The hyperornithinemia–hyperammonemia-homocitrullinuria syndrome

The biochemical role of ORC1 is complex and highly relevant for the different tissues where it is expressed. ORC1 transports ornithine, lysine and arginine into the mitochondrial matrix of peripheral tissues and pericentral hepatocytes; in periportal hepatocytes, in which UC enzymes are expressed, it catalyzes a very efficient ornithine/citrulline exchange reaction [65], connecting the enzyme activities of urea synthesis in the cytosol to those in the mitochondria. ORC1 plays therefore a key role in the UC (Figure 3). ORC1 catalyzes the transport of the L-isomers of ornithine, citrulline, lysine and arginine by a 1:1 substrate exchange reaction and less efficiently exchanges a basic amino acid for an H+ [65-67]. Two human isoforms of the mitochondrial ornithine carrier, ORC1 and ORC2, have been identified so far. Despite having a high sequence identity (87%) with ORC1, ORC2 is less active, presents a lower affinity for ornithine and citrulline, and shows a broader substrate specificity because of its capability to transport histidine and homocitrulline as well as the D-isomers of ornithine, lysine and arginine [68]. Both isoforms are mainly expressed in liver, pancreas, lungs, and testis, although ORC2 to a much lesser extent than ORC1 in all tissues investigated [68]. The total mitochondrial ornithine/citrulline exchange activity per whole organ in vivo is unknown; it has been suggested that the late onset and the variable clinical phenotype of HHH syndrome may be due to the redundancy of this exchange activity [8,37]. This is catalyzed either by ORC2 [68] or by the SLC25A29 gene product (previously reported to be a mitochondrial carnitine/acylcarnitine- or ornithine-like carrier called ORNT3 [69]), which is able to rescue the ornithine metabolism deficiency in fibroblasts of HHH patients [69,70] and to transport basic amino acids as well as ornithine into proteoliposomes [71]. The residual ornithine transport in cultured fibroblasts and liver of affected individuals supports this hypothesis of gene redundancy in HHH syndrome [8,37].

ORC1 deficiency reduces the mitochondrial availability of ornithine, which increases in the cytosol causing hyperornithinemia. The accumulation of ornithine in cytosol leads also to increased levels of polyamines as spermine and spermidine [72]. In the periportal zone of the liver lobuli, the lower level of mitochondrial ornithine reduces the UC rate since the matrix ornithine is required by the ornithine transcarbamylase (OTC) to prime the UC. Ammonia and carbamyl-phosphate (CP) levels increase, explaining hyperammonemia. Increased CP may have two destinations: it may bind lysine, forming homocitrulline which gives rise to homocitrullinuria or enter in the pyrimidine pathway, leading to increased excretion of orotic acid in urine (Figure 3). Plasma glutamine is taken up from the systemic circulation into the gut and then processed to glutamate, pyrroline-5-carboxylate, ornithine and citrulline. As the liver extracts only a small quantity of citrulline, this aminoacid is mainly transported to the kidney and metabolized to arginine in the tubules of the cortical zone and used for guanidino acetate synthesis by arginine-glycine amidotransferase (AGAT), the first step of creatine synthesis, with ornithine as by-product [73]. In ORC1 deficiency, the high cytosolic ornithine concentrations inhibit AGAT leading to secondary creatine deficiency [21,44,74]. Secondary creatine deficiency may be also due to low cellular arginine availability due to the block in UC flow. Moreover, ornithine excess may inhibit creatine biosynthesis as observed in ornithine delta-aminotransferase (OAT) deficiency [75,76] (Figure 3). This is in line with a previous observation of two patients with HHH syndrome, in whom urinary creatine excretion was normalized by citrulline and arginine but not by ornithine supplementation [21].

Since ORC1 is highly expressed in the brain and in particular in astrocytes, one could speculate that an 7absent or dysfunctional protein may affects the metabolism of neurons and of glial cells. Hyperammonemia has a toxic effect on the astrocytes, causing mitochondrial dysfunction, cellular swelling and a change in cellular bioenergetics [77]. Moreover, Braissant et al. [78] have shown through in vitro experiments with reaggregated mixed brain cells (neurons, astrocytes, oligodendrocytes and microglia) primary cultures, impaired axonal growth and abnormal localization and phosphorylation of the intermediate neurofilament M-protein after ammonia exposure. Addition of creatine to the media seems to protect against this effect of ammonia on the neural cytoskeleton; this protective effect depends on the presence of glial cells and cannot be observed in neuron enriched cell cultures (78). These findings suggest that in ORC1 deficiency AGAT inhibition due to high ornithine levels reduces the endogenous creatine production in cerebrum and cerebellum and thus renders the brain more vulnerable to a local increase of ammonia [78].

Recently, Viegas et al. have shown that excessive ornithine and homocitrulline levels can cause protein and lipid oxidation and may negatively interfere in oxidative phosphorylation and Krebs cycle function of the rat brain, with secondary oxidative stress [79,80]. Impairment of brain bioenergetics and the oxidative damage induced by these metabolites may possibly contribute to the neurological symptoms affecting patients with HHH syndrome.

HHH syndrome is a genetic autosomal recessive disease caused by mutations in the SLC25A15 (solute carrier family 25, member 15) gene [37]. This gene maps on chromosome 13q14.11, spans about 23 kb and contains 7 exons encoding for the isoform 1 of the ornithine carrier ORC1, a member of the mitochondrial carrier family [65]. The open reading frame is encoded by exons 2 through 7 [53]. Exon 1 encodes part of the 5’UTR. The normal product of SLC25A15 gene is a 301 amino acid protein composed, like other mitochondrial carriers, of six α-helices that traverse the inner mitochondrial membrane with the C- and N-termini exposed to the cytosolic side of the membrane. ORC1 shows a tripartite structure with three similar domains each with two transmembrane helices connected by a long hydrophilic matrix loop [65]. There is experimental evidence for the direct involvement of the ORC1 residues E77, R179 and E180 in substrate binding, whereas W224 and R275 seem to be important in triggering the substrate-induced conformational changes that leads to substrate translocation; N74 and N78 residues are part of the substrate binding pocket [81] (Figure 4).

Several SLC25A15 mutations have been associated with the clinical phenotype of the HHH syndrome. From 1999 to 2014, 35 mutations have been identified: 18 missense mutations, 7 small insertion, 2 small deletions, 4 nonsense mutations, 1 gross deletion, 1 micro-rearrangement, 1 intronic rearrangement (Table 1). All mutations occurred in the coding region. Two common mutations, p.F188del and p.R179*, are reported. The first accounts for about 30% of patients with the HHH syndrome and it is characteristic (but not exclusive) of patients of French-Canadian descent because of a founder effect [38]. The second mutation (15% of HHH patients) appears to be prevalent in patients of Japanese and Middle Eastern origin [27,47,57].

The functional effects of some SLC25A15 mutations on substrate transport have been investigated using in vitro cell culture and liposome reconstitution studies [53,57,68], revealing that some SLC25A15 missense and nonsense mutations (p.T32R, p.F188del, p.G190D, p.M273K, p.T272I, p.G113C, p.L71Q and p.L283F) cause reduced transport activity while others (p.G220R, p.R179*, p.G27R, p.R275Q and R275*) completely abolish the function. Interestingly, some patients with null SLC25A15 alleles did not show neonatal hyperammonemia [57].

The 18 pathogenic missense mutations alter residues phylogenetically conserved in the wild-type protein, which may reflect that they have specific important roles for function. However, the majority of the single-residue mutations causing the HHH syndrome are non-conservative substitutions that in many cases introduce a change in the charge of amino acid side chain (p.A15E, p.G27R, p.G27E, p.T32R, p.M37R, p.P126R, p.E180K, p.G190D, p.G220R, p.M273K, p.R275G and p.R275Q) [22,37,57], prolines (p.L193P and p.A264P) or residues with different side chain size or polarity (p.A15V, p.G113C, p.G216S, p.T272I, p.L71Q, p.F188L and p.L283F) [57,60], that alter the protein structure and function. Missense mutations can be mapped onto the 3D structural homology model of ORC1 [81], which is based on the structure of the carboxyatractyloside inhibited bovine ADP/ATP carrier (PDB ID 1OKC), to display the location of each mutation (Figure 4). In a simple analysis, the mutations may be divided into three groups depending on their location in the model: residues close to the proposed binding site, residues close to the substrate entrance/exit on the cytosolic side and residues close to the substrate exit/entrance on the matrix side. The majority of the HHH syndrome mutations are found in residues that have side chains protruding into the internal pore of ORC1 where the substrate is translocated, suggesting that these mutations could interfere with substrate translocation. Mutations located in residues of the substrate binding site p.E180K, p.R275G and p.R275Q are presumably directly deleterious to substrate binding and other mutations in proximity, such as p.G27R, p.G27E, p.T272I and p.G113C, probably have an effect in deforming the substrate-binding pocket. Mutations on the cytosolic side, such as p.F188L, p.G190D, p.G216S and p.G220R, could interfere with the substrate entering or exiting its binding site, or with the closing/opening of the substrate-binding cavity towards the cytosol. A similar explanation could be given for the effect of p.T32R and p.A264P mutations that interfere with the closing/opening of the substrate-binding cavity but on the matrix side. The residues A15, M37, L71, P126, L193 and M273 have side chains on the external surface of ORC1 and therefore these mutations cannot directly obstruct substrate translocation. Instead these mutations may indirectly impair function, probably by causing local perturbations in the ORC1 structure, e.g., the proline of the p.P126R mutation is located in one of the signature motifs that are highly conserved in all mitochondrial carriers and is likely to be crucial for the structure and conformational changes during substrate translocation [72].

The metabolic triad of hyperammonemia, hyperornithinemia, and urinary excretion of homocitrulline establishes the diagnosis of HHH syndrome. However, some patients may present with an incomplete biochemical phenotype. The biochemical data in individuals with HHH syndrome at the time of diagnosis are summarized in Table 2 and in Additional file 1: Table S1. Of note, HHH syndrome is characterized by a lower degree of hyperammonemia if compared with other UCDs [1] and plasma ammonia level usually normalizes in response to pharmacological treatment.

Plasma concentration of ornithine can range from 216 to 1915 μmol/L (normal: 30–110 μmol/L). Despite protein-restricted diet in combination with pharmacological treatment, ornithine concentration in plasma remains elevated with only a very few patients reported with normal levels at long-term follow-up [2-4,15,48,56].

Homocitrullinuria is a hallmark of the disease, however some patients may show absent or only minimal excretion of homocitrulline in urine [50] (Additional file 1: Table S1).

Similarly to other UCDs [1], plasma glutamine concentrations and urinary orotic acid may be elevated (Additional file 1: Table S1). Occasional organic aciduria, with increase excretion of Krebs Cycle intermediates (succinate, citrate, fumaric, α-ketoglutaric) and lactate was reported [50,52].

The diagnosis of HHH syndrome may be confirmed by the evaluation of cellular mitochondrial transport of radiolabelled ¹⁴C-ornithine in cultured skin fibroblasts [9]. Cultured fibroblasts from patients with null mutations usually show an approximately 75%-80% reduction in ornithine transport: this may suggest a potential role of redundant transporters in achieving a residual transport [37,69]. There is no correlation between ornithine transport capacity, genotype and phenotype [37,53].

As detected by computer tomography (CT) scan and magnetic resonance (MRI) studies, brain abnormalities include mild cerebral and cerebellar atrophy [16,23,31,27,57], white matter changes [4,57,59], subdural hematoma [C. Dionisi-Vici, personal observation], cystic lesions and calcifications [4], and diffuse brain edema, evident at ultrasound scan [35]. However, normal brain CT scan or MRI findings were reported in several patients, mainly in those who did not experience an overt hyperammonemic coma [36,40,60]. Interestingly, multiple stroke-like lesions were reported in a 4 year-old patient presenting irritability, vomiting, highly elevated liver enzymes, hyperammonemia, and coagulopathy [56].

Neurophysiologic studies show abnormal motor evoked potentials affecting pyramidal tract of lower limbs, generally without upper limb involvement [24,36]. Somatosensory evoked potentials are normal; electroneuromyography can show abnormal nerve conduction velocity [24,36,37,51] and may reveal signs of spinal anterior horn cells degeneration [34].

Blood coagulation studies may be abnormal with deficiency of factor VII, X, XI, and antithrombin III [7,15,21,38,39,55-57].

Liver structural (light microscopy) and ultrastructural changes (electron microscopy) are commonly observed [7,9,26,40,57,82] and include vacuolated hepatocytes with intracytoplasmic glycogen deposition, small nuclei, dense chromatin, and fat droplets without fibrosis. At electron microscopy, mitochondria appear abnormally shaped and sized with lamellar cristal-like inclusions [11].

HHH syndrome enters in the differential diagnosis of any patient presenting with hyperammonemia at any age. The main causes of hyperammonemia associated with inborn errors of metabolism include [1,83]: UCDs (they generally present along with elevation in plasma ammonia concentration, hyperglutaminemia and metabolic alkalosis); organic acidemias (they show in addition metabolic acidosis, ketonuria and normal or reduced plasma glutamine levels); fatty-acid oxidation defects (hyperammonemia is associated with hypoglycaemia, hypertransaminasemia, increase of plasma creatine-phosphokinase levels); lysinuric protein intolerance (characterized by low concentrations of plasma ornithine, lysine, and arginine and persistent urinary excretion of lysine, ornithine, and arginine); hyperinsulinism-hyperammonemia syndrome (characterized by severe hypoglycaemia); pyruvate carboxylase deficiency (presenting with lactic acidosis and hypoglycaemia); and the recently reported defect of mitochondrial carbonic anhydrase VA (presenting with lactic acidosis, hypoglycaemia and a characteristic organic aciduria) [84].

A complete routine chemistry panel [i.e. blood ammonia, glucose, lactate, liver function tests (including transaminases, gamma-GT, coagulation, bilirubin, albumin) creatine kinase, uric acid, arterial blood gases, and blood cell counts, urinalysis], and metabolic investigations with plasma amino acids, urine amino acids,organic acids and orotic acid measurements allow the differential diagnosis.

Hyperornithinemia is also a characteristic feature of OAT deficiency responsible for gyrate atrophy of the choroid and retina (OMIM #258870) [85]. This disorder is caused by defect in OAT gene, located at 10q26. The fasting plasma ornithine in OAT deficiency ranges from 400 to 1400 μmol/L [85]. Initial symptoms include myopia and night blindness, starting in early to mid-childhood; patients then develop reduction of visual fields, posterior subcapsular cataracts and abnormal electroretinogram [85]. The fundoscopic aspect of the chorioretinal atrophy in gyrate atrophy is highly specific. Patients have generally a normal cognitive level, although one large series suggests an increased incidence of intellectual impairment [76]. A few OAT patients experienced neonatal hyperammonemia [86]. Interestingly, and similarly to some HHH patients, they were all found to have normal plasma ornithine concentration at birth which progressively increased in the following weeks when the diagnosis was fully defined [86].

Other conditions in which homocitrullinuria can be observed should be listed in the differential diagnosis of HHH syndrome. These include lysinuric protein intolerance (MIM# 222700) or OTC deficiency (MIM# 311250), which can present in some cases with low quantity of homocitrulline in urine [87,88]. Furthermore, canned food or heated milk products may represent a source of detectable homocitrulline in urine [88].

In patients with early childhood onset of gait abnormalities and spasticity, the differential diagnosis includes cerebral palsies and inherited spastic paraplegias. Interestingly among the inherited disorders of the UC, HHH syndrome and argininemia share the peculiar neurological feature of pyramidal dysfunction [1,88,89].