Biallelic variants in NOS3 and GUCY1A3, the two major genes of the nitric oxide pathway, cause moyamoya cerebral angiopathy

Genealogical trees of the six consanguineous sporadic cases are shown in Additional file 1: Figure S1. Four of the consanguineous probands originated from Maghreb (M030, M035, M084 and M101) and two were of Middle-East origin (M038 and M116). All but one were females. Four probands had their first symptoms during infancy or adolescence, including two probands with a very early onset during their first year of life (M035 and M084).

Identification in three of the six probands of rare homozygous coding variants in NOS3 and GUCY1A3

Thirty-four homozygous rare coding non-synonymous variants, belonging to 33 distinct genes, were identified in the 6 consanguineous probands (Table 1 and Additional file 3: Table S1).

Table 1 ES filtering data obtained for the six consanguineous MMA probands

Variants of NOS3, the gene which encodes eNOS, were identified in M035 and M084 probands. It was the only gene to be mutated in more than one of the 6 consanguineous proband analyzed. One of these variants, the c. 1502 + 1G > C substitution identified in M084, was a disruptive variant. The other one, a c.1942 T > C substitution identified in M035, was a missense variant leading to the replacement of the cysteine 648 by an arginine (p.C648R) in the flavodoxine reductase domain of the protein (Fig. 1).

Fig. 1figure 1

Schematic representation of the NOS3 and GUCY1A3 variant identified in M084, M035 and MP038 probands. Upper panels: NM_000603 and NM_000856 transcripts are the canonical transcripts for NOS3 and GUCY1A3 respectively, and are the ones that are referred in the present paper. Three shorter NOS3 transcripts are referenced in Refseq (NM_001160109, NM_001160110 et NM_001160111), and encode for shorter protein isoforms that have been demonstrated to be non-functional [7]. Seventeen additional GUCY1A3 transcripts are referenced in Refseq. Lower panels: Representation of the human eNOS (NP_000594) and the alpha subunit of sGC (NP_000847), that are referred in the present paper [8, 9]. The variants identified in NOS3 and GUCY1A3 in the present article are positioned on their respective transcript and protein. Legend: H-NOX: heme-NO/O2–binding domain; PAS: Per/Arnt/Sim domain, H: helical domain.

GUCY1A3, the gene that encodes a subunit of the sGC enzyme, the major NO receptor in the vascular wall, was found to be mutated at homozygous state in M038 proband. This c.1778G > A substitution (rs370478508) is a missense variant leading to the replacement of arginine 593 by histidine in the catalytic domain of the protein.

All three variants were confirmed by Sanger sequencing.

CADD scores for these three variants are respectively 27, 24 and 35. The two NOS3 variants are absent from ExAC and GnomAD (v3.1.2) public databases and from the Greater Middle East (GME) database (http://igm.ucsd.edu/gme/data-browser.php), that lists variants identified in around 2500 Mediterranean and Middle East controls. Of note, interrogation of the GnomAD v3.1.2 public database found no homozygous NOS3 disruptive variant carrier (i.e., nonsense, splice-site and frameshift insertions or deletions) after removing low quality variants. Cumulative Allele Frequency for heterozygous NOS3 disruptive variants in GnomAD is near of 1/3000, establishing the rarity of such loss-of-function (LOF) variants in the NOS3 gene. The GUCY1A3 rs370478508 variant was absent from ExAC and present in a heterozygous state in only three carriers (2 African/African American and 1 European non-Finnish) in GnomAD (v3.1.2), with an allele frequency of 2 X10−5. It was absent from the GME database.

In the 6 consanguineous probands analyzed, ES data revealed no additional candidate variant neither in RNF213 nor in genes known to be involved in Mendelian forms of MMA.

Functional consequences of the NOS3 c.1502 + 1G > C splice variant

The c.1502 + 1G > C variant (NM_000603) identified in NOS3 in a homozygous state in M084 was predicted to disrupt a canonical splice donor at RNA level (r.spl). Analysis of the cDNA prepared with RNA extracted from circulating blood cells of M084 showed that the c.1502 + 1G > C splice-site variant caused an intron 12 retention and led to a premature termination codon in exon 13 (Figs. 1; 2 Panel A, a et b; Additional file 4: Figure S2 Panel A.). This change in the open reading frame led to a truncated predicted protein (p.Ala502Trpfs*71).

Fig. 2figure 2

The two variants of NOS3 detected in M035 and M084 probands are loss-of-function mutations. Panel A: The c.1502 + 1G > C splice-site variant identified in M084 causes a total loss of eNOS protein. (a) The c.1502 + 1G > C variant disrupts the canonical donor GT splice-site and causes intron 12 retention into the mRNA, resulting in a frameshift and in a premature termination codon in exon 13; Light grey arrows represent the primers used for cDNA amplification. Abbreviations: nt = nucleotides; PTC = premature termination codon. (b) Agarose gel electrophoresis migration of cDNA PCR amplicons from control (lane 2) and M084 (lane 3) using the primers shown in figure A (sequences provided on request). Lane 1: 100 base pairs ladder. The size of the amplicons obtained for M084 cDNA is about 200 nucleotides longer that those got from wild-type cDNA. Sequencing of amplicons showed a 202 nucleotides insert corresponding to the intron 12 retention into the mRNA. Detection of the mutated mRNA in M084 blood cells suggests that this mutant mRNA is spared by nonsense mRNA-mediated decay. (c) Western-blot performed on lysates form EPC derived from M084 proband and controls. Labelling with the monoclonal B-5 antibody directed against the N-terminal part of human and murine eNOS (Santa Cruz) showed a total loss of expression of eNOS in M084 proband (absence of signal on lane 5). Lane 1: NOS3 -/- knock-out mouse. Lane 2: wild-type mouse. Lane 3: EPC from healthy control 1. Lane 4: EPC from healthy control 2. Lane 5: EPC from M084 proband. Lane 6: EPC from healthy control 3. Panel B: The mutated p.C648R eNOS protein (c.1942 T > C variant) is unstable. HEK-293 cells were transfected with vectors containing the WT and mutated M035 full-length NOS3 cDNA. Western-blot performed on lysates from transfected cells showed a strong reduction of eNOS protein amount in three independent clones (lanes 1–3) in comparison to the clone transfected with the wild-type cDNA (lane 4). The primary antibody used is the monoclonal B-5 (Santa-Cruz). Lane 1: c.1942 T > C mutated overexpression clone 1. Lane 2: c.1942 T > C mutated overexpression clone 2. Lane 3: c.1942 T > C mutated overexpression clone 3. Lane 4: wild-type overexpression clone

Western-blot performed with EPC protein lysates from M084 showed that the c.1502 + 1G > C leads to a total lack of the mutant eNOS protein in circulating endothelial progenitor cells. The use of a monoclonal antibody directed against the N-terminal part of the protein allowed to exclude the expression of a truncated protein (Fig. 2 Panel A, c).

Functional consequences of the NOS3 c.1942 T > C (p.C648R) missense variant

The c.1942 T > C substitution of NOS3 identified in a homozygous state in M035 leads to the p.C648R missense variant; this variant is located in the flavodoxine reductase domain of the protein (Fig. 1; Additional file 4: Figure S2 Panel B). As we could not get EPC from M035, we transfected HEK293 cells with a c.1942 T > C mutant cDNA in order to explore the functional consequences of this variant on eNOS stability. Western-blot showed a clear decrease in the eNOS protein amount detected in mutant HEK293 transfected cells (Fig. 2 Panel B), contrasting with a conserved mRNA expression in qRT-PCR experiments (Additional file 5: Table S2). Altogether, these data strongly suggest that the p.C648R mutated protein is unstable.

Structural analysis of the p.C648R mutated eNOS

A multiple sequence alignment in the region of the mutation is shown in Fig. 3A, an overview of the full-length human eNOS AlphaFold2 model is presented in Fig. 3B while in the insert 3 C, the experimental structures of the human oxygenase module and of the predicted human reductase module based on the experimental structure of the rat protein are shown with the same orientation. A zoom in the region of residue C648 is presented in Fig. 3D. The exact orientation of the different domains is still not fully known but residue C648 can be analyzed as it is not directly at the interface with the other domains. C648 is located in the reductase module, in the middle of a β-strand, it is strictly conserved in the sequences (or replaced by a small Serine amino acid that has about the same volume and related properties although the polarity of the oxygen and sulfur atoms are different with an enhanced ability for Serine to form hydrogen bonds, Fig. 3A) and is fully buried. Its relative per-residue solvent accessible surface area (rSASA), for the side chain atoms was computed to be 0% [10]. This residue is in a tightly packed area and surrounded by hydrophobic and aromatic side chains. There is no space to accommodate a long and potentially positively charged arginine residue in this region of the protein. Independently of the rotamer selected, when replacing the cysteine by an arginine residue, severe clashes were noticed that could not be fixed by energy minimization (Fig. 3E). This substitution should thus induce local structural changes and is expected to be destabilizing given the environment and the type of amino acid substitution (i.e., small to large and potentially positively charged at least during some steps of the folding process). Stability predictions (computed with DUET) indicate that the p.C648R substitution should be destabilizing (ΔΔ G = − 1.0 kcal/mol).

Fig. 3figure 3

Structural modeling and in-silico analysis of the effect of missense variants identified in NOS3 and GUCY1A3. A and F: Multiple Sequence Alignments (MSA) showing that the residues C648 of eNOS and R593 of alpha sCG are evolutionary conserved residues. B and C: 3D-structures of the wild type eNOS protein (experimental and 3D models). The C648 residue is located in the reductase domain of the protein. D and E: A zoom in the region of the C648 residue is presented in D (wild-type protein) and E (mutated p.C648R protein). The substitution p.C648R is predicted to destabilize the 3D-structure of the domain (DUET prediction ΔΔG = − 1.0 kcal/mol). There is no space to accommodate the larger and potentially positively charged arginine side chain in the region of the protein (steric clashes are represented by red and green cylinders).G and H: 3D-structures of the wild type sCG protein in an inactive (G) and NO-activated (H) states. sCG is a heterodimer composed of an α-subunit and a β-subunit. The R593 residue mutated in M038 is carried by the α-subunit and is located in the catalytic domain of sCG protein. I and J: A zoom in the region of the R593 residue is presented in I (wild-type inactive state) and J (wild-type NO-activated state). R593 takes part in a salt-bridge network that involves the evolutionary conserved residues E526 (α -subunit), R539 and E473 (β-subunit). E473 interacts with the GTP substrate when the sCG is activated (GMPCPP: GTP binding pocket). The substitution p.R593H is predicted to destabilize the 3D-structure of the inactive (ΔΔ G = -1.5 kcal/mol) and active forms (ΔΔG = − 1.60 kcal/mol) of sGC, through perturbation of non-covalent interactions in the catalytic domain, and negatively impact the formation of the GTP binding pocket

These data strongly suggest that the p.C648R substitution is expected to be destabilizing and to perturb proper folding of this region of the protein. These data, in addition with the instability of the mutated eNOS detected on western blotting experiments, strongly suggest that this p.C648R variant is a loss of function variant.

Structural analysis of the mutated p.R593H sGC alpha1 subunit

The cryo-electron microscopy structures of the human sGCα1β1 heterodimer are available for both the inactive state and the NO-activated state.

The 3D structure of the inactive sGC heterodimer is shown in Fig. 3 G while a close-up view in the area of R593 is presented in Fig. 3I. R593 is located on the α -subunit, in a loop structure and in the catalytic module. It is strictly conserved in the MSA (Fig. 3F) and is buried in the 3D structure; its rSASA was computed to be 13%. R593 is involved in a buried salt-bridge network involving E526 from the α -subunit (strictly conserved, located in a loop structure, rSASA = 28%) which also forms a salt-bridge with R539 from the β-subunit (strictly conserved, located in the N-terminal region of a β-strand, rSASA = 9%), that also forms a salt-bridge with E473 from the β-subunit (strictly conserved, located in a loop, rSASA = 14%). Of importance, the E473 side chain has polar interactions with the GTP substrate once the protein is activated (see below). Interactive amino-acid substitution of R593 α to histidine suggests that the newly introduced side chain could either significantly clashes into E526 α inducing local structural changes and destabilize the salt-bridge network or, alternatively, if some other rotamers are selected, positions the H side chain away from E526α, again destroying part of the salt-bridge network. According to the DUET stability computation, the p.R593H is predicted to be destabilizing (ΔΔG = − 1.50 kcal/mol).

The NO-activated structure is shown in Fig. 3 H and the region of R593 on the α-subunit is seen in Fig. 3J. Some important structural changes are noticed as compared to the inactive form and in the region of R593. For instance, in that region, some residues located in loop structures in the inactive state are now in a β-strand or the opposite and many residues in this region become even more shielded from the solvent. The salt-bridge network discussed above is also significantly modified while the binding pocket for the GTP substrate and cofactor Mg +  + ions is now fully formed. In the NO-activated form, R593 (α-subunit) is located in a loop structure and fully buried (rSASA = 2%). It has favorable hydrophobic-aromatic contacts with part of the side chain of R539 (β-subunit, now located in a loop, rSASA = 3%) and is part of a buried salt-bridge network involving both E526 (α-subunit, located in a loop, rSASA = 11%) and E473 (β-subunit, now located in a β-strand, rSASA = 12%). R539 (β-subunit) forms a salt-bridge with E526 (α-subunit) and E473 (β-subunit) has polar contacts with the GTP substrate. Assuming that an activated form of the mutant protein could still be formed, a histidine at position 593 should perturb the hydrophobic-aromatic-electrostatic interactions as compared to arginine independently of the exact position of the H side chain. According to the DUET stability prediction, the p.R593H in the activated form is predicted to be destabilizing (ΔΔG = − 1.60 kcal/mol).

Altogether, these data strongly suggest that the p.R593H substitution impacts the 3D structure in this region, the dynamics of the protein and its stability and thus affect the flow of information along the transducer module between the sensor module and the catalytic module.

Clinical description of the probands harboring variants in NOS3 and GUCY1A3

Proband M084 is a 31-year-old female born from healthy consanguineous parents (first cousins) originating from Morocco. Her familial and prenatal history were unremarkable. She had normal psychomotor development and graduated from university. From 9 months to 9 years old, she had repeated episodes of transient left hemiparesis especially during hyperventilation when crying. At 8 years of age, cerebral Magnetic Resonance Imaging (MRI) showed two old cortical infarcts in the right posterior cerebral artery (PCA) and middle cerebral artery (MCA) territories. A conventional angiography revealed an occlusion of the right terminal ICA bifurcation, a tight stenosis of the P1-P2 segment of the left PCA, and a long stenosis of the left anterior cerebral artery (ACA). A fine vascular network was observed close to the occluded arteries, thus confirming the diagnosis of MMA (Fig. 4). No abnormality was detected on trans-thoracic echocardiography and aorta CT angiography, and blood pressure was normal. A low-dose aspirin treatment was introduced. At 12 years of age, bilateral ocular hypertension, thin corneas, myopia, and a left homonymous hemianopia related to the posterior cerebral infarct were detected during a routine ophthalmological examination. The diagnosis of juvenile glaucoma was confirmed. After failure of medical treatment, a filtration surgery was finally performed at the age of 14 (both eyes) and 17 (left eye) years old, and the patient was subsequently treated with local ophthalmologic treatments. Thereafter, she remained stable from a neurological and ophthalmological point of view.

Fig. 4figure 4

Imaging features of M035, M084 and M038 probands. A: MRI Fluid-attenuated inversion recovery (FLAIR) images. B: 3D Time-Of-Flight MR angiography (MRA). C and D: Digital subtracted conventional angiography. M084 proband: A: old cortical ischemic lesion (star) in the right MCA territory. B: Absence of right MCA (arrow). C (profile view, early contrast opacification time): Occlusion of the terminal right ICA downstream to the origin of posterior communicating artery (arrow) associated with bilateral deep collateral vascular network (arrowhead). D (profile view, late contrast opacification time): Note the presence of leptomeningeal collaterals from distal branches of the fetal right PCA participating in the blood supply of right MCA territory (dotted circle). M035 proband: A: Ischemic lesions (stars) in the right and left MCA territories; arterial hyperintensities (arrows) suggestive of Ivy sign. B: absence of the terminal segment of both ICAs and of the proximal segment of both MCAs (white arrows), ACAs (thin arrows) and PCAs (black arrows). C (frontal view): Steno-occlusive changes of the terminal ICAs bifurcation (arrows) associated with moyamoya vessels (arrowheads). D (profile view): Occlusion of both PCAs in their proximal segment (arrow), also associated with a collateral vascular network (arrowhead). M038 proband: A: Ischemic lesions (stars) in the right and left MCA territories. B: stenosis of the terminal part of right ICA (arrowhead), occlusion of the proximal segment of right MCA (arrow), and of left ACA (thin arrow). Terminal part of left ICA and proximal part of left MCA are fed by the left posterior communicating artery (black arrow). C (frontal view) and D (profile view): Multistage stenosis of the terminal part of right ICAs (arrows) associated with moyamoya vessels (arrowheads)

Proband M035 was the third child of consanguineous parents (first cousins) originating from Tunisia. No pathological condition was reported in his two older brothers. Prenatal history was notable for intra-uterine growth retardation diagnosed at 27 weeks of gestation and premature delivery threats requiring steroid treatment. He was born late preterm (34 weeks of gestation) and delivery required caesarean section due to abnormal fetal heart rate. Birth weight was 1615 g (5th percentile), birth length 40.5 cm (3rd percentile) and occipito-frontal circumference 29.5 cm (20th percentile). Post-natal infancy was remarkable for neonatal hypoglycemia, short stature (< 3rd percentile) facial and extremities dysmorphism (face: short columella, long philtrum, fingers: spatulate fingers, fetal pads), pigmented skin spots and abdominal patches, cryptorchidism, and psychomotor retardation. At 6 months of age, he presented with a first ischemic stroke revealed by hypotonic seizures during an upper respiratory infection. MRI showed recent ischemic lesions in the left MCA territory and intracranial arterial stenosis. He experienced a second ischemic stroke in the peri-operative period of cryptorchidy surgery (orchidopexia) at the age of 12 months associated with new ischemic lesions on MRI in the left MCA and ACA territories. Conventional angiography showed stenosis of terminal ICA bifurcations and deep neovessels suggestive of a bilateral MMA (Fig. 4). No abnormality was detected on trans-thoracic echocardiography, renal arteries Doppler examination, and ophthalmologic examination. Blood pressure was normal. Because of the syndromic presentation, a large chromosomal rearrangement was ruled out with the completion of a karyotype and a 730 K array analysis.

A cerebral revascularization using indirect techniques (bilateral multicraniostomy) was performed at the age of 17 months. Post-operative period was marked by a lack of transdural collateral development leading to a second targeted indirect surgery when 30-month-old. After cerebral revascularization, the patient presented a total of 6 ischemic strokes, 2 of them prompted by surgery (cryptorchidism) or crying, and 4 of them prompted by viral upper respiratory infections (associated respectively with VRS, HSV, and influenza B). At last examination (6 years old), he presented with asymmetrical bilateral motor deficit, severe oral dyspraxia and mental retardation. He was able to walk but could not speak. MRI performed during follow-up showed worsening of pre-existing arterial steno-occlusive lesions and involvement of posterior circulation on right and left PCAs (Fig. 4).

Proband M038 is a 52-year-old female born from healthy consanguineous parents (first cousins) originating from Turkey. Her familial history was marked by several cases of sudden death of unknown origin on both paternal and maternal sides. Her father died at 63 years after cardiac surgery. The patient developed high blood pressure at 25 years of age, requiring a triple antihypertensive therapy. At 43 of age, she presented a transient numbness of the right leg, followed two months later by a sudden episode of sensitivity disorder on the left hemibody. Cerebral MRI showed recent ischemic lesions in the superficial right MCA territory and in watershed areas of the left hemisphere and old ischemic lesions in the deep right MCA territory. Conventional angiography revealed an imaging pattern suggestive of MMA including steno-occlusive lesions of right intracranial ICA bifurcation associated with a deep right collateral network and an occlusion of the proximal part of left ACA (Fig. 4). Trans-thoracic echocardiography as CSF study didn’t detect any abnormality. Blood tests ruled out dyslipidemia, diabetes, inflammatory syndrome, but showed an isolated persistent IgG anticardiolipin antibody. Aorta CT angiography showed a 80% stenosis of the superior mesenteric artery ostium. A coronary computed tomography angiography was normal. A low dose Aspirin treatment was then started. The following months were marked by several transient episodes of left sensory-motor deficits revealing bilateral new punctiform ischemic lesions in the superficial right and left MCA territories. A treatment by Clopidogrel was then added to Aspirin. One year later, she suffered a transient left hemiparesis related to a hemodynamic transient ischemic attack (TIA) following a vasovagal syncope. Brain MRI and magnetic resonance angiography (MRA) showed a new ischemic lesion in the right ACA territory and a worsening of arterial steno-occlusions. Basal and acetazolamide brain perfusion SPECT with 99mTc-hexamethylpropyleneaminoxime (HMPAO) images showed a misery perfusion pattern in the right MCA and ACA territories. A cerebral revascularization surgery was then performed on the right side. This STA-MCA bypass was complicated by a cerebral hemorrhage related to a reperfusion syndrome associated with elevated blood pressure values leading to a persistent left hemiplegia.

During the following months, she presented with partial seizures which was treated par Levetiracetam and several hemodynamic TIAs. During the 7 following years of follow-up, she had no further cerebrovascular event.

Clinical and radiological features of the three mutated probands are summarized in Table 2, where are also listed the main characteristics observed in the GUCY1A3 mutated MMA patients reported in literature [5, 11].

Table 2 Main clinical and neuroimaging features of the NOS3 and GUCY1A3 mutated probands reported in the present study and in literature

留言 (0)

沒有登入
gif