Novel HPD mutation p.A244V compound with p.T219M causing tyrosinemia type III in a Chinese girl and review of the genotype–phenotype spectrum

Abstract

Background

Hereditary tyrosinemia type III (HT III) is an extremely rare form of tyrosinemia, characterized by autosomal recessive inheritance and biallelic mutations in the HPD gene. The clinical presentation of HT III is variable and poorly understood, with symptoms ranging from developmental delay and intellectual impairment to seizures and intermittent ataxia. This study aimed to provide further insights into the clinical and genetic characteristics of HT III.

Methods

A 3‐year‐old girl, identified through newborn screening, was diagnosed with HT III using targeted next‐generation sequencing. A comprehensive literature review was conducted, and the clinical, biochemical, and genetic findings of previously reported HT III patients were summarized and analyzed.

Results

The genetic analysis of the proband revealed compound heterozygous mutations in the HPD gene such as c.731C>T (p.A244V) and c.656C>T (p.T219M). Notably, the HPD p.A244V mutation had not been previously documented in public databases or the scientific literature. Bioinformatics analysis classified both variants as pathogenic variants. The patient exhibited persistent tyrosinemia, elevated levels of related metabolite derivatives, confirming the diagnosis of HT III. The review of previously published cases contributed to a better understanding of the clinical and genetic characteristics associated with HT III.

Conclusion

Early diagnosis and prompt treatment in infancy are crucial for managing HT III effectively. Dietary therapy, particularly during childhood, plays a significant role in disease management. The findings from this study enhance our understanding of the genotype–phenotype associations in HT III and emphasize the importance of early intervention for improved patient outcomes.

Molecular analysis of the HPD variants. (a) HPD gene sequencing of the patient and her parents. The patient carried compound heterozygous PANK2 gene mutations of c.656C>T and c.731C>T, the father carried c.656C>T, and the mother carried c.731C>T. Arrows indicate the location of the variants. (b) Schematic illustration of HPD mutants and their interactions with surrounding amino acids. Green, the wild‐type amino acids; red, the mutant amino acids; blue, their surrounding amino acids; and yellow, the hydrogen bond. The stability of mutant HPD proteins was calculated using the I‐Mutant suite and denoted as DDG (DDG <0 means decreased stability; DDG >0 means increased stability). (c) Multiple sequence alignments of HPD from different organisms. Residues are colored based on conservation. The white letters in red boxes represent the highest conservation grade, the black letters in yellow boxes represent the second‐highest conservation grade, and no color indicates the least conserved grade. The purple triangle indicates variants p.T219M and p.A244V.

1. INTRODUCTION

Hereditary tyrosinemia type III (HT III, MIM #276710) is an extremely rare autosomal recessive disorder first reported in 1982 (Endo et al., 1982). HT III results from a deficiency in the enzyme 4‐hydroxyphenylpyruvate dioxygenase (HPPD) caused by biallelic mutations in the HPD gene (MIM *609657) (Rüetschi et al., 2000). The enzyme catalyzes the conversion of 4‐hydroxyphenylpyruvate to homogentisic acid, the second reaction in tyrosine catabolism (Hager et al., 1957). Impaired enzyme activity leads to the accumulation of tyrosine in plasma and the massive excretion of tyrosine derivatives, such as 4‐hydroxyphenylpyruvate, 4‐hydroxyphenyllactate, and 4‐hydroxyphenylacetate, into urine (Tomoeda et al., 2000).

The clinical phenotype of HT III patients remains variable and unclear because of the low number of diagnosed cases; however, characteristic findings include mild mental retardation, seizures, developmental delay, and intermittent ataxia (Ellaway et al., 2001; Jones et al., 2020). To date, HT III research has received little attention worldwide, with only 20 patients and one case in China having been reported (Ellaway et al., 2001; Heylen et al., 2012; Kahraman et al., 2022; Najafi et al., 2018; Szymanska et al., 2015; Tong et al., 2019; Vakili et al., 2021). Some patients with enzymatic deficiency did not undergo genetic analyses. The genotype–phenotype relationship in HT III patients is not established.

In the present study, we described the clinical phenotypes, biochemical features, and genetic findings of a Chinese girl with HT III who carried the compound heterozygous mutations of c.656C>T (p.T219M) and c.731C>T (p.A244V) in the HPD gene. Furthermore, a review of the reported variants of the HPD gene and HT III patients was performed to understand better the genotype–phenotype relationship and treatment necessity of HT III.

2. MATERIALS AND METHODS

2.1. Study participants and ethical considerations

This study was approved by the Clinical Research Ethics Committee of Changzhi Maternal and Child Health Care Hospital. Written informed consent was obtained from the parent/legal guardian of the patient included in this study. The subject was a girl with increased levels of tyrosine detected via newborn screening. She underwent a comprehensive clinical assessment, a complete medical history investigation, blood and urine biochemistry tests, and genetic testing to diagnose the disease.

2.2. Genetic analysis

Target sequencing was performed in the patient using a genetic diagnostic panel for hereditary metabolic diseases covering 86 genes (including the HPD gene) (MyGenostics Inc., Beijing, China) according to the manufacturer's protocol. Sequencing of captured DNA fragment was carried out with 150 bp paired‐end reads on Illumina HiSeq × Ten platforms. Detailed sequence‐data analysis was carried out in accordance with the previously reported protocol (Quenez et al., 2021; Tao et al., 2023).

2.3. Bioinformatic analysis and statistical analysis

Amino acid substitutions were studied in silico to predict the pathogenic effect of the change through VarSome (https://varsome.com/), which utilizes multiple bioinformatic algorithms (Kopanos et al., 2019). Single‐nucleotide polymorphisms were queried from gnomAD (https://gnomad.broadinstitute.org/). Multiple sequence alignment was performed using COBALT (Papadopoulos & Agarwala, 2007), and the alignments were visualized using ESPRIT 3.0 (Robert & Gouet, 2014). HPD structural three‐dimensional (3D) modeling was performed based on the Protein Data Bank (PDB) accession 3ISQ. The structure of the wild‐type (WT) and mutant HPD protein was assessed using the Iterative Threading Assembly Refinement (I‐TASSER) software. The model with the highest C‐score (0.72) was selected. The 3D structural images were visualized using PyMOL1.7. The protein stability of the mutations based on the predicted structure was predicted with the online tools I‐Mutant3.0 (http://gpcr2.biocomp.unibo.it/cgi/predictors/I‐Mutant3.0/) (Capriotti et al., 2005). The R software (version 3.5.3; https://www.r‐project.org/) was used for statistical analysis and to visualize the results.

3. RESULTS

3.1. Clinical features

The girl was the firstborn and only child of healthy and non‐consanguineous Chinese parents. During pregnancy, the mother had no history of exposure to teratogenic pathogens or drugs. On week 39 of gestation, the birth length was 51 cm (75th percentile), the birth weight was 3900 g (95th percentile), and the occipitofrontal circumference was 35.3 cm (90th percentile). She did not manifest any dysmorphic features. The neonatal screening performed on day 4 revealed an initial plasma tyrosine concentration of 667.76 μmol/L (normal range: 30–400 μmol/L), whereas all other amino acids and succinylacetone were within normal ranges. Subsequent control samples revealed the persistence of elevated tyrosine levels (1113.04 μmol/L on day 18, 1034.48 μmol/L on day 24, and 856.44 μmol/L on day 28). The urinary organic acid profile through GC/MS analysis revealed elevated levels of 4‐hydroxyphenylpyruvate and 4‐hydroxyphenyllactate without marked elevation of succinylacetone. Persistently elevated tyrosine levels suggested tyrosinemia. At 1 month of age, the girl received a phenylalanine‐ and tyrosine‐free formula (Mead Johnson). The ratio of formula milk to breastfeeding was determined based on previous reports (Gu, 2015). Comprehensive details concerning the patient's plasma tyrosine concentration, relevant metabolites in urine, and formula intake are available in Table 1. The physical examination revealed no evidence of ocular, liver, or skin involvement. Renal and liver functions were normal. No feeding difficulties or dysphagia were found. At 1 year of age, she was suspected of having COVID‐19 but only presented with a fever for 4 days at the highest temperature of 41°C. At the age of 14 months, she underwent an assessment using the Children Neuropsychological and Behavior Scale (CNBS‐R2016). Her language development exhibited a slight lag (DQ = 72), while her gross motor skills, fine motor skills, adaptive behavior, and personal–social abilities were all within the normal range (DQ = 85, 87, 90, and 86, respectively). At the age of 20 months, the patient was admitted to the hospital due to recurrent fever for 1 month, which was attributed to a urinary tract infection. Throughout this time, she consistently displayed no indications of developmental issues.

TABLE 1.

Serum and urine biochemical markers, and dietary composition for the patient.

Age	5 days	20 days	1 month	1.5 months	3 months	6 months	12 months	14 months	18 months	20 months	Reference range
Tyrosine level (μmol/L)	667.76	1113.04	856.44	301.39	276.92	404.00	404.94	/	235.67	176.30	30–400
4‐hydroxy‐phenyllactic acid a	/	/	636.79	/	/	260.75	/	/	190.98	/	1.80–12.51
4‐hydroxy‐phenylpyruvic acid a	/	/	131.08	/	/	89.21	/	/	58.42	/	0.20–1.90
Weight (kg)	3.9	/	4.6	/	6.5	8.3	10.5	11.5	12.1	12.3	/
Height (cm)	51.3	/	54.5	/	61.7	68.6	77.2	80.0	83.7	85.1	/
Diet	Breast nursing	Breast nursing	Formula: 1.5 g/kg/days Breast nursing: 1.5 g/kg/days				Formula: 0.8 g/kg/days Breast nursing: 1.5 g/kg/days				/

^a The urinary organic acids were detected by semiquantitative GC/MS analysis.

3.2. Genetic findings

At 1 month of age, the patient underwent targeted next‐generation sequencing (NGS) using a genetic diagnostic panel of hereditary metabolic diseases, which revealed two variants in the HPD gene, c.656C>T (p.T219M) and c.731C>T (p.A244V) (Figure 1a). Her parents carried the variants. The variant p.T219M was recorded at a frequency of 4/152,108 in the GnomAD database, while the variant p.A244V has not been reported in the literature and is absent from the general population. VarSome's in silico pathogenicity scores, including the combined annotation‐dependent depletion score (CADD) 28.7 for p.T219M and CADD 28.4 for p.A244V, support the potential pathogenicity of both variants (Rentzsch et al., 2019). As shown in Figure 1b, the two variants were located in vicinal oxygen chelate (VOC) domain 2 (residues 180–338) and were predicted to decrease the stability of the HPD protein through I‐Mutant3.0. The variant p.T219M disrupted the original hydrogen bonds and destabilized the amino acid interactions. The variant p.A244V changed the length and angle of the hydrogen bond between residues A244 and S223. These variants may disrupt correct folding and abolish the function of VOC domain 2. Moreover, the two missense mutations were located on a site that is highly conserved among species (Figure 1c). These findings suggest that the two variants could cause HPD dysfunction. Although according to the variant classification guidelines of ACMG (Richards et al., 2015), both of the two variants are currently categorized as variants of uncertain significance (PM2_supporting+PP3), we have compelling grounds to believe that these variants are associated with HT III, considering the clinical and biochemical presentations of the affected children.

FIGURE 1.

Molecular analysis of the HPD variants. (a) HPD gene sequencing of the patient and her parents. The patient carried compound heterozygous PANK2 gene mutations of c.656C>T and c.731C>T, the father carried c.656C>T, and the mother carried c.731C>T. Arrows indicate the location of the variants. (b) Schematic illustration of HPD mutants and their interactions with surrounding amino acids. Green, the wild‐type amino acids; red, the mutant amino acids; blue, their surrounding amino acids; and yellow, the hydrogen bond. The stability of mutant HPD proteins was calculated using the I‐Mutant suite and denoted as DDG (DDG <0 means decreased stability; DDG >0 means increased stability). (c) Multiple sequence alignments of HPD from different organisms. Residues are colored based on conservation. The white letters in red boxes represent the highest conservation grade, the black letters in yellow boxes represent the second‐highest conservation grade, and no color indicates the least conserved grade. The purple triangle indicates variants p.T219M and p.A244V.

4. DISCUSSION

Individuals with elevated tyrosine levels detected through newborn screening confront a diverse array of potential conditions, encompassing transient tyrosinemia of the newborn, as well as HT types I, II, and III, among other liver‐related disorders. Given the complex considerations surrounding drug interventions such as nitisinone and orfadin, it is difficult for primary healthcare practitioners to address the requirements of this particular population. Importantly, there have been documented instances of these medications causing adverse reactions in HT III patients (Vakili et al., 2021). The primary diagnosis in our case was HT III, which was supported by normal succinylacetone levels and the absence of oculocutaneous manifestations, effectively ruling out HT I and II. This highlights the importance of meticulous biochemical assessments and comprehensive clinical evaluations when traversing the landscape of differential diagnoses for hypertyrosinemia patients. In addition, this prompts the consideration of a more cautious approach, involving controlled tyrosine intake during the initial periods of treatment, pending the genetic analysis definitive findings. This pragmatic approach intends to prevent unwarranted adverse effects of medical interventions.

To our current knowledge, the existing literature has documented only 21 patients (including our case) that have received a definitive diagnosis of HT III (Capalbo et al., 2019; Cerone et al., 1997; Ellaway et al., 2001; Heylen et al., 2012; Kahraman et al., 2022; Praece et al., 1996; “Prevalence and Architecture of de Novo Mutations in Developmental Disorders,” 2017; Rüetschi et al., 2000; Szymanska et al., 2015; Tomoeda et al., 2000; Tong et al., 2019; Turner et al., 2019; Turro et al., 2020; Vakili et al., 2021; Wang et al., 2019; Zhang et al., 2021; Zhao et al., 2020) (Table S1). Among them, 57.14% (12/21) exhibited neurological manifestations, such as intellectual disability and seizures. Notably, instances of intellectual disability were notably diminished in those who received intervention before their sixth month (11.1% vs. 58.33%) (p = 0.0349), indicating that a low‐tyrosine regimen might exert an influence on the developmental trajectory of HT III. Moreover, seven cases exhibited no overt clinical signs, primarily identified via neonatal screening, and managed systematically (Ellaway et al., 2001; Heylen et al., 2012; Rüetschi et al., 2000; Szymanska et al., 2015; Tong et al., 2019; Vakili et al., 2021). In our case, the patient received a diet restricted in tyrosine and phenylalanine at the initiation of her first month. Following this dietary intervention, the serum tyrosine levels of the patient consistently adhered to the accepted normal range. While the presence of 4‐hydroxy‐phenyllactic acid and 4‐hydroxy‐phenylpyruvic acid in urine continued to surpass established benchmarks, these concentrations displayed a noticeable decrease when contrasted with their levels prior to treatment commencement. As the patient approached the age of 20 months, no further aberrations were discerned. Drawing from these collective findings, along with our own clinical observations, we emphasize the pivotal role of initiating interventions during infancy and recommend the implementation of dietary restrictions targeting tyrosine and phenylalanine intake throughout childhood.

After reviewing the existing literature, a total of 11 variants were identified in HT III patients (including our patient), corresponding to six missense, three splice sites, and two nonsense variants (Figure 2). Genotypes were unique in most families, and only one variant—p.Y160C—was observed in two families. Notably, the majority of the variants (9/11, 81.82%) were concentrated in the region that might affect the VOC domain 2, a critical component for enzymatic functionality. In this study, we identified two HPD variants, p.T219M and p.A244V, which predicted to impede correct folding and eliminate VOC domain 2 function, and are therefore harmful. However, further functional studies are needed to validate their pathogenicity.

FIGURE 2.

Reported variants in the HPD gene. The intron–exon structure of the HPD gene is shown, along with previously reported variants. The variants found in this study are shown in purple within exon 10. Disease‐associated variants in the literatures are shown in red, while the heterozygous or indeterminate variants are shown in brown. Pathogenic or likely pathogenic variants recorded in the ClinVar database which were specifically associated with HT III are indicated in red italic, while others are shown in blue.

HT III is inherited autosomal recessively; however, heterozygous variations including c.248delG (p.G83Afs5), c.460G>A (p.G154S), and c.784G>A (p.A262T) can likewise raise blood tyrosine levels (Wang et al., 2019; Zhang et al., 2021; Zhao et al., 2020). Several possibilities exist are given as follows: (i) Targeted NGS may have missed harmful mutations on the opposite allele, such as intronic variants or larger fragment deletions/duplications; (ii) certain HPD variations may cause enzyme activity to drop below a threshold, resulting in higher blood tyrosine levels; and (iii) HT III may be inherited in both dominant and recessive manners, similar to hypermethioninemia (Panmanee et al., 2020). Unfortunately, the family members of our case declined to undergo biochemical testing, thereby limiting our ability to obtain their tyrosine levels. The unavailability of this data due to the family's refusal represents a limitation in our study. As universal neonatal screening programs are adopted internationally, larger cohorts of HT III patients could test this idea.

In conclusion, we identified a patient with HT III through newborn screening, who was successfully treated with a low‐phenylalanine and low‐tyrosine diet. A novel missense variant, p.A244V, in the HPD gene was detected, expanding our understanding of the genetic variants associated with HT III. Furthermore, this study provides insights into known HPD gene variants, aiding in our understanding of the correlation between genotypes and phenotypes, and the potential underlying pathogenic mechanisms. However, further research with larger cohorts and multicenter collaborations are necessary to fully elucidate the genetic mechanisms and better characterize this exceptionally rare disease.

AUTHOR CONTRIBUTIONS

Dong Han conceived, designed, and performed the experiment. Yilun Tao conducted genetic data acquisition and interpretation, reviewed the published cases, and wrote the manuscript. Chen Zhao and Juan Li performed the patient follow‐up. Lihong Wang and Xiaoze Li provided the clinical treatment guidance and performed in patient management. Haiwei Wang and Chenggang Huang revised the manuscript. Wenxia Song supervised the study. All authors have read and approved the final manuscript.

FUNDING INFORMATION

This research did not receive any specific grant from funding agencies in the public, commercial, or not‐for‐profit sectors.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

ETHICS STATEMENT

This study was approved by the Clinical Research Ethics Committee of Changzhi Maternal and Child Health Care Hospital. Written informed consent was obtained from the parent/legal guardian of the patient included in this study.

CONSENT FOR PUBLICATION

The patient's parents provided written informed consent for the publication of this report.

Supporting information

Table S1.

Table S2.

Han, D. , Wang, L. , Zhao, C. , Li, J. , Huang, C. , Song, W. , Wang, H. , Li, X. , & Tao, Y. (2024). Novel HPD mutation p.A244V compound with p.T219M causing tyrosinemia type III in a Chinese girl and review of the genotype–phenotype spectrum Molecular Genetics & Genomic Medicine, 12, e2298. 10.1002/mgg3.2298

DATA AVAILABILITY STATEMENT

All data described in this study are provided within the article and supplementary material. Raw sequencing data and de‐identified clinical data are available from the corresponding authors upon request.