Clinical, biochemical and molecular characteristics in 11 Czech children with tyrosinemia type I

Overview

Hereditary tyrosinemia type 1 (HT1) is rare autosomal recessive inborn error of metabolism caused by deficiency of fumarylacetoacetate hydrolase. HT1 manifests with sever liver and kidney impairment and associates with an increased risk of liver cancer development. The aim of our study is to present a detailed clinical picture and results of biochemical and molecular genetic analyses in 11 Czech patients with HT1 diagnosed in our clinic since 1982.

Results:
In 9 patients the disease manifests between 1.5-7 months of age with refusal to eat, failure to thrive and vomiting. In 4 children HT1 progresses to acute liver failure. One clinically healthy boy was diagnosed because of affected sister. In one boy with liver cirrhosis the diagnosis was delayed until the age of 5.5 years. In all children the biochemical investigation showed elevated liver enzymes, α1-fetoprotein and hypophosphatemic rickets. Metabolic investigation revealed increased plasma tyrosine level, urinary excretion of succinylacetone and in 8 measured patients also increased urinary δ-aminolevulinic acid concentration. Three patients born before 1988 died due to liver cancer development (two of them) or liver failure. The average age of our 8 living patients is 10.7±8.3 years. Mutation analysis of FAH gene confirmed the HT1 in these patients and three novel mutations were found in FAH gene: c.579C>A, c.680G>T and c.1210G>A. Clinical status in six patients is favourable on strict low protein diet combined with Orfadin® therapy. However, in two children despite of the maximal available therapy lasting 2 and 10 years resp., the disease progressed towards liver cancer development and necessity of liver transplantation.

Conclusion:
Early diagnostics of HT1 as a part of extended newborn screening is the only possibility to further improve the prognosis of the patients. Moreover, available molecular-genetic analysis of the FAH gene enables prenatal diagnostics in affected families.

Key words:
tyrosinemia, liver failure, rickets, succinylacetone, fumarylacetoacetate hydrolase, FAH gene

Introduction

Hereditary tyrosinemia type 1 (HT1) is autosomal recessive inborn error of metabolism caused by deficiency of fumarylacetoacetate hydrolase (FAH). The FAH enzyme is expressed mainly in the liver (90%) and the kidney (10%). In addition to HT1, there are two other forms of hereditary tyrosinemia with more favourable outcome (1). The prevalence of HT1 throughout the world is rare. It is estimated to be no greater than 1 in 100,000 live birth with several exceptions. There is a high prevalence in some regions of Finland and Quebec (1:63 000, 1:2 000 respective) due to founder allele effect (2, 3). The marked elevation of plasma tyrosine and increased urinary excretion of succinylacetone (SAA), maleylacetoacetate (MAA) and fumarylacetoacetate (FAA) cause liver impairment and renal-tubular dysfunction (4). SAA is a potent inhibitor of the δ-aminolevulinic acid dehydratase step in porphyrin synthesis, causing the porphyria-like neurological crises (5).

The clinical manifestations of HT1 are very variable and affected individual can present at any time from the neonatal period to adulthood. There is considerable variability of presentation even between members of the same family (6, 7). The reason of milder phenotype is probably „self-induced“ correction of the fumarylacetoacetase defect in hepatocytes. (8-12). Clinically, HT1 may be classified based on the age at onset of symptoms, which broadly correlates with disease severity: an „acute“ form manifests before 6 months of age with acute liver failure; a „subacute“ form presenting between 6 months and 1 year of age with liver disease, failure to thrive, coagulopathy, hepatosplenomegaly, rickets and hypotonia; and a „chronic“ form that present after the first year of life with chronic liver disease, renal disease, rickets, cardiomyopathy and/or porphyria-like syndrome (13-16).

In symptomatic patients, biochemical tests of liver function are usually abnormal. In particular, liver synthetic function is severely affected, coagulopathy and/or hypoalbuminemia are often present even if other tests of liver function are normal. In most acutely ill patients, α₁-fetoprotein (AFP) levels are greatly elevated. A Fanconi-type tubulopathy is often present with aminoaciduria, phosphateuria and glucosuria, and radiological evidence of rickets may be present. Increased urinary excretion of succinylacetone and δ-aminolevulinic acid together with elevated plasma tyrosine level are pathognomonic of HT1 (17, 18). Confirmation of the diagnosis required either enzyme (FAH) assay or mutation analysis (FAH gen). FAH assays may be performed in liver biopsy or in fibroblasts.

Historically, HT1 was treated with a tyrosine and phenylalanine restricted diet, with or without liver transplantation. In 1992 a new drug Orfadin^®, 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyklohexanedione (NTBC, nitisinon), a potent inhibitor of 4-hydroxyphenylpyruvate dioxygenase (HPPD) was introduced; it has revolutionised the treatment of HT1 and is now the mainstay of therapy (19-21).

The aim of our study is to present a detailed clinical picture and results of biochemical and molecular genetic analyses in 11 Czech patients with HT1 diagnosed in our clinic since 1982.

Patients and methods:

Our study group comprises 11 patients (5 boys – P3, P4, P5, P8, P11, 6 girls – P1, P2, P6, P7, P9, P10), from ten non-consanguineous families, in whom HT1 was diagnosed on biochemical level between 1982 and 2006. Mutation analysis of FAH gene was preformed in 8 living patients between 2008 and 2009. Three patients already died (P1-3) at the age of 6 months, nearly 8 years and 11.5 years, respectively. The average age of surviving patients (P4-11) is 10.7±8.3 years (the average r, the standard deviation SD) within the range of 3-27 years.

Biochemical analyses

Tyrosine in plasma and  δ-aminolevulic acid in urine were analyzed by ion exchange chromatography on automatic amino acids analyzer AAA 400 (Ingos, Czech Republic) with ninhydrine detection at 440 nm and at 570 nm for amino acids with secondary amino group and for amino acids with primary amino group, respectively. Amino acids were identified by retention time.

Succinylacetone in urine was measured by spectrophotometric method. Method is based on succinylacetone inhibition of δ-aminolevulinic acid dehydratase from hemolyzate. δ-aminolevulinic acid dehydratase catalyzed biosythesis of porfobilinogen from d-aminolevulinic acid. The absorbance of porfobilinogen conjugate with dimetylaminobenzaldehyd, measured at 555 nm, is proportionate to the succinylacetone concentration in the sample.

Molecular-genetic analysis

Total genomic DNA and RNA were isolated from blood leukocytes. A phenol-chloroform extraction was used for DNA isolation. Total RNA was isolated with TriReagent^® according to manufacturer’s instructions (MRC, Great Britain). 1000 ng of total RNA was transcribed into cDNA by reverse transcriptase SuperScript III (Invitrogen) and Oligo(dT) primer (Promega). cDNA of FAH gene was amplified by PCR in 3 overlapping fragments and sequenced on genetic analyzer ABI 3100 Avant (Applied Biosystems). Based on the type and localization of the observed mutation, corresponding exons and adjacent intronic region of FAH gene were sequenced from genomic DNA. All found mutations were confirmed by PCR-RFLP analysis in DNA of patients and their parents. 3 novel mutations in the FAH gene were excluded by PCR-RFLP in 100 control DNA samples.

Results

Three patients, who were born between 1982 and 1988, have already died. The disease manifested by acute liver failure in two of them. The female patient (P1) born in 1985 died due to liver failure at the age of 6 months. Although the female patient (P2) born in 1988 had survived the acute liver failure at the beginning of the disease, she died because of liver cancer development at the age of nearly 8 years. The diagnosis of HT1 in male patient (P3) born in 1982 was confirmed after accidentally found hepatosplenomegaly at the age of 5.5 years. The progression of rickets was already documented by X-ray examination and the fibrosis was found in the liver biopsy. The boy died because of liver cancer development at the age of 11.5 years.

Two living patients (P4-5) were ascertained also for the acute liver failure with ascites formation at the age 2-7 months. The liver functions were stabilized in male patient P4 born in 2006, but because of liver cancer development the liver transplantation was indicated at the age of 31 months. Patient P5 is without any clinical symptoms at the age of 6.5 years, presently. The disease manifested by refusal to eat, failure to thrive and vomiting after the introduction of the infant formula between the second and seventh month of life in other five patients with HT1 (P6-P10). Male patient P11 was without any clinical symptoms when diagnosed at the age of 3 months. He was ascertained because of the presence of the disease in his sister (P10). The average age at the time of diagnosis was 6 months in these 6 children (from 2 to 18 months). The signs of rickets were already documented in patient P10 at the time of diagnosis at the age of 18 months.

Elevated liver enzymes (ALT 1.0±0.9, AST 1.4±0.9 μkat/l, r±SD), highly increased α₁-fetoprotein (AFP) level (the median 79959 μg/l for detected levels 19.2 - 509705 μg/l) and laboratory signs of hypophosphatemic rickets (ALP 17±7 μkat/l, phosphorus 1.2±0.36) were present in all early diagnosed patients. The AFP level was not increased in P3, who was diagnosed at the age of 5.5 years. The diagnosis was confirmed on biochemical level by markedly increased plasma tyrosine concentration in blood (650±258 μmol/l), SAA (335±170 μmol/l) and 5-ALA (155±119 mg/g creatinine) in urine, respectively. The enzymatic activity of FAH in lymphocytes (<2% of controls) was measured in the cooperation with abroad laboratory in patients diagnosed before year 2000.

The low protein diet (0.5±0.1 g/kg/day) with the supplementation of essential amino acids mixture without phenylalanine, tyrosine, and L-carnitine was introduced in all children immediately after the diagnosis was confirmed. The NTBC treatment (1mg/kg/day) began immediately after the diagnosis confirmation in P4-6 and P10-11, and with the interval of 4 months after the diagnosis in P7. The NTBC treatment in the oldest patients P8 and P9 (presently 20.5 and 27 years) was given only at the age of 11 and 18 years, respectively. The NTBC treatment could not be administrated in patients P1-3, who died before 1995. In five patients who were on the low protein diet and NTBC treatment since the beginning, the 5-ALA excretion normalized in 6±4 days, SAA in 11±8 days and the phosphate blood level in 10±7 weeks, the alkaline phophatase in 1.5±1.3 months, and coagulopathy resolved in 2.3±1.2 months, respectively. The resolving of elevated liver enzymes occurred only in four patients (P5-7, P11) 14±5 months after the treatment administration. The AFP level decreased markedly after the treatment introduction (Figure 1A), but this was fully normalized only in patients P5 and P6 treated with low protein diet and NTBC immediately after the diagnosis confirmation. The normalization of elevated liver enzymes and AFP level was not observed in patient P4. The liver transplantation was indicated due to liver cancer development at the age of 31 months. The transplantation was successful and the boy is without any diet restriction with NTBC treatment decreased to 10%, presently.

The normalization of SAA excretion and only trace 5-ALA detection was seen in the patient P7 treated by diet (since 5 months of age), but the marked AFP decrease occurred only after the NTBC administration (3396 µg/l in 8 months to 42 µg/l in 14 months). The intermittent high excretion of SAA (220±90 µmol/l) in urine was documented at the age of 10 and 11 years in patient P8, who was diagnosed in 1989 at the age of 4 months, although the compliance in diet was good. The introduction of NTBC treatment could not avoid the liver cancer development with an indication to liver transplantation (20.5 year old patient is in the waiting list, presently). The AFP time course in P8 is demonstrated (Figure 1B). We observed the AFP level increase going together with liver cancer development also in two patients who died due to this complication (the values of thousands at the carcinoma manifestation with progression to the hundreds of thousands closely before exitus). Although the NTBC treatment (and the definitive SAA normalization) in patient P9 was commence only in 18 years of life, the health state is well, now.

The present clinical state and relevant laboratory data of 8 living patients with HT1 (P4-11) are shown in table 1. The phospo-calcium metabolism parameters and coagulations are in normal range in all patients. Neither of 5-ALA or SAA has been detected in urine in any patient in the long-term follow up.

We have indentified the mutations in FAH gene in all eight living patients (Table 2). The patients P10-11 are homozygotes for the mutation c.554-1G>T, P7 is homozygote for the c.1062+5G>A mutation. Other patients are compound heterozygotes (Table 2). Three novel not previously described mutations were found: mutation c.579C>A introducing premature stop-codon C193X, the c.680G>T mutation leading to G227V substitution and the c.1210G>A mutation leading to G404S substitution. None of these three mutations were found in the control group of 100 DNA samples.

Discussion

Hereditary tyrosinemia type 1 (HT1), a rare inborn error of tyrosine metabolism caused by deficiency of fumarylacetoacetate hydrolase (FAH), was diagnosed in 11 patients from 10 families in our department within last 27 years. The diagnosis was confirmed either on biochemical or molecular level. The aim of our study was to describe natural cause of the disease, summarize our experience with treatment and present three novel mutations found in FAH gene.

FAH is a cytosolic protein active as a homodimer. FAH monomer consists of 419 amino acids, structurally and functionally divided into the N-terminal and C-terminal domain. The C-terminal domain forms the active site, the N-terminal domain is considered to be a functional regulatory site. The Mn²⁺ and Na⁺ ions necessary for the proper structure and function of the enzyme are localized in the active site of the protein (22-24). There is 88% sequence homology between the human and murine FAH enzyme. The amino acids constituting the active site are strictly interspecies conserved. Human FAH gene is located in the chromosome 15q23-25 and consists of 14 exons (25). 43 different pathogenic mutations have been identified in the FAH gene so far (26).

In our group, 3 novel mutations were found. The C193X (c.579C>A) mutation introduces premature stop-codon and thus leading to the loss of the active site in FAH protein. The mutation G227V is located close to Asp233, which is important for anchoring of both ions of the active site. The substitution G227V may possibly interfere with the spherical constitution of active site and may affect non-covalent interactions between the subunits of the FAH homodimer. The G404S mutation might result in the conformation change of the N-terminal domain, which is important for the activity regulation. Both of 227 and 404 positions are insterspecies conserved for the glycine amino acid. The E6/I6del26 mutation has been characterized only at the genome DNA level so far (27). The analysis of PCR products amplified from cDNA of patient P9 ([c.554-1G>T]+[c.548_553+20]) has shown, that the c.548_553+20 mutation possibly interfere the splicing site of intron 6, as the rest of intron 6 shorter of first 10 bp remained in cDNA.

Hereditary tyrosinemia type 1 is characterized by early presentation with liver and kidney involvement (28, 29), and neuropathic crisis attacks (30) in the absolute majority of patients. The age of manifestation of HT1 in our patients (range 1.5-7 months) corresponds to those described previously (31). P3 was diagnosed with delay at the age of 5.5 years, when the liver cirrhosis had been already present. Kidney involvement was present in 55% of our patients at the time of diagnosis. Only 36% of our children (4/11) manifested by acute liver failure, which is in contrast to 75% in the study of van Spronsena et al. (29) and 43% in the study of Santra et al. (31), respectively. No neurological crises attacks were observed in our group of patients. The sudden onset of peripheral neuropathy of unknown etiology occurred in patient P5. This resolved in course of few following months.

The prognosis of HT1 was poor till 1992, when the treatment with Orfadin^® (NTBC, nitisinon) was introduced. The survival of 2 years in children with early onset (under 2 months) was only 29% (29). The main mortality cause was the liver insufficiency and the development of liver cancer. After introducing of Orfadin^® into the general practice (20) the prognosis improved dramatically in children with HT1. Its administration to patients suffering from HT1 with signs of acute liver failure led to improvement of liver functions and coagulopathy in 90% of cases already within the first week of treatment. The more rapid decrease of AFP levels was documented (32) and resolution of tubular functions (31) with average normalization till the age of 12 months was observed. No nefrocalcinosis develops after the treatment introduction in most cases. This was present in 33% HT1 patients before the NTBC era treatment (31). The alleviation of nearly all the neurological crises attacks is apparent (33). The risk of the liver cancer development is more than three times lower in HT1 patients, in whom the NTBC treatment began under the age of two years (34, 35).

The treatment with NTBC is available in the Czech Republic since 1996. Three patients, who were born between 1982 and 1988, died before the NTBC treatment could be administered. The early NTBC treatment was introduced in 6 our patients. In concordance with literature, we observed a rapid normalization of coagulopathy and tubular functions, as well as the normal excretion of 5-ALA and SAA in urine, respectively. The complete normalization of elevated liver enzymes and AFP occurred only in 4 and 2 children, respectively. Despite early diagnosis and treatment administration in patient P4 (1/6), the liver cancer developed and the liver transplantation was necessary. The similar course of the disease was present also in patient P8 treated since the age of 11 years, in whom the liver cancer developed at the age of 20 years. It is not clear yet, why the liver cancer developed already between the second and third year of life in P4 despite of adequate and compliant treatment, while in P8 the liver cancer developed not before the age of 20 years with NTBC treatment since the age of 11 years. Also our oldest female patient P9 treated by NTBC since the age of 18 years has normal laboratory results and without any clinical complications. The FAH activity is extremely low in patients P8 and P9 (under 1% of controls). The role of extrinsic factors, different genetic background and epigenetic mechanisms are supposed.

Conclusion

Hereditary tyrosinemia type 1 manifests by liver and kidney involvement in particular. Although the prognosis improved dramatically under combined dietary and pharmacological treatment with NTBC, patients are at risk of the liver cancer development with the necessity of the liver transplantation. The only one possibility, how to decrease the risk of possible complications in patients with HT1, is the early diagnostics in the extended newborn screening of hereditary metabolic diseases. The HT1 diagnosis was possible only on biochemical level without possibility of genetic counseling and prenatal diagnosis till the year 2008. The molecular-genetic analysis is available in our department, presently.

Acknowledgments
This work was supported by project MZ0VFN2005 a MSM0021620806.

Abbreviations

• AFP - α₁-fetoprotein
• ALP - alkalic phosphatase
• ALT - alanine aminotransferase
• AST - aspartate aminotransferase
• 5-ALA - 5-δ-aminolevulinic acid
• FAA - fumarylacetoacetate
• FAH - fumarylacetoacetate hydrolase
• MAA - maleylacetoacetate
• NTBC - 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyklohexandion
• r - average
• SAA - succinylacetone
• SD - standard deviation