Clinical presentation and long-term outcomes of type 1 tyrosinemia in Finland: a nationwide study

Background: Introduction of nitisinone and newborn screening (NBS) have transformed the treatment of type 1 tyrosinemia, but the effects of these changes on the long-term outcomes remain obscure. Also, the predictors for later complications, the signicance of drug levels and the normalization of laboratory and imaging ndings are poorly known. We investigated these issues in a nationwide study. Results: Type 1 tyrosinemia was diagnosed in 22 children in 1978–2019 in Finland. Incidence was 1/90,102, with a signicant enrichment in South Ostrobothnia (1/9,990). Median age at diagnosis was 5 (range 0.5-36) months, 55% were girls and 13 had homozygotic Trp262X mutation. Four patients were detected through screening and 18 clinically, their main ndings being liver failure (50% vs. 100%, respectively, p=0.026), ascites (0% vs. 53%, p=0.104), renal tubulopathy (0% vs. 65%, p=0.035), rickets (25% vs. 65%, p=0.272), growth failure (0% vs. 66%, p=0.029), thrombocytopenia (25% vs. 88%, p=0.028) and anaemia (0% vs. 47%, p=0.131). One patient was treated with diet, seven with transplantation and 14 with nitisinone. Three late-diagnosed (6-33 months) nitisinone treated patients needed transplantation later. Kidney dysfunction (86% vs. 7%, p=0.001), hypertension (57% vs. 7%, p=0.025) and osteopenia/ osteoporosis (71% vs. 14%, p=0.017) were more frequent in transplanted than nitisinone-treated patients. Blood/serum alpha-fetoprotein decreased rapidly on nitisinone in all but one patient, who later developed intrahepatic hepatocellular carcinoma. Liver values normalized in 31 months and other laboratory values except thrombocytopenia within 18

Liver transplantation was the only cure for TT1 until the discovery of nitisinone [14]. Although the drug is effective in preventing the production of toxic metabolites, the accumulation of tyrosine must be prevented by a protein restriction [11]. Furthermore, although data is scarce, nitisinone-treated patients may also develop complications [6,15,16], particularly in the case of delayed diagnosis or persistently high tyrosine levels [6]. However, optimal long-term concentrations of plasma nitisinone and tyrosine are unclear. Newborn screening (NBS) has improved the short-term outcomes of TT1 patients [16,17], but long-term results are still lacking [12]. Altogether, natural history studies of TT1 in the era of nitisinone and NBS are scant and have concentrated on a few geographical areas [16].
In Finland, the treatment of TT1 is centralized, with systemically maintained medical records and NBS programmes launched in 2014. This has enabled us to evaluate the incidence, changing features and long-term outcomes of TT1 in a nationwide setting.

Incidence, family background and genetics
Twenty-one patients were diagnosed in the period 1987-2018, and one in 1978. Additional search resulted in > 2000 metabolic/liver patients, none of whom had TT1. Incidence was 1/90,102 with signi cant enrichment in South Ostrobothnia (Fig. 1A), the area from which most patients originated ( Fig. 1B). Two patients were of non-Finnish origin, and one of these had consanguineous parents. Three families had a history of TT1. Thirteen patients had a homozygous Finnish type c.786G > A, (p.Trp262X) mutation. Four patients had a compound heterozygous mutation, including Trp262X + c.1062 + 5G > A (p.?), Trp262X + unidenti ed other mutation, c.205del (p.Ser69fs) + c.554-1G > T (p.?) and c.191delA (p.Gln64fs) + c.191delA (p.Gln64fs). The latter two patients were originally from Kosovo and Iraq.

Characteristics at diagnosis
Twelve (55%) patients were girls and the median age was 5 (range 0.5-36) months. Eighteen patients were found clinically, the main ndings including acute abdomen (n = 8), septicaemia (n = 4), ascites (n = 3), rickets (n = 2) and intestinal bleeding (n = 1). Three were found by NBS and one by family screening. They had fewer clinical (Table 1), laboratory (Table 2) and imaging abnormalities (Table 3) and higher calcium and prothrombin time (PT) and lower conjugated bilirubin (DBil) levels ( Table 2) than those detected clinically. Median height was − 1.3 (-1.8-0.1) SD in screened and − 1.7 (-3.1-0.7) SD in clinically-detected patients (p > 0.05) respectively. Three clinically-detected children had bilateral and two unilateral inguinal hernia and two also scrotum hernia; they all had ascites.   The conducted imaging studies included wrist X-ray, abdominal, cardiac and cranial ultrasound and liver and brain magnetic resonance imaging or computer tomography. Cardiac ultrasound was available for 19 patients, Central nervous system (CNS) imaging was done for 16 patients and the other imaging studies for all 21 patients. 1 Two cases with resolving cerebral atrophy and one craniopharyngioma; 2 One mild mitral regurgitation and one atrium septum defect Initial treatment and short-term outcomes

Long-term outcomes
Median follow-up time of all patients was 16 (range 3-32) years and 12 (3-24) years on nitisinone. One patient died (at the age of 7 years) before the era of transplantation, one (29 years) with transplant due to unknown cause and one (15 years) later transplanted patient for intracerebral haemorrhage. Median age of the surviving patients was 12 (3-24) years on nitisinone and 30 (29)(30)(31)(32) with initial liver transplant.
Kidney dysfunction, hypertension and reduced bone mineral density were more common in transplanted than among nitisinone-treated patients, but not after adjusting for current age (Table 4). Four patients needed re-transplant(s) and four had surgical complications. Three late-diagnosed (6-33 months) patients on nitisinone also needed transplant due to elevation of alpha-fetoprotein (AFP) and new/persisting nodules in imaging studies raising suspicion of intrahepatic malignancy. One of them had intrahepatic hepatocellular carcinoma and two cirrhosis. One nitisinone-treated patient had porphyria crises while having undetectable urine SA but low plasma nitisinone (13 µmol/l). None had eye complications. Three screen-detected patients also had subsequent health problems (developmental delay, learning di culties, osteopenia). Median adult height was − 1.4 (-0.5 to -3.9) SD in nitisinonetreated and − 1.5 (0.2 to -2.0) SD in transplanted patients (p = 0.639).

Normalization of the laboratory and imaging results
Serum AFP decreased steadily on nitisinone except in one patient with later malignancy ( Fig. 2A), while tyrosine and nitisinone levels varied markedly ( Fig. 2B/C). In transplanted patients, AFP persisted ( Fig. 2A) and tyrosine was kept low until operation (Fig. 2B). DBil normalized in 10-550 days, albumin in 58-60 days, and haemoglobin in 3-90 days on nitisinone. Thrombocytopenia normalized in 3-90 days, except in three patients, of whom two were later transplanted. Acidosis and tubulopathy disappeared within three months and urine SA within one month. Normalization of the other values is presented in Figs. 3 and 4.
Liver imaging abnormalities persisted until transplantation. On nitisinone, they normalized in 10/13 patients within 56 months; two of the three with persistent ndings were later transplanted. Splenomegaly normalized within 56 months except in two transplanted patients and one (diagnosed at 8 months) nitisinone-treated patient. It also appeared later in two transplanted and three nitisinone-treated (diagnosis 5-33 months) patients of whom four developed liver complications. Kidney, central nervous system (CNS) and cardiac ndings and rickets normalized within 56 months. However, 5/6 transplanted and 1/6 nitisinone-treated patients with rickets later developed osteopenia/osteoporosis. None of the NBS patients had persistent laboratory or imaging abnormalities and thus none needed transplantation.
Long-term nitisinone treatment, tyrosine levels and protein intake The median nitisinone dose (n = 13) was 1.00 (range 0.69-1.83) mg/kg/day and the mean plasma level of 212 measurements 56 (12-97) µmol/l. There was no correlation between the levels and dosing or taking the drug once (n = 3) or twice a day (n = 10). Urine SA remained constantly negative even with low nitisinone. Median protein intake during nitisinone was 2.2 (1.0-3.0) and 2.0 (1.3-3.0) mg/kg/day before and after one year of age respectively, and the ratio between natural and tyrosine/phenylalanine-free
The number of screen-detected patients was low, but their milder phenotype compared with those detected clinically was evident. Moreover, in line with earlier short-term studies [6,13,22,29], early detection seemed to improve the prognosis; all subjects needing transplantation despite nitisinone had late diagnosis. As regards the bene ts of NBS, the current evidence is again based mainly on short-term reports [6,17,29], an exception being a Canadian study which found none of the NBS patients to have signi cant liver problems after 5-10 years [16]. However, like some other groups [6,17,29] we observed signs of hepatic dysfunction which, together with the aforesaid neonatal symptoms, suggests disease progression already in utero [17]. Risk of later health problems emphasizes the need for careful follow-up also for screen-detected patients [6].
Basic laboratory values and liver function tests normalized rapidly in most cases, while this took longer in case of transaminases and biliary parameters. This is mostly in line with earlier reports [17,19,23,25], although the follow-up time has usually been shorter. Transaminase levels were higher and normalized more slowly than described before [16,23], but this did not predict later complications. Some children also presented with high ammonium ion (NH4+), this being feared to predict early need for transplantation [11], but the values decreased promptly on nitisinone. Slow decline of AFP is a physiological phenomenon in infancy [30], but our ndings con rm that nondecreasing or rerising values predict malignancy [16,19,22]. Persistent thrombocytopenia, a sign of a chronic liver disease, was also a strong predictor of subsequent need for transplantation.
The kidney, CNS, cardiac and bone imaging ndings normalized within a few years, but some patients had persistent liver abnormalities and/or splenomegaly. Comparable gradual improvement in renal ndings has also been reported by a British group [25] whereas clinically important data about the disappearance of the other ndings or signi cance of their perseverance has been limited. Persistent liver abnormalities have been reported in 19-57% of patients, but the follow-up times have been shorter than in the present study [6,16,17,19,23,29]. Here the nonresponsive ndings and reappearance of splenomegaly were major warning signs for subsequent hepatic transplantation.
Long-term complications of TT1 were mostly analogous with those reported in the literature [15-17, 19, 25, 29, 31, 32]. Neurological problems and poor growth were more common here, but the follow-up times of these earlier studies may have been too short to detect these late-appearing issues. The former have been suggested to be partially attributable to the side effects of nitisinone [32,33], but this is debatable [31,34] and here low levels seemed more harmful. However, early disease onset seems to increase the risk for neurological complications [6], again supporting the idea of in utero progression of TT1. Poor growth has been sparsely reported 28 , but the possible risk for short stature observed here calls for further studies. There is also limited and inconsistent data about the prevalence and appropriate follow-up of bone issues in TT [16,12], but our results suggest that surveillance at least for cases with rickets at diagnosis is warranted. In contrast, as in prior short-term studies [17,19,23,25,29], persistent renal involvement seems to be rare.
We found no signi cant association between long-term complications and tyrosine levels, but low plasma nitisinone, even with negative urine SA, was associated with learning di culties, growth delay and a need for transplantation. The reported nitisinone target range has varied 20-80 µmol/l [6,11,35] and the dosing usually aims to achieve negative urine SA [6,24]. Optimal levels remain somewhat unclear, but frequent monitoring of the levels with a target of 40-60 µmol/l has been recommended [12].
Interpretation of the markedly uctuating nitisinone values can be di cult, but our results underline the importance of su cient levels, which may be even higher than previously suggested.
The main strengths of the present study were the nationwide coverage and availability of comprehensive medical data. Detailed registers also provided information on long-term outcomes and enabled us to assess risk factors for later complications, although weaknesses in the retrospective design remain. As a further limitation, an intensi ed register search was conducted in only one district, which in theory could have led to a few earlier cases being missed. Some patients may also have died before the transplantation era and were thus lost since medical records are deleted 12 years posthumously.

Conclusions
The overall prognosis of TT1 has improved in the nitisinone era and NBS seems to provide further bene ts. However, clinicians should realize that a risk of complications persist even among screendetected patients. Intensi ed surveillance is warranted, especially in patients with delayed diagnosis and persistent laboratory or imaging abnormalities. In addition, maintaining su cient nitisinone levels is important.

Study design and patients
Treatment of metabolic disorders in Finland is centralized in the university hospitals of Tampere (TAYS), Helsinki, Turku, Oulu and Kuopio. Patients with TT1 were searched from these centres by contacting the physicians responsible and by applying International Classi cation of Diseases code E70.2. In addition, since TAYS was known to diagnose most of the TT1 patients due its location near South Ostrobothnia, an intensi ed search among children with metabolic disease or liver failure diagnosed since 1960 was conducted there. Some follow-up visits took place in the central hospitals of Seinäjoki and Satakunta, and this data was also obtained. After patient identi cation, comprehensive medical data on each case was collected from birth until Sept. 2019.

Clinical characteristics and family background
The date of diagnosis was de ned as the rst positive urine SA, also in the case of NBS performed initially from dried blood spot [36]. The collected data comprised a diagnostic approach (clinical suspicion vs. screening), demographic data, symptoms and clinical ndings, developmental stage and growth parameters [37,38] and relevant data about pregnancy and delivery. In addition, the origins of patients' grandparents or the birthplace of the patient, presence of other TT1 cases in the family and possible consanguinity were documented.

Laboratory and imaging ndings
The collected blood values included alanine aminotransferase (ALT), albumin, ALP, AFP, NH4+, blood count, calcium, γ-GT, glucose, PT and total (TBil) and conjugated bilirubin. The presence of liver or kidney dysfunction, anaemia, thrombocytopenia and hypoglycaemia [11,26,[39][40][41] were also recorded. During nitisinone treatment, the above-mentioned laboratory values, blood tyrosine and nitisinone levels and urine SA were monitored until current date. In transplanted patients, AFP and tyrosine values were monitored until transplantation.
The presence of ascites, abnormalities of liver, spleen, kidney, heart and CNS and the presence of rickets and osteopenia/osteoporosis were evaluated using X-rays, ultrasound, computer tomography, magnetic resonance and bone densitometry. Besides diagnostic imaging, at least annual abdominal surveillance was conducted in all patients. The disappearance of the abnormalities and the appearance of new ndings were also documented.

Treatment
Possible treatment modalities included sole dietary restriction, liver transplantation and nitisinone. Histology of the explants and possible transplantation complications were recorded, as were the compliance to and dosing (mg/kg/day) of nitisinone.

Statistical analysis
Categorical variables are reported as numbers and percentages, and numerical data as medians with quartiles or ranges. Comparisons were made with Mann-Whitney test, Chi-square or Fisher's exact test as appropriate. Associations between diagnostic ndings and later complications were analysed with Spearman's correlation and binary logistic regression, which was used for age adjustments, and those between nitisinone and tyrosine levels and complications with ROC [43] and crosstabulation. Incidence was the number of TT1 patients divided with all live births. General birth data was provided by Statistics Finland [44].