutagenic primers; ligase- PCR, HOT cleavage; denaturing gradient gel electrophoresis (DGGE), temperature denaturing gradient gel electrophoresis (TGGE), single-stranded conformational polymorphism (SSCP) and denaturing high performance liquid chromatography (Kuklin et al., 1997). Direct sequencing may be accomplished by any method, including without limitation chemical sequencing, using the Maxam-Gilbert method ; by enzymatic sequencing, using the Sanger method ; mass spectrometry sequencing ; sequencing using a chip-based technology; and realtime quantitative PCR. Preferably, DNA from a subject is first subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers. However several other methods are available, allowing DNA to be studied independently of PCR, such as the rolling circle amplification (RCA), the Invader TMassay, or oligonucleotide ligation assay (OLA). OLA may be used for revealing base polymorphisms. According to this method, two oligonucleotides are constructed that hybridize to adjacent sequences in the target nucleic acid, with the join sited at the position of the polymorphism. DNA ligase will covalently join the two oligonucleotides only if they are perfectly hybridized to one of the allele.

Therefore, short DNA sequences, in particular oligonucleotide probes or primers, according to the present invention include those which specifically hybridize the regions including the pathogenic variants of the invention .

Oligonucleotide probes or primers may contain at least 10, 15, 20 or 30 nucleotides. Their length may be shorter than 400, 300, 200 or 100 nucleotides.

According to the invention, the determination of the presence or absence of said NVs may also be determined by detection or not of the NPR1 protein(s) (i.e. marked reduction of NPR1 protein or NPR1 protein not detected in the sample) by any method known in the art. The presence of the protein of interest may be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labelled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, etc. The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith. Labels are known in the art that generally provide (either directly or indirectly) a signal. As used herein, the term “labelled” with regard to the antibody or aptamer, is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indocyanine (Cy5), to the antibody or aptamer, as well as indirect labelling of the probe or antibody (e.g., horseradish peroxidise, HRP) by reactivity with a detectable substance. An antibody or aptamer may be also labelled with a radioactive molecule by any method known in the art. For example, radioactive molecules include but are not limited radioactive atom for scintigraphic studies such as 1123, 1124, Ini 11, Rel86 and Rel88. The aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound. Solid supports which may be used in the practice of the invention include substrates such as nitrocellulose (e g., in membrane or microtiter well form); polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, etc.

More particularly, an ELISA method may be used, wherein the wells of a microtiter plate are coated with an antibody against the protein to be tested. A biological sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate (s) can be washed to remove unbound moieties and a detectably labelled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.

Alternatively, an immunohistochemistry (IHC) method may be used. IHC specifically provides a method of detecting a target in a biological sample or tissue specimen in situ. The overall cellular integrity of the sample is maintained in IHC, thus allowing detection of both the presence and location of the target of interest. Typically a biological sample is fixed with formalin, embedded in paraffin and cut into sections for staining and subsequent inspection by light microscopy. Current methods of IHC use either direct labelling or secondary antibodybased or hapten-based labelling. Examples of known IHC systems include, for example, EnVision™ (DakoCytomation), Powervision® (Immunovision, Springdale, AZ), the NBA™ kit (Zymed Laboratories Inc., South San Francisco, CA), HistoFine® (Nichirei Corp, Tokyo, Japan). In particular embodiment, a tissue section (e g. a tissue sample) may be mounted on a slide or other support after incubation with antibodies directed against (the) protein(s) encoded by NPR1 gene(s) with SNVs located at the different exon. Then, microscopic inspections in the sample mounted on a suitable solid support may be performed. For the production of photomicrographs, sections comprising samples may be mounted on a glass slide or other planar support, to highlight by selective staining the presence of the protein of interest. Therefore IHC samples may include, for instance: (a) preparations comprising cell samples (b) fixed and embedded said cells and (c) detecting the protein of interest in said cell samples. In some embodiments, an IHC staining procedure may comprise steps such as: cutting and trimming tissue, fixation, dehydration, paraffin infiltration, cutting in thin sections, mounting onto glass slides, baking, deparaffination, rehydration, antigen retrieval, blocking steps, applying primary antibodies, washing, applying secondary antibodies (optionally coupled to a suitable detectable label), washing, counter staining, and microscopic examination.

Accordingly additional object of the invention relates to an in vitro method for diagnosing of Neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock disease in a subject, said method comprising determining, in a sample obtained from said subject, the presence or absence of nucleotide variants (NVs) located in NPR1 gene said SNV leading to (or being associated with) NPR1 loss of function.

In a specific embodiment the nucleotide variant (NV) located in NPR1 gene leads to a dramatic reduction of NPR1 function either by marked reduction of RNA levels through frameshift, stop gain or spliced variants or marked loss of function of the NPR1 protein as the consequence of missense variants.

In a specific embodiment the nucleotide variant (NV) located in NPR1 gene is a homozygous stop gain variant or frameshift variant or missense variants.

In particular embodiment the NV is selected from the group consisting of NM_000906.4:c.l 159OT [p.Arg387Ter]) and (NM_000906.4:c.l75del [p.Val59TrpfsTer8 ]) and wherein :

- the presence of homozygous variants (NM_000906.4:c.l l59C>T [p.Arg387Ter]) and/or (NM_000906.4:c. l75del [p.Val59TrpfsTer8]) indicates an increased risk of having or developing a neonatal systemic hypertension (NSH) and/orNSH associated cardiogenic shock;.

The “diagnosis” means the identification of the condition or the assessment of the severity of the disease or that the diseases will evolve in worse manner. Kit of the invention

A second object of the invention is a kit for identifying whether a subject has or is at risk of having or developing an Neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock disease, comprising:

- at least a means for detecting bi-allelic nucleotide variant (NV) located in NPR1 gene associated with NPR1 loss of function and

- instructions for use.

In one embodiment of the invention the kit for identifying whether a subject has or is at risk of having or developing an Neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock disease, comprising:

- at least one primer and/or at least one probe for amplification of a sequence comprising a nucleotide variant (NV) located in NPR1 gene associated with NPR1 loss of function and

- instructions for use.

- at least a means for detecting the bi-allelic nucleotide variant (NV) selected from the group consisting of consisting of NM_000906.4:c.l l59C>T [p.Arg387Ter]) and (NM_000906.4:c.l75del [p.Val59TrpfsTer8 ]) and

- instmctions for use.

In one embodiment of the invention, the kit for identifying whether a subject has or is at risk of having or developing an Neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock disease, comprising:

- at least one primer and/or at least one probe for amplification of a sequence comprising a nucleotide variant (NV) consisting of consisting of NM_000906.4:c.l 159OT [p.Arg387Ter]) and (NM_000906.4:c.l75del [p.Val59TrpfsTer8 ]) and

- instructions for use.

In one embodiment of the invention, the primer or probe may be labelled with a suitable marker. In another embodiment of the invention, the primer or probe may be coated on an array.

Example of NPR1 primer is described in Table 2 Therapeutic method

As previously mentioned mutations in new disease gene (NPR1) were identified by the inventors and for the first time it is demonstrate that biallelic loss of function of NPR1 is responsible for isolated neonatal onset systemic hypertension in humans, which represents a new autosomal genetic cause of isolated infantile systemic hypertension or cardiogenic shock.

Accordingly a third object of the present invention is a nuclease for use in treating a neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock diseases and/or preventing progression of a neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock diseases in a patient, wherein the presence of a SNV at NPR1 gene associated with NPR1 loss of function, in a sample previously obtained from said patient, have been detected by a method of the invention previously described.

In a specific embodiment the nucleotide variant (NV) located in NPR1 gene leads to a marked reduction of NPR1 function either by marked reduction of RNA levels through frameshift, stop gain or spliced variants or marked loss of function of the NPR1 protein as the consequence of missense variants.

In a specific embodiment the single nucleotide variant (SNV) located in NPR1 gene is a homozygous stop gain variant or frameshift variant.

In particular embodiment the bi-allelic SNV is selected from the group consisting of NM_000906.4:c.l 159OT [p.Arg387Ter]) and (NM_000906.4:c.l75del [p.Val59TrpfsTer8 ]) and wherein :

A man skilled in the art, know as to design a specific nuclease in order to repair of genetic point mutations like SNVs located at NPR1 gene, namely NM_000906.4:c.l 159OT [p.Arg387Ter]) and (NM_000906.4:c.l75del [p.Val59TrpfsTer8 ]).

The term “nuclease” or “endonuclease” means synthetic nucleases consisting of a DNA binding site, a linker, and a cleavage module derived from a restriction endonuclease which are used for gene targeting efforts. The synthetic nucleases according to the invention exhibit increased preference and specificity to bipartite or tripartite DNA target sites comprising DNA binding (i.e. TALE recognition site(s)) and restriction endonuclease target site while cleaving at off-target sites comprising only the restriction endonuclease target site is prevented.

Restriction endonucleases (also called restriction enzymes) as referred to herein in accordance with the present invention are capable of recognizing and cleaving a DNA molecule at a specific DNA cleavage site between predefined nucleotides. In contrast, some endonucleases such as for example Fokl comprise a cleavage domain that cleaves the DNA unspecifically at a certain position regardless of the nucleotides present at this position. Therefore, preferably the specific DNA cleavage site and the DNA recognition site of the restriction endonuclease are identical. Moreover, also preferably the cleavage domain of the chimeric nuclease is derived from a restriction endonuclease with reduced DNA binding and/or reduced catalytic activity when compared to the wildtype restriction endonuclease.

According to the knowledge that restriction endonucleases, particularly type II restriction endonucleases, bind as a homodimer to DNA regularly, the chimeric nucleases as referred to herein may be related to homodimerization of two restriction endonucleases subunits. Preferably, in accordance with the present invention the cleavage modules referred to herein have a reduced capability of forming homodimers in the absence of the DNA recognition site, thereby preventing unspecific DNA binding. Therefore, a functional homodimer is only formed upon recruitment of chimeric nucleases monomers to the specific DNA recognition sites. Preferably, the restriction endonuclease from which the cleavage module of the chimeric nuclease is derived is a type IIP restriction endonuclease. The preferably palindromic DNA recognition sites of these restriction endonucleases consist of at least four or up to eight contiguous nucleotides. Preferably, the type IIP restriction endonucleases cleave the DNA within the recognition site which occurs rather frequently in the genome, or immediately adjacent thereto, and have no or a reduced star activity. The type IIP restriction endonucleases as referred to herein are preferably selected from the group consisting of Pvull, EcoRV, BamHl, Bcnl, BfaSORF1835P, Bfil, Bgll, Bglll, BpuJl, Bse6341, BsoBl, BspD6I, BstYl, CfrlOl, Ecll8kl, EcoO1091, EcoRl, EcoRll, EcoRV, EcoR1241, EcoR12411, HinPl l, Hindi, Hindlll, Hpy991, Hpyl881, Mspl, Muni, Mval, Nael, NgoMIV, Notl, OkrAl, Pabl, Pad, PspGl, Sau3Al, Sdal, Sfil, SgrAl, Thai, VvuYORF266P, Ddel, Eco571, Haelll, Hhall, Hindll, andNdel.

Other nuclease for use in the present invention are disclosed in WO 2010/079430, WO2011072246, W02013045480, Mussolino C, et al (Curr Opin Biotechnol. 2012 Oct;23(5):644-50) and Papaioannou I. et al (Expert Opinion on Biological Therapy, March 2012, Vol. 12, No. 3 : 329-342) all of which are herein incorporated by reference. Another object of the present invention is a method of treating a neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock disease in a subject comprising the steps of : a) providing a biological sample from a subject, b) detecting in a biological sample obtained at step a) the presence or absence of an allelic variant of a nucleotide variant (NV) located in NPR1 gene, said NV leading to NPR1 loss of function; and if a NVs is detected, treating the subject with an nuclease.

In particular embodiment the SNV is selected from the group consisting of In a specific embodiment the nucleotide variant (NV) located in NPR1 gene leads to a marked reduction of NPR1 function either by marked reduction of RNA levels through frameshift, stop gain or spliced variants or marked loss of function of the NPR1 protein as the consequence of missense variants.

In a specific embodiment the single nucleotide variant (SNV) located in NPR1 gene is a homozygous stop gain variant or frameshift variant or missense variant.

In particular embodiment the SNV is selected from the group consisting of NM_000906.4:c.l 159OT [p.Arg387Ter]) and (NM_000906.4:c.l75del [p.Val59TrpfsTer8 ]) and wherein :

The invention also relates to a method for treating neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock with an antihypertensive drug in a subject wherein the presence or absence of bi-allelic nucleotide variant (NV) located in NPR1 gene, said NV leading to NPR1 loss of function, obtained from a sample of said subject, have been detected, by one of the methods of the invention.

In a specific embodiment the bi-allelic single nucleotide variant (SNV) located in NPR1 gene leads to a dramatic reduction of NPR1 RNA levels.

In a specific embodiment the bi-allelic single nucleotide variant (SNV) located in NPR1 gene is a homozygous stop gain variant or frameshift variant or missense variant.

In a preferred embodiment, the SNV is selected from the group consisting of NPR1 gene (NM_000906.4:c.l l59C>T [p.Arg387Ter]) and (NM_000906.4:c.l75del [p.Val59TrpfsTer8]) - the presence of the allele (NM_000906.4:c.l l59C>T [p.Arg387Ter]) and/or (NM_000906.4:c.l75del [p.Val59TrpfsTer8]) indicates an increased risk of having or developing a neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock;

In the context of the invention, the term "treating" or "treatment", as used herein, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of the disorder or condition to which such term applies.

The term “antihypertensive drug” means a class of a drug that are used to treat hypertension (high blood pressure). Antihypertensive therapy seeks to prevent the complications of high blood pressure, such as stroke, hearth failure, kidney failure and myocardial infarction..

There are many classes of antihypertensives, which lower blood pressure by different means. Among the most important and most widely used medications are thiazide diuretics, calcium channel blockers, ACE inhibitors, angiotensin II receptor antagonists (ARBs), and beta blockers..

Another object of the present invention is a method of treating neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock in a subject comprising the steps of: a) providing a biological sample from a subject, b) detecting in a biological sample obtained at step a) the presence or absence of biallelic nucleotide variant (NV) located in NPR1 gene associated with NPR1 loss of function; and if NVs is detected, treating the subject with an antihypertensive drug.

In a specific embodiment the nucleotide biallelic variants (NV) located in NPR1 gene leads to a marked reduction of NPR1 function either by marked reduction of RNA levels through frameshift, stop gain or spliced variants or marked loss of function of the NPR1 protein as the consequence of missense variant.

In a specific embodiment the biallelic nucleotide variant (NV) located in NPR1 gene is homozygous stop gain or frameshift variants or missense variants predicted to be pathogenic .

In particular embodiment the NV is selected from the group consisting of NM_000906.4:c.l 159OT [p.Arg387Ter]) and (NM_000906.4:c.l75del [p.Val59TrpfsTer8 ]) and wherein : - the presence of the allele (NM_000906.4:c.l l59C>T [p.Arg387Ter]) and/or (NM_000906.4:c.l75del [p.Val59TrpfsTer8]) indicates an increased risk of having or developing a neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock.

Another object of the present invention relates to a method of treating neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a vector which comprises a nucleic acid molecule encoding for a NPR1 polypeptide, wherein the presence or absence of bi-allelic nucleotide variant (NV) located in NPR1 gene, said NV leading to NPR1 loss of function, obtained from a sample of said subject, have been detected, by one of the methods of the invention.

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.

FIGURE:

Figure 1- Homozygous loss of function variants in NPR1 in two families with neonatal onset systemic hypertension and transcript analysis.

(A) Pedigrees with Sanger sequencing results for families 1 (Fl) and 2 (F2) are shown. Arrows indicate mutant nucleotide positions. The affected individuals carry homozygous NPR1 variants. The nucleotide and amino acid changes based on NM_000906.4 and NP_000897.3 reference sequences respectively are indicated. Open symbols: unaffected; filled symbols: affected. (B) Quantitative-RT-PCR analysis (Q-RT-PCR) of NPR1, P-actin and GAPDH was performed from RT of control (Cl) and affected individuals (Fl; 11:1 and 11:2). The ratio of NPR1 to P-actin or GAPDH RNA quantity is markedly reduced in both patients (<13%) when compared to control. In contrast, the ratio of P-actin to GAPDH is similar in control and patients.

EXAMPLE:

METHODS The parents of all affected individuals provided written informed consents for genetic analysis of their children or fetuses and themselves in accordance with the ethical standards of our institutional review boards (CEERB: 2019-035).

Whole exome sequencing

Genomic DNA for each individual was extracted from blood or frozen tissue (lung sample in case of fetal death) with the use of a QiaAmp DNA midi or mini Kit respectively (Qiagen). WES was performed using a completed Twist Bioscience Human Core Exome (Consensus CDS) kit for library preparation and exome enrichment in 10 affected individuals from 6 unrelated families (Integragen). Sequencing was performed on a Genome Analyzer Hiseq4000 Illumina instrument in paired-end mode with a read length of 2x 80bp (Integragen). The median coverage was 80X.

Bioinformatics analysis

Reads were aligned to the human reference genome sequence (UCSC hgl9, NCBI build 37.3) via the BWA program.³ Variants were selected using the SAMtools⁴ then annotated using Annovar softwares.⁵ Variants in coding regions (including non-synonymous and nonsense variants), intron-exon junctions (<10bp) or short coding insertions or deletions were selected when the minor allele frequency (MAF) was less or equal to 0.005 (using 1000G, ExAC, TopMed and GnomAD). Prediction of pathogenicity of missense variants was performed using Polyphen-2 (with score>0.5)⁶ or Sift softwares (with score<0.05)⁷ and splice variants using Human splicing finder.⁸

Sanger sequencing

Selected variants identified through whole exome sequencing were validated by Sanger sequencing. PCR primer pairs were designed from genomic DNA to amplify and sequence each NPR1 exon (Table SI). PCR amplification was carried out as previously described.⁹ PCR products were then purified then sequenced using the forward or reverse primers (Eurofins Genomics). The obtained DNA sequences were compared with published sequences (BLAST, NCBI). Sanger sequencing was also performed to establish the genotype of each family member and to analyze the segregation of the variant within each family. The coding regions of NPR were sequenced in a cohort of 11 additional affected individuals from 10 unrelated families.

Real time PCR amplification of genomic DNA

Real time PCR amplification was conducted using genomic DNA on a CFX96 Touch Real-Time PCR Detection System (BIO-RAD) using the SsoAdvanced Universal SYBR Green Supermix (BIO-RAD). Genomic deletion was defined when the ratio of tested DNA to control DNA was equal to or less than 0.5. Real time PCR amplification of each sample was performed in duplicate using primers of each exon of NPR1 (Table SI). ALB (albumin) was used as internal control (Table SI).

Reverse Transcription-PCR Amplification

Total RNAs were extracted from lymphoblastoid cell lines of two controls (healthy individuals unrelated to affected families, Cl and C2) and two affected individuals from family 1 (II: 1 and 11:2) by using TRI Reagent LS method (Sigma). One hundred ng RNA was used to synthesize cDNA by using random primers following the manufacturer’s manual (SuperScript III reverse Transcriptase, Invitrogen) in a final volume of 20 pL. PCR amplification was carried out as previously described.⁹ PCR amplification analysis from single strand cDNA was performed using primers chosen in exons 3 and 5 of NPRJ gene (Table SI). As internal control for PCR amplification, P-Actin cDNA was coamplified (Table SI). RT-PCR products were separated by agarose gel electrophoresis and labelled with ethidium bromide.

Quantitative- Reverse Transcription-PCR amplification

After RNA extraction from lymphoblastoid cell lines (see above), reverse transcription was performed using 10 ng RNA by using IScript Reverse Transcription Supermix (BIO-RAD) in a final volume of 20 pl. Quantitative RT-PCR amplification was conducted using 1 pl of RT on a CFX96 Touch Real-Time PCR Detection System (BIO-RAD) using the SsoAdvanced Universal SYBR Green Supermix (BIO-RAD). Quantitative RT-PCR amplification of each sample (control Cl, patients II: 1 and 11:2 of Family 1) was performed in duplicate using primers of NPR1 (Table 2), P-actin and GAPDH cDNA (not shown). A standard curve of quantitative RT-PCR was performed from the RT of control sample for the three cDNA targets. Quantitative RT-PCR amplification of P-actin and GAPDH cDNA was used as internal controls for quantification.

RESULTS

In family 1 (Fl), four affected children were born to consanguineous parents from African origin (Figure and Table 1). In the first male fetus (FI JI.1), an increased nuchal translucency (NT) of 3.5 mm was detected at the first-trimester based on ultrasound examination. Karyotype was normal. Echocardiography at 20 weeks of gestation (w.g.) was normal. Ultrasound examinations at the second and third trimester were normal. The child was bom at 39 w.g. with normal weight, size and head circumference. Before postnatal day 10, the child developed polypnea associated with heart failure and systemic hypertension (systolic blood pressure: 128 mmHg, above the 99^th percentile)². Echocardiography, renal ultrasound with doppler and renal angiography were normal. Serum electrolytes, plasma active renin concentration, plasma and urine aldosterone, thyroid hormone levels were normal. For the - l' l - second female fetus (F1.II.2), an increased NT of 3.8 mm was detected at the first-trimester. Ultrasound examination and echocardiography performed at 19, 22, 28 and 33 w.g. detected isolated right ventricular and ventricular septum hypertrophy. Karyotype and 22ql l FISH analysis were normal. The child was born at 39 w.g. Systemic hypertension was detected before postnatal day 5 (systolic blood pressure: 107 mm Hg, above the 99^th percentile). Echocardiography was initially normal and renal ultrasound with doppler were normal. After the age of one year, systolic pulmonary pressure was estimated 40 mmHg at ultrasonography and remained stable during follow up. A right heart catheterization confirmed that pulmonary arterial pressure was 47/17/31 mmHg. For the third female fetus (Fl. II.3), NT was normal (2 mm) at the first trimester as well as ultrasound examinations at 22, 25, 29 and 32 w.g. and echocardiography at 30 w.g. The child was born at 41 w.g. Before postnatal day 10, the newborn developed respiratory distress associated with cardiogenic shock and systemic hypertension (systolic blood pressure: 139 mm Hg, above the 99^th percentile). Echocardiography and renal ultrasound with doppler were normal. For the fourth female fetus (F 1.11.4), an increased NT of 7.4 mm was detected at the first-trimester with normal echocardiography and ultrasound examinations at 14, 16, 22, 27, 32 and 36 w.g. Karyotype, microarray comparative genomic hybridization (aCGH) and RASopathy associated genes analyses were normal. The child was bom at 40 w.g. and systemic hypertension was detected before postnatal day 5 (systolic blood pressure: 108 mm Hg, above the 99^th percentile). Echocardiography detected a patent foramen ovale. Psychomotor development, height and weight growth were normal in the three children aged over 7 years (Fl. Hl; II 2; II.3) and over 1 year for the youngest (Fl .11.4). The mother has no medical history but the father is treated for systemic hypertension discovered after 30 years of age (systolic/diastolic blood pressure: 160/110 mm Hg).

Whole exome sequencing (WES) was performed in three out of the four affected children of family 1 (11:2, 11:3 and 11:4). Predicted deleterious variants in genes shared by the three affected individuals were selected as candidates. An homozygous variant in NPRJ gene was selected as the best candidate. It is an homozygous stop gained variant located in exon 4 of NPR1 gene (NM_000906.4:c.ll59C>T; NP_000897.3:p.Arg387Ter). This variant was annotated in dbSNP155 (rsl313788512) with a very low MAF of 0.000004 (TOPMED) and 0.000007 (GnomAD). In addition, based on WES data of the three affected individuals, this variant is located within a homozygous region of 11 Mb from genomic position chrl : 145826991 to 156923966 (GRCh38[hg38]) indicating a large region of homozygosity by descent (data not shown, available on request). The variant was confirmed through Sanger sequencing of PCR products using primers flanking the variant (Figure, Table SI). The variant was found homozygous in the four affected children and heterozygous in both parents (Figure). Reverse Transcript! on-PCR amplification (RT-PCR) products of NPR1 cDNA from lymphoblastoid cells RNA of two affected individuals (family 1 : 11: 1 and 11:2) compared to unrelated healthy controls (Cl, C2) revealed a dramatic reduction of NPR1 RNA levels in affected individuals compared to controls and P-actin expression (Figure, Table SI). To quantify the effect of the homozygous NPRJ stop gained variant on RNA expression, quantitative-RT-PCR analysis of NPR1, P-actin and GAPDH was performed from RT of control (Cl) and affected individuals 11:1 and 11:2 of Family 1. Based on the standard curves from control, the ratio of NPR1 to P-actin or GAPDH RNA expression was found to be markedly reduced in both patients (<13%) indicating a dramatic reduction of NPR1 RNA levels (Figure). The ratio of P-actin to GAPDH was similar in control and patients 11: 1 and 11:2 (Figure). These results demonstrate the deleterious effect of NPRJ homozygous stop gained variant on RNA stability. As the father developed systemic hypertension during adulthood, Sanger sequencing and quantitative PCR of the coding regions of NPRJ were performed on the father’s DNA to determine whether an allelic variant in NPRJ was present. No variant other than the heterozygous stop gain variant (NM_000906.4:c.l 159C>T) was identified (data not shown).

NPRJ was therefore selected as high candidate for the early onset systemic hypertension-causing gene. Importantly, in three out of the four affected children of family 1, increased nuchal translucency was observed at the first ultrasound examination between 11 and 14 w.g. For this reason, NPRJ genetic analysis included affected individuals with either isolated increased fetal nuchal translucency thickness with normal karyotype, aCGH and RASopathy associated genes or early onset isolated systemic hypertension. Whole exome sequencing or Sanger sequencing and quantitative PCR of the coding regions of NPRJ (Table SI) were performed on the DNA samples of a cohort of 15 additional unrelated families with either increased fetal nuchal translucency thickness (n=9) or childhood onset isolated systemic hypertension (n=6) using the same criteria for variant selection. In two out of 6 families, systemic hypertension was detected before 1 month of age and in 4 families, systemic hypertension was observed from 1 month to 7 years of age.

This approach allowed the identification of an additional family (family 2 [F2], Figure and Table 1). In this family, two affected children were born to parents from the same geographic origin of Africa. In the first male fetus (F2.II.1), the pregnancy was reported as normal. The child was born at 38 w.g. with normal weight, size and head circumference. Before postnatal day 15, the newborn developed cardiogenic shock. Echocardiography revealed left ventricular hypertrophy. He died before postnatal day 15 right after admission in the intensive care unit. Metabolic defect or infectious diseases were excluded. For the second female fetus (F2.II.2), the pregnancy was reported as normal including NT at the first trimester (1.4 mm). The child was born at 39 w.g. with low weight (2500 gr), but normal size and head circumference. Before postnatal day 20, she developed cardiogenic shock and systemic hypertension (systolic blood pressure: 116 mm Hg, above the 99^th percentile). Echocardiography revealed left ventricular myocardial dysfunction secondary to systemic hypertension. Renal ultrasound with Doppler and renal angiography were normal. aCGH and thyroid hormone, plasma renin and aldosterone levels were normal. Sanger sequencing of the coding regions of NPR1 identified homozygous frameshift variant in exon 1 of NPR1 in both affected children (Figure). This variant (NM_000906.4:c.l75del; p.Val59TrpfsTer8) was not annotated in all available databases and leads to a frameshift and premature stop codon. Lymphoblastoid cell lines were not available in the affected children of the second family. This frameshift variant was inherited from healthy parents who were both heterozygous for the variant. Their arterial blood pressures were normal.

DISCUSSION

We report herein the identification of biallelic NPR loss of function variants in severe isolated neonatal systemic hypertension in two unrelated multiplex families indicating that biallelic NPR mutations are responsible for this condition. Our data indicate an autosomal recessive mode of inheritance of this condition. Importantly, cardiogenic shock or heart failure was observed in four out of the six affected individuals (Table 1). In the first family, we showed that the homozygous stop gained variant in NPRJ gene leads to a dramatic reduction of NPR1 transcripts. NPR1 is a member of the guanylate cyclase family of receptors, which produce cGMP in response to ligand binding. NPR1 elevates intracellular levels of cGMP when the atrial natriuretic peptide (ANP) or brain natriuretic peptide (BNP) binds to the extracellular domain of the receptor and allosterically activates its guanylate cyclase catalytic domain. This binding induces an increase in intracellular cGMP and initiates natriuresis, diuresis, and vasodilation, all of which contribute to lowering blood pressure.^10,11 Disruption of the Nprl gene in mice leads to chronic elevations of blood pressure (by 35-45 mmHg as compared with wild-type mice), marked cardiac hypertrophy, ventricular enlargement and sudden death in homozygous mouse mutants.¹² The hearts, kidneys and vasculature of the homozygous mouse mutants (aged less than 5 months) were normal when examined by histological methods. Systemic hypertension was accompanied by marked cardiac hypertrophy and ventricular enlargement with no evidence of myocardial dysfunction.¹³ In addition, Nprl expression affects the sensitivity of blood pressure to dietary salt. Indeed, heterozygous knock out mice have saltsensitive hypertension compared to wild type mice.^{14, 15}

Increased 1^st trimester nuchal translucency during the pregnancy was observed in 3 of the 4 affected children of family 1. Interestingly, a slight reduction in the expected number of homozygous Nprl knockout mutant mice was statistically significant and is related to fetal hydrops observed in approximately 10% of homozygous embryos.¹² Therefore, increased 1^st trimester nuchal translucency is likely another consequence of biallelic NPRJ loss of function. Even if this symptom is not constant as for other diseases associated with increased nuchal translucency such as trisomy 21, Turner or Noonan syndromes, arterial pressure should be carefully followed in postnatal period.

In addition, cardiogenic shock or heart failure were observed in 4 out of the 6 affected children carrying pathogenic variants in NPRJ. The severity of the phenotype of the affected children was also reported in homozygous Nprl knock out mice with sudden death¹² and indicates a major role of NPR1 in regulating blood pressure in humans.

In human, genome-wide association studies, exome studies or direct sequencing of the NPRJ gene were performed in large cohorts of patients with systemic hypertension and revealed some variants with high MAF in the untranslated region of NPRJ associated with higher blood pressure.¹⁶'²⁰ However, these analyses were performed in patients with onset of systemic hypertension after 15 years of age while in the two families reported here, systemic hypertension was detected within the first month of age.

Our data should lead to analyze NPRJ gene in neonatal isolated systemic hypertension or cardiogenic shock of unknown origin. In addition, in isolated increased nuchal translucency with normal karyotype, aCGH and RASopathy associated genes, arterial pressure should be carefully followed in postnatal period and systemic hypertension should lead to NPRJ genetic investigation. The benefits of an accurate genetic diagnosis include more tailored management of systemic hypertension, heart involvement and improved surveillance.^{2, 21} A precise genetic diagnosis enables an accurate genetic information to the affected individuals and their family members.

Table 2

REFERENCES:

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12 Lopez MJ, Wong SK, Kishimoto I, et al. Salt-resistant hypertension in mice lacking the guanylyl cyclase-A receptor for atrial natriuretic peptide. Nature 1995;378:65-8.

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15 Pandey KN. Genetic Ablation and Guanylyl Cyclase/Natriuretic Peptide Receptor- A: Impact on the Pathophysiology of Cardiovascular Dysfunction. Int J Mol Sci 2019;20:3946. 16 Pitzalis MV, Sarzani R, Dessi-Fulgheri P, et al. Allelic variants of natriuretic peptide receptor genes are associated with family history of hypertension and cardiovascular phenotype. J Hypertens 2003;21 :1491-6.

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Genetic Variants in the Natriuretic Peptide Receptor 1 Gene Modulate Guanylate Cyclase Activity. Circ Genom Precis Med 2019;12:e002472.

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Claims

CLAIMS;

1. A method of identifying a subject having or at risk of having or developing a neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock, comprising determining, in a sample obtained from said subject, the presence or absence ofbi-allelic nucleotide variant (NV) located in NPR1 gene, said NV leading to NPR1 loss of function.

2. The method according to claim 1, wherein the bi-allelic NV located in NPR1 gene is a homozygous stop gain or frameshift or missense variant.

3. The method according to claim 2, wherein the bi-allelic NV located in NPR1 gene is selected from the group consisting of NM_000906.4:c.l l59C>T [p.Arg387Ter] and NM_000906.4:c.l75del [p.Val59TrpfsTer8] and

4. The method according to anyone of claim 1 to 3, wherein the sample is a blood, amniotic fluid or chorionic villi sample.

5. The method according to anyone of claim 1 to 4, wherein the presence or absence of said SNV is determined by nucleic acid sequencing or by PCR analysis.

6. A kit for identifying whether a subject has or is at risk of having or developing a neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock, comprising :

- at least a means for detecting the bi-allelic nucleotide variant (NV) located in NPR1 genes associated with NPR1 loss of function and

- instructions for use.

7. A kit according to claim 6, comprising :

- - at least a means for detecting the bi-allelic nucleotide variant (NV) selected from the group consisting of consisting of NM_000906.4:c.l l59C>T [p.Arg387Ter]) and/or NM_000906.4:c.l75del [p.Val59TrpfsTer8 ] and

- instructions for use.

8. A kit according to claim 7, comprising:

- at least one primer and/or at least one probe for amplification of a sequence comprising a SNV consisting of consisting of NM_000906.4:c.l l59C>T [p.Arg387Ter]) and (NM_000906.4:c.175del [p.Val59TrpfsTer8 ]) and

- instructions for use.

9. A nuclease for use in treating a neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock and/or preventing progression of a neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock in a patient, wherein the presence of bi-allelic nucleotide variant (NV) in NPR1 gene, said NV leading to NPR1 loss of function, in a sample previously obtained from said patient, have been detected by a method according to anyone of claim 1 to 5.

10. A nuclease for use according to claim 9, wherein the SNV is selected from the group consisting of NM_000906.4:c.l l59C>T [p.Arg387Ter] and NM_000906.4:c.l75del [p.Val59TrpfsTer8] and wherein :

- the presence of the allele (NM_000906.4:c.l l59C>T [p.Arg387Ter]) and/or (NM_000906.4:c.175del [p.Val59TrpfsTer8]) indicates an increased risk of having or developing a neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock.

11. A method for treating neonatal systemic hypertension (NSH) and/or NSH associated cardiogenic shock with an antihypertensive drug in a subject wherein the presence or absence of bi-allelic nucleotide variant (NV) located in NPR1 gene, said NV leading to NPR1 loss of function, obtained from a sample of said subject, have been detected, by the methods of to anyone of claim 1 to 5.