Update on Lysinuric Protein Intolerance, a Multi-faceted Disease Retrospective cohort analysis from birth to adulthood

Four systems have been described for cationic amino-acids transport through plasma membrane in mammals: y⁺, b^0,+, B^0,+ and y⁺L. These systems differ in terms of specificity for amino-acids, tissue localisation and dependency on sodium [10]. The y⁺LAT-1 and 4F2hc subunits heterodimerise to form the part of the y⁺L system that is responsible for the exchange of cationic amino-acids for neutral amino-acids plus sodium at the basolateral plasma membrane of polarised cells, such as epithelial cells of the proximal renal tubules, small bowel, lung and leukocytes. Only mutations in the SLC7A7 gene encoding the y⁺LAT-1 subunit have been identified in LPI patients, but no mutation has been reported in SLC3A2 encoding 4F2hc. Interestingly, cystinuria (OMIM 220100) is also caused by defects in cationic amino-acids transport in the b^0,+system [13‐15].

Initially described by Perheetupa and Visakorpi in 1965, LPI has been reported worldwide [15]. There is a higher prevalence in Finland reaching 1/60 000. However, LPI has also been documented in Japan, Turkey, Italy and Maghreb [3, 4, 12, 16]. As expected with an autosomal recessive disease, inbreeding was important in our cohort.

Since the first description of LPI, a great deal of clinical heterogeneity has been observed among patients. LPI is often revealed by the appearance of chronic digestive symptoms and failure to thrive after presumptive diagnoses of coeliac disease, other causes of malabsorption or “food allergies” [3, 4]. However, the onset can also be sudden with hyperammonemic encephalopathy. All but one patient had hepatosplenomegaly at diagnosis, a feature that should alert practitioners to the possibility of LPI.

As observed in our cohort, patients from the same family can have entirely different evolution of symptoms, which may hinder the ability to provide a definitive prognosis (Table 1). Inter-familial clinical heterogeneity (alveolar proteinosis, cerebral impairment, cardiac event) in patients displaying the same genotype (families 1 and 4, c.726G > A homozygous) is also common. To date, 51 mutations in SLC7A7 have been reported [13‐15]. Here, we report 5 different mutations, 4 of which were previously described and 1 was a novel mutation. With the lack of a clear genotype-phenotype relationship, the identification of mutations in patients may permit confirmation of the diagnosis but is not useful in predicting the severity of the clinical course [16]. In a Finnish transcriptomic study on 13 patients, the expression of 926 genes differed significantly from controls, involving basic cellular functions such as cell cycle, signalling, ion transport or apoptosis. The extent of transcriptional effects has been hypothesised to explain the variety of symptoms observed in LPI [13]. Although 12/16 of our patients were males, this overrepresentation was not statistically significant (Fisher’s exact test) and has not been described in other cohorts [17, 18].

Pulmonary involvement appears to play a major role in the prognosis of the disease. The characteristics of the respiratory disease in our cohort were recently published [8] and consist clinically, radiologically and histologically of an early and persistent PAP, which, in some cases, can be associated with fibrosis. The respiratory complications have no obvious association with the clinical or radiologic severity of the PAP. Radiological findings usually show an interstitial or alveolo-interstitial pattern with intralobular lines, interlobular septa thickening and frequent cysts and ground glass opacities. Fibrosis may develop independently from PAP. The early onset of pulmonary disease is associated with a severe prognosis as previously described [18]. There were more asymptomatic patients in the Finnish study (65%), in which chest X-ray was used to diagnose the disease [18]. Santamaria et al. observed that 8/9 patients were asymptomatic despite the presence of infiltrative lung disease on the HRCT-scan [17]. To date, the HRCT-scan is recommended to detect lung involvement [8].

PAP is a result of defects in the surfactant homeostasis composed of proteins and phospholipids that are synthesised by type 2 pulmonary epithelial cells (PECs). PECs are also responsible for the recycling of 70 to 80% of the surfactant, whereas the rest is phagocyted by alveolar macrophages (therafter, AM) or caught in the lymphatic circulation. PAP can be either acquired or inherited. In children, genetic causes of PAP include LPI, methionyl-tRNA synthetase (MARS), and mutations in Granulocyte-macrophage colony stimulating factor (GM-CSF) receptors genes CSF2RA and CSF2RB [19]. In adults, the most frequent form of PAP is autoimmune and mediated by anti-GM-CSF antibodies. In this latter form, GM-CSF injections have been shown to be effective [20]. GM-CSF is essential for the differentiation of AMs by enhancing their ability to uptake and catabolise remaining surfactant components. Although SLC7A7 gene expression is enhanced by GM-CSF, AM differentiation is not impaired in LPI patients [21], and GM-CSF appears to be inefficient or even dangerous in hemophagocytic-lymphohistiocytosis (HLH) related to LPI.

In alveolar macrophages (AMs), y + LAT-1/4F2hc is responsible for the influx of arginine [22]. y + LAT-1/4F2hc defects lead to a decrease of intracellular arginine, which is the main substrate for nitric oxide synthase (NOS), the enzyme responsible for the production of nitric oxide (NO) [23]. NO plays a critical role in the lytic processes of macrophages as well as in vasoregulation, platelet aggregation and neurotransmission [24]. The actual paradigm for PAP-LPI is that AMs present a defect in phagocytic function that impairs the homeostasis of surfactant [25]. Moreover, LPI-related PAP is clearly aggravated by infections. All of the children who died did so from bacterial or viral pneumonias that complicated PAP. It has been recently described that the Toll Like Receptor 9 (TLR 9) pathway, which is essential for bacterial and viral defence, is impaired in macrophages of patients with LPI, with decreased interferon type I production [23]. Intracellular NO accumulation secondary to defects in arginine transport has been proposed to explain the pro-inflammatory pattern in LPI, but Kurko recently showed that the NO levels were on the contrary decreased in patients with LPI [3, 4, 23]. In addition, the pro-inflammatory pattern could result from the upregulation of TLR2/1 and TLR4 pathways leading to higher levels of tumor necrosis factor alpha (TNF alpha) and interleukin-12 [23]. The role of corticosteroids also remains controversial. There is no pathophysiological rational or clear benefit that justifies promoting this treatment, which is associated with an increased risk of infections. A prompt antibiotic regimen should therefore be initiated after microbiological documentation of an infection. Vaccination and physiotherapy for airway clearance are also important in the management of this chronic respiratory complication.

Interestingly, we were surprised to find some evidence for a higher plasma concentration of lysine in patients with a poor prognosis. The role of lysine or cationic amino-acids in PAP has not clearly been determined. Because the intra-alveolar cationic amino-acid levels and the possible role of pulmonary epithelial cells are unknown, further elucidation of their roles has been hampered. Of note, a 13-year-old boy died 26 months after a heart-lung transplantation for LPI-AP and recurrent PAP on the graft, which suggests that macrophages play a major role in the pathophysiology of the disease [26]. It was recently reported that the defect of SLC7A7/y⁺LAT-1 leads to the increased activity of another cationic amino-acid transporter such as SLC7A1/CAT1, as suggested by Tringham and al., who used a transcriptomic approach [13]. Nevertheless, this transporter is expressed in the basolateral plasma membrane of epithelial cells, but apparently not in AMs [22, 23, 27]. We speculate that the lack of SLC7A1 in AMs may increase susceptibility to lysine depletion in this cell type thereby possibly leading to both higher risk of alveolar proteinosis and moderate increase of lysine levels in plasma of patients with poorer outcome. Nevertheless, other factors would be involved to explain why plasma arginine and ornithine are not similarly involved. Alternatively, the association between moderate increases in lysine levels and poorer outcome might reflect putative downstream regulatory mechanisms, for instance a need to more strongly repress lysine oxydation in order to maintain protein synthesis. Such a putative mechanism would lead to increased lysine during catabolic stresses.

Except for the presence of early and persistent tubular involvement, renal disease primarily affects older patients (mean age of onset 17.7 years; SD 9.73 years). When kidney dysfunction is suspected, GFR should be actually measured rather than just estimated [28]. Glomerular involvement with kidney failure was reported in half of our patients after the age of 20. Despite a common genotype, the 3 available renal biopsies showed different histological patterns, such as glomerular amyloidosis, interstitial fibrosis or mesangial sclerosis, consistent with a previous report by Tanner, who evaluated a Finnish cohort [28]. The mechanisms remain obscure. Like in our patient with lupus nephritis, some autoimmune nephropathies associated to LPI have benefited from classical immunosuppressive therapy [9, 29]. Proximal tubulopathy seems to be an early hallmark of the disease as it was observed in all of our patients. Fanconi syndrome has also been reported, though the underlying mechanism remains hypothetical [30]. An elevation of intracellular lysine concentrations in the tubule would increase tubular permeability and enhance apoptosis through NADPH oxidase [31]. Therapeutic concerns focus on the early introduction of ACE-blockers to diminish proteinuria and possible hypertension [28]. Interestingly, 3 patients have successfully benefited from renal transplantation, as reported in the literature [28].

Various inflammatory and auto-immune manifestations including vasculitis can complicate LPI [32]. Interestingly, defects of phagocytosis have been observed in circulating LPI-associated monocytes and non-alveolar macrophages, as described in auto-immune diseases [25, 33]. Moreover, classical immunosuppressive treatment improves LPI inflammatory complications [32]. Whereas the T-cell response is normal, the humoral immune response is defective in some patients with LPI and an increased risk of infection [34]. However, we observed only one case of severe sepsis in our cohort.

HLH is another frequent immune complication observed in LPI [3, 4]. Primary or secondary HLH is due to an uncontrolled and self-sustained hyper-inflammatory response involving NK-cells and cytotoxic T-lymphocytes that leads to the uncontrolled activation of macrophages and pro-inflammatory cytokine secretion [35]. Such cytokine secretion has been reported in 4 patients with LPI with notably high levels of soluble CD25 [36]. In our cohort, haemophagocytosis was histologically documented in only 6 patients through bone marrow aspiration. Most of the time, HLH symptoms were limited to characteristic splenomegaly, hypertriglyceridemia, elevations in ferritin levels and low fibrinogen, none of which lead to further investigation because they were not life-threatening. Nevertheless, hypertriglyceridemia was associated with recurrent acute pancreatitis in 2 patients who did not respond to treatment with statins, fibrates or gemfibrozil. Hypertriglyceridemia is considered a risk for pancreatitis when the levels are >1000 mg/dL (11.2 mmol/L) [37], but the question arises of whether moderate levels can induce pancreatitis in such patients. Interestingly, an association between bone marrow abnormalities suggesting HLH and pancreatitis in LPI patients has already been described [38], underlining this hypothesis. Nevertheless the existence of predisposing factors for hypertriglyceridemia such as Asn9 variant of lipoprotein lipase discovered in a brotherhood among patients should remind the probable role for other genes in the pathogenicity of the different symptoms observed in the disease.

Failure to thrive is a hallmark of LPI and is associated with protein depletion intrinsically associated to LPI but also secondary to malabsorption. Growth hormone supplementation was effective in patients with associated growth hormone insufficiency as already reported [39]. Its use has not been evaluated yet in LPI. We did not find any obvious benefit from oral supplementation with citrulline, lysine or arginine. Although criteria for osteopenia were limited to bone radiography, osteopenia was reported in the majority of the patients. Parto et al. also described histologic signs of osteoporosis in 8/9 patients. Collagen synthesis in skin fibroblasts was decreased. The mechanism involved in the development of osteopenia seems to be more associated with synthesis defects secondary to protein depletion than increase of degradation by osteoclasts or inflammation [40]. Finally, multiple nutritional deficiencies are observed in patients with LPI in the area of global nutritional depletion. Selenium deficiency can lead to myocardiopathy and myopathy [41]. Targeted nutritional monitoring is therefore essential.

A secondary urea cycle disorder with hyperammonemia has been well described in LPI. Indeed, low arginine and ornithine is thought to primarily lead to the functional depletion of the urea cycle intermediates [10]. More than half of the patients presented with cognitive disorders. Though this complication has not been reported in the literature, it can be easily explained by chronic hyperammonemia. With regard to chronic dyslipidaemia, we did not observe any cerebral strokes, though an unexpected myocardium infarction did occur. This latter was definitively associated with diffuse coronaropathy. We are the first to report a cardiac involvement but another vascular disease, moyamoya vasculopathy, was recently described in LPI [42]. The cycle of low plasma levels of arginine resulting in NO depletion, decreased arterial and coronary vasodilatation and ultimately ischemia has already been described [43]. Cardiovascular monitoring is thus necessary in young adults with LPI who present hypertriglyceridemia, recurrent kidney failure and other risk factors for arterial disease. Moreover, we also report a conduction disorder whose mechanism is obscure. Based on renal and hepatic biopsies from 2 different patients, we noted that amyloidosis can complicate LPI. Whether amyloid lesions were responsible for the conduction block is unclear because echocardiography was not as suggestive. Nonetheless, this potential correlation urges caution.

General therapeutic management remains empirical with 3 major axes: prevention of hyperammonemia, nutritional supplementation and prevention of specific complications. There is a general consensus that a hypoproteinemic regimen should be initiated with an objective of 1 g/kg/d. associated with L-citrulline supplementation, L-carnitine 20-50 mg/kg/d., vitamins and other nutritional supplementation, if necessary [3, 4]. Ammonia scavengers such as sodium benzoate or phenylbutyrate are used based on glutamine levels. L-citrulline is used to correct the intracellular defect of arginine through arginosuccinate-synthase and argininosuccinate-lyase. Because arginine was initially thought to be increased in macrophages, the dosage of L-citrulline dosage was a subject of controversy because of the possibility that it could increase inflammatory damages through its conversion to arginine and NO. L-Citrulline supplementation is thus usually limited to 100 mg/kg/d [3, 4]. Nevertheless, it was shown that arginine and NO were actually lacking in macrophages and that low plasma levels of arginine may be associated with cardiovascular disease. Therefore, the dose may need to be reassessed. Also, an alimentation restricted into long chain fatty acids and supplemented with medium chain fatty acids is questionable, in order to decrease the levels of triglycerides and prevent the risk of pancreatitis. Our data show a borderline significant association between relatively increased lysine levels (within normal to low values) and poor prognosis, calling for new investigations on the mechanisms of lysine transport and metabolism in LPI patients. Finally, this study reveals the importance of careful renal and cardiovascular monitoring. ACE-blockers and hypolipidemic agents may play an essential role in the therapeutic management and prevention of several complications.