Abstract
Alkaptonuria (AKU) is a rare autosomal recessive disorder of amino acid metabolism caused by defects in the HGD gene. The diagnosis of AKU is often delayed or missed due to its insidiousness. AKU is far from well-known in China. This study aims to provide an epidemiological synthesis of Chinese AKU patients. Firstly, we reported a Chinese pediatric AKU patient who visited because of short stature. Her urinary organic acid analysis revealed a small amount of homogentisic acid (HGA). Whole exome sequencing and Sanger sequencing revealed that the patient carried a c.343-1G > A/c.1027 A > C(p.Met343Leu) compound heterozygous variant in the HGD gene, which was inherited from her parents, respectively. During 4-year follow-up, a small amount of HGA was persistently present in her urine, whereas her height exhibited catch-up growth and subsequently normalized. According to the literature, 91 Chinese AKU patients, including the present one, were included. Among them, the male-to-female ratio was approximately 2:1, and the average age at diagnosis was 36.99 ± 23.05 years. The most common clinical feature was darkened urine (100.00 %), followed by arthropathy (69.23 %) and pigmentation of the skin or sclera (58.24 %). Pigmentation of the skin or sclera and arthropathy occurred more frequently in adult patients. Eleven different variants in the HGD gene were identified, including 5 known variants and 6 not listed in HGMD. This study describes a new Chinese AKU patient and summarizes the clinical and molecular features of Chinese AKU patients, which will help enrich the knowledge of AKU and contribute to early recognition, diagnosis and treatment.
Keywords: Alkaptonuria; HGD gene; Homogentisate 1,2-dioxygenase; Homogentisic acid; Urinary organic acid analysis; Whole exome sequencing; Sanger sequencing
Highlights
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This study describes a new Chinese patient with AKU.
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This study summarizes the clinical and molecular characteristics of Chinese AKU patients.
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This study suggests a candidate approach for newborn screening for AKU.
1. Introduction
Alkaptonuria (AKU), also known as darkened urine disease or ochronosis in China, is a disorder of amino acid metabolism caused by defects in the HGD gene (OMIM * 607474) with an autosomal recessive inheritance pattern. AKU is an extremely rare disease with an estimated incidence of 1/250,000–1/1,000,000 [1].
The HGD gene, located on human chromosome 3q13.33, encodes homogentisate 1,2-dioxygenase (HGD) comprised of 445 amino acids. HGD, mainly found in the liver, plays an important role in phenylalanine and tyrosine metabolism by catalyzing the degradation of an intermediate product, homogentisic acid (HGA) [2]. The deficiency of HGD in AKU patients leads to the accumulation of HGA in the blood, subsequent excretion in the urine, and eventually deposition in bones, connective tissues, and other organs, which cause multi-system symptoms including darkened urine, ochronosis of the skin and connective tissues, and damage to bone, liver, kidneys, and other organs [3,4].
The onset of AKU is insidious with darkened urine as the first manifestation, which is also the primary manifestation occurred in childhood, while significant clinical symptoms typically do not appear until adulthood. As darkening of urine color may not occur right after voiding, the diagnosis of AKU is often delayed or missed [5,6].
Due to the rarity and insidiousness, AKU is far from well-known in China. Consequently, it is probable that a number of AKU cases are underdiagnosed. While Chinese AKU patients have been reported as single cases or in small series, there is a lack of large cohort studies in Chinese population. This gap prevents the establishment of population-specific clinical knowledge necessary for effective diagnosis and management of AKU in China.
In the present study, we summarized the clinical and genetic characteristics of Chinese AKU patients by presenting a new pediatric case and reviewing the literature, in order to provide an epidemiological synthesis and improve the understanding of this disease.
2. Materials and methods
2.1. Patient
A pediatric patient with AKU was enrolled in Guangzhou Women and Children's Medical Center. Her parents are non-consanguineous and unaffected. The patient and her parents are from southern China and are Han ethnicity. The clinical data comprising medical history, family history, physical examinations, imaging examinations, and laboratory examinations were collected and evaluated retrospectively.
2.2. Molecular analyses
DNA extraction: DNA samples of the patient and her parents were extracted from EDTA-anticoagulated peripheral blood samples using QIAGEN DNeasy Blood and Tissue Kit.
Whole exome sequencing (WES): To identify the genetic cause, the patient-parents trio was first subjected to WES using Agilent SureSelect Human All Exon V6 kit, Illumina NovaSeq 6000 platform, and PE150 sequencing mode. The SNP databases, including 1000Genomes, ESP6500, ExAC, and GnomAD, were employed to exclude the polymorphic alleles, while HGMD (Professional 2025.3) and ClinVar were engaged to confirm the known pathogenic variants. For novel variants, in-silico tools, including PROVEAN, SIFT, PolyPhen-2, MutationTaster, FATHMM, NetGene2, NNSPLICE 0.9, and RNA Splicer, were used to predict the functional consequences. The pathogenicity of variants was evaluated according to the guidelines of American College of Medical Genetics (ACMG) [7].
Sanger sequencing: To verify the suspected variants identified by WES, Sanger sequencing was subsequently conducted. The exons 6 and 13 together with their adjoining intron boundaries in the HGD gene (NG_011957.1, NM_000187.4) were amplified by PCR reactions and sequenced using an ABI 3730xl DNA Analyzer.
2.3. Protein 3-D structure analysis
The 3-D structure of the HGD protein was obtained from the Uniprot database. The wild-type and mutated 3-D structures of the HGD protein were drawn by PyMOL software, while the length of hydrogen bonds was calculated using Discovery Studio software.
2.4. Urine analyses
Urine color observation: The urine samples of the patient and a healthy control were collected and left standing at room temperature with exposure to air. The urine color was observed at days 0, 1, 2, 3, 4, 7, and 10, respectively.
Urinary organic acid analysis: As described previously [8], the urine samples of the patient were collected and standardized to 0.25 mg creatinine, derivatized with 100 μL bis-(trimethylsilyl)trifluoracetamide+1 % trimethylchlorosilane, and allowed to react at 60 °C for 10 min. The metabolites were analyzed using an Agilent 5975C/7890A gas chromatography-mass spectrometer.
2.5. Follow-up
From January 2021 to April 2025, the patient maintained follow-up with an interval varying from 1 month to 16 months. Her height and weight were measured at every visit, while liver and kidney function, serum insulin-like growth factor 1 (IGF-1), and urinary organic acid analysis were monitored periodically.
2.6. Literature review
Publications from 1959 to date regarding Chinese AKU patients were retrieved from China National Knowledge Infrastructure, Chinese Medical Association Journal Database, VIP Database, Wanfang Data Knowledge Service Platform, and PubMed database, respectively, with the use of Chinese terms “尿黑酸尿症”, “黑酸尿症”, “黑尿症”, “褐黄病”, “尿黑酸”, or “HGD基因”, and English terms “alkaptonuria and Chinese”, “alkaptonuria and China”, “homogentisic acid and Chinese”, “homogentisic acid and China”, “Ochronosis and Chinese”, “Ochronosis and China”, “Ochronotic and Chinese”, “Ochronotic and China”, “HGD gene and Chinese”, or “HGD gene and China”. All acquired publications were further reviewed. For AKU patients, the inclusion criterion was a clinical diagnosis of alkaptonuria or ochronosis. Duplicate cases across different publications were identified by comparing patients' clinical information and authors' affiliations, and thus merged. The exclusion criteria were as follows: 1) Lack of family history; 2) Absence of any confirmatory laboratory examinations including HGA qualitative test, HGA quantitative test, histopathological examinations, and HGD gene testing. Patients were excluded only if they met both criteria. Therefore, only patients with complete clinical data were included in the subsequent statistical analyses.
2.7. Statistical analyses
SPSS Statistics 17.0 software was used for statistical analyses. The means, standard deviation (SD), and median were calculated for measurement data, while percentage for enumeration data. For cross-tables, Pearson Chi-square test was conducted to compare clinical manifestations among different age groups. A statistically significant difference was defined as p < 0.05.
3. Results
3.1. Clinical manifestations of the pediatric patient
The female pediatric patient was the second child born to non-consanguineous parents at full-term with a birth length of 48 cm (−1.0 SD) and a birth weight of 3.1 kg (−0.3 SD) [9]. There was no prenatal issue, birth injury or asphyxia. The patient first visited our clinic because of short stature in January 2021 when she was 3 years and 3 months of age. She had normal intelligence and motor function, but her physical development was delayed with a height of 89.5 cm (−2.2 SD) and a weight of 13.4 kg (−0.8 SD) [9]. Neither her parents nor her elder sister had similar symptoms.
Her ultrasonography of abdominal organs and urinary system had no abnormality, whereas pituitary magnetic resonance imaging (MRI) showed a Rathke's cyst (data not shown). No abnormality was found in her blood cell count, liver and kidney function, blood gas analysis, thyroid function, adrenal cortical function, growth hormone, IGF-1, insulin-like growth factor binding protein-3, parathyroid hormone, routine urine examination, urine mucopolysaccharide electrophoresis, and chromosome karyotype (data not shown), whereas urinary organic acid analysis indicated the presence of a small amount of homogentisic acid (HGA) (Fig. 1B).
Fig. 1.
The urinary organic acid chromatograms of the pediatric patient. A. The urinary organic acid chromatogram of a normal control; B-E. The urinary organic acid chromatograms of the patient at the age of 3 years and 3 months, 4 years and 2 months, 6 years and 2 months, and 7 years and 6 months, respectively. The blue arrow indicates internal standard, green arrow indicates external standard, and red arrow indicates HGA.
Based on the clinical phenotypes, imaging examinations, and laboratory tests, the patient was diagnosed with AKU and short stature.
3.2. Molecular findings of the pediatric patient
WES revealed that the patient carried a c.343-1G > A/c.1027 A > C(p.Met343Leu) compound heterozygous variant in the HGD gene, which was inherited from her parents, respectively. No definitive pathogenic variants associated with short stature were detected. Sanger sequencing subsequently confirmed the paternal c.343-1G > A variant and maternal c.1027 A > C(p.Met343Leu) variant in the HGD gene of the patient (Fig. 2).
Fig. 2.
The sequencing diagrams of the enrolled family. The red arrow indicates the c.343-1G > A variant in the HGD gene, while blue arrow indicates c.1027 A > C(p.Met343Leu) variant.
The c.343-1G > A variant is located in the canonical −1 splice site and probably alters the mRNA splicing of the HGD gene. To date, it has not been documented in the SNP databases or HGMD. This variant was first reported in a Chinese patient with AKU and IgA nephropathy in 2023. However, this report was published in Chinese and is not indexed in PubMed. According to the ACMG guidelines, this variant was categorized as pathogenic (PVS1 + PM2_Supporting + PP4) [7].
The c.1027 A > C(p.Met343Leu) variant (rs755078457) is a missense variant leading to the substitution of methionine with leucine at position 343 of the HGD protein. It has a very low global population frequency of 0.00004894 in gnomAD, but a much higher frequency of 0.001626 in East Asian population. Although annotated as likely benign in ClinVar, it was predicted to be damaging by in-silico tools. According to the ACMG guidelines, this variant was categorized as uncertain significance (PM3 + PP2 + PP3 + PP4 + BS1 + BP6) [7].
Methionine at position 343 of the HGD protein is conserved across multiple species (Fig. 3A). It is located on a β-helix and forms hydrogen bonds with serine at position 363, with lengths of 2.85 Å and 3.04 Å, respectively. Although the mutated leucine is a hydrophobic amino acid similar to the wild-type methionine, its side chain exhibits reduced steric hindrance. Consequently, the hydrogen bond lengths change to 2.84 Å and 3.16 Å, respectively, resulting in weakened hydrogen bonding and affecting the protein structure (Fig. 3B). This alteration in the Protein 3-D structure suggests that the c.1027 A > C (p.Met343Leu) variant probably had a damaging effect on the HGD protein.
Fig. 3.
Analyses of HGD protein. A. The alignment of HGD protein sequences from 14 representative organisms. The red frame indicates the mutated position. B. 3-D structure of the HGD protein. Wild type methionine at position 343 is shown with green color in the left panel, whereas mutated leucine is shown with red color in the right panel. The serine at position 363 is shown with yellow color and forms hydrogen bonds with wild type methionine or mutated leucine. The yellow dotted line indicates hydrogen bond.
3.3. Four-year follow-up of the pediatric patient
The patient was followed up in our clinic for 4 years, from January 2021 to April 2025. To avoid interfering with her growth and development, neither a low-protein diet nor medication was administered. The management consisted of observational follow-up and growth monitoring.
During follow-up, the patient's urine turned brown-black after being exposed to air for more than 1 day, whereas that from the healthy control remained light yellow (Fig. 4A). Urinary organic acid analysis showed that a small amount of HGA was persistently present in the patient's urine (Fig. 1C-1E). The patient's height caught up to the normal reference range (Fig. 4B), while her serum IGF-1 levels remained normal (Fig. 4C). By the last visit at 7 years and 6 months of age, her height had reached 118 cm (−1.5 SD), a value that does not support the diagnosis of short stature [9].
Fig. 4.
The follow-up of the pediatric patient. A. Photos of the urine samples from the patient and a normal control after exposure to air for 0, 1, 2, 3, 4, 7, and 10 days, respectively. The control's urine is left standing in the left, whereas the right one is from the patient. B. The height curve of the patient. C. The IGF-1 levels patient.
3.4. Characteristics of Chinese patients with AKU
One hundred and twelve publications, from 1959 to date, regarding Chinese AKU patients were reviewed, including 96 Chinese publications and 16 English publications [5,6,[10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24]], and other 95 Chinese publications]. After merging duplicate cases, 116 Chinese AKU patients have been identified. Ninety-one patients with complete clinical data, including the present patient, were included in the subsequent statistical analyses (Fig. 5, Table 1).
Fig. 5.
The flow diagram of the Chinese AKU patient cohort. CNKI, China National Knowledge Infrastructure; CMAJD, Chinese Medical Association Journal Database; Wanfang, Wanfang Data Knowledge Service Platform.
Table 1.
Compilation of clinical features of 91 Chinese AKU patients.
| Patient | Gender | Family history | Age at diagnosis (years) |
Clinical manifestations |
Laboratory examinations |
|||||
|---|---|---|---|---|---|---|---|---|---|---|
| Darkened urine | pigmentation of the skin or sclera | Arthropathy | HGA qualitative test | HGA quantitative test | Histopathological examinations | HGD gene testing | ||||
| 1 | Female | + | 45 | + | − | + | / | / | Kidney+ | c.343-1G > A, heterozygous |
| 2 | Male | − | 47 | + | + | + | + | / | / | / |
| 3 | Male | − | 2 | + | + | − | + | / | / | / |
| 4 | Female | − | 10 | + | − | − | + | / | / | / |
| 5 | Female | − | 8 | + | − | − | + | / | / | / |
| 6 | Female | − | 6.75 | + | − | + | + | / | / | / |
| 7 | Male | + | 47 | + | + | + | + | / | / | / |
| 8 | Male | + | 51 | + | + | + | + | / | / | / |
| 9 | Female | − | 3 | + | − | − | + | / | / | / |
| 10 | Male | − | 40 | + | + | + | + | + | / | / |
| 11a | Male | + | 16 | + | + | − | + | / | / | / |
| 12a | Male | + | 9 | + | − | − | + | / | / | / |
| 13 | Female | − | 17 | + | + | + | + | / | / | / |
| 14 | Female | − | 9 | + | − | − | + | / | / | / |
| 15 | Female | − | 1.67 | + | − | − | + | / | / | / |
| 16 | Male | − | 66 | + | + | + | + | / | / | / |
| 17 | Female | − | 1.5 | + | − | − | + | / | / | / |
| 18 | Female | − | 19 | + | − | − | + | / | / | / |
| 19 | Male | − | 42 | + | + | + | + | + | / | / |
| 20b | Male | + | 17 | + | + | − | + | / | / | / |
| 21b | Male | + | 9 | + | − | − | + | / | / | / |
| 22c | Male | + | 6 | + | − | − | + | / | / | / |
| 23c | Female | + | 9 | + | − | − | + | / | / | / |
| 24c | Male | + | 11 | + | − | − | + | / | / | / |
| 25 | Female | − | 40 | + | − | + | + | / | / | / |
| 26 | Female | − | 6 | + | − | − | + | / | / | / |
| 27 | Male | − | 0.75 | + | − | − | / | + | / | 1) c.221 A > T(p.Glu74Val), heterozygous; 2) c.1095_1100dupCAGCAC(p.Ser366_Thr367dup) ⁎, heterozygous |
| 28 | Female | − | 0.17 | + | − | − | / | + | / | 1) c.124delG(p.Glu42Serfs⁎69), heterozygous, maternal; 2) c.342 + 3 A > C, heterozygous, paternal |
| 29 | Male | − | 48 | + | + | + | / | / | Skin+ | / |
| 30 | Male | − | 55 | + | + | + | / | / | Bone+ | |
| 31 | Male | − | 55 | + | + | + | / | / | Bone+ | c.469 + 1G > C, homozygous |
| 32 | Female | − | 67 | + | − | + | / | / | Bone+ | |
| 33 | Male | − | 70 | + | + | + | / | / | Bone+ | / |
| 34 | Male | − | 62 | + | − | + | / | / | Bone+ | / |
| 35 | Male | − | 41 | + | + | + | + | / | / | / |
| 36 | Male | − | 45 | + | − | + | + | / | / | / |
| 37d | Male | + | 57 | + | + | + | + | / | / | / |
| 38d | Male | + | 49 | + | − | + | + | / | / | / |
| 39 | Female | − | 40 | + | − | + | + | / | / | / |
| 40 | Male | − | 45 | + | − | + | + | + | Bone+ | / |
| 41 | Male | − | 49 | + | + | + | + | / | Skin+ | / |
| 42 | Male | − | 45 | + | + | + | + | / | / | / |
| 43 | Male | − | 52 | + | − | + | + | / | Bone+ | / |
| 44 | Male | − | 47 | + | + | + | + | + | / | / |
| 45 | Male | − | 70 | + | + | + | / | / | Bone+ | / |
| 46 | Male | − | 49 | + | + | + | + | / | / | / |
| 47 | Male | − | 68 | + | + | + | + | / | / | / |
| 48 | Male | − | 55 | + | + | + | + | + | / | / |
| 49 | Female | − | 60 | + | − | + | / | / | Bone+ | / |
| 50 | Female | − | 52 | + | + | + | / | / | Bone+ | / |
| 51 | Male | − | 64 | + | − | + | + | / | Bone+ | / |
| 52 | Male | − | 55 | + | + | + | + | / | Bone+ | / |
| 53 | Male | − | 66 | + | + | + | + | / | Bone+ | / |
| 54 | Male | − | 47 | + | − | + | + | / | / | / |
| 55 | Male | − | 43 | + | + | + | + | / | / | / |
| 56 | Male | − | 40 | + | + | + | + | / | / | / |
| 57e | Male | + | 60 | + | + | + | + | / | / | / |
| 58e | Female | + | 55 | + | + | + | + | / | / | / |
| 59 | Male | − | 44 | + | + | + | + | / | / | / |
| 60 | Male | + | 56 | + | + | + | + | + | / | / |
| 61 | Female | − | 0.08 | + | − | − | / | + | / | 1) c.34 A > C(p.Asn12His), heterozygous, maternal; 2) c.910 A > G(p.Lys304Glu), heterozygous, paternal |
| 62 | Female | − | 3.75 | + | − | − | / | + | / | / |
| 63 | Male | − | 18 | + | + | + | + | / | / | / |
| 64 | Female | − | 0 (1 day) | + | − | − | + | / | / | / |
| 65 | Male | − | 28 | + | − | − | + | / | / | / |
| 66 | Female | − | 2.33 | + | + | − | + | / | / | / |
| 67 | Male | − | 38 | + | + | + | + | / | / | / |
| 68f | Male | + | 60 | + | + | + | + | / | / | / |
| 69f | Female | + | 55 | + | + | + | + | / | / | / |
| 70 | Male | − | 44 | + | + | + | + | / | / | / |
| 71 | Female | − | 57 | + | + | + | + | / | / | / |
| 72 | Male | − | 38 | + | + | + | + | + | / | / |
| 73 | Male | − | 38 | + | + | + | + | / | / | / |
| 74 | Male | − | 48 | + | + | + | + | / | Skin+ | / |
| 75 | Male | + | 42 | + | + | + | + | / | / | / |
| 76 | Male | + | 49 | + | + | + | / | / | Bone+ | / |
| 77 | Male | + | 60 | + | + | + | / | / | Bone+ | / |
| 78 | Male | − | 65 | + | + | + | + | / | / | / |
| 79 | Female | − | 56 | + | + | + | + | / | Bone+ | / |
| 80 | Male | − | 0.83 | + | − | − | + | + | / | / |
| 81 | Female | − | 0.25 | + | − | − | / | + | / | 1) c.469 + 1G > C, heterozygous, paternal; 2) c.986 T > G(p.Phe329Cys), heterozygous, maternal |
| 82 | Female | − | 63 | + | + | + | / | / | Bone+ | / |
| 83 | Male | + | 6 | + | − | − | + | + | / | c.15G > A(p.Lys5=), homozygous, parental |
| 84 | Male | + | 0.08 | + | − | − | + | + | / | c.15G > A(p.Lys5=), homozygous, parental |
| 85 | Male | − | 52 | + | − | + | + | / | Bone+ | / |
| 86 | Female | − | 62 | + | + | + | / | + | / | / |
| 87 | Male | − | 54 | + | + | + | + | / | Bone+ | / |
| 88 | Female | − | 61 | + | + | + | / | / | Bone+ | / |
| 89 | Male | − | 56 | + | + | + | + | / | Bone+ | / |
| 90 | Male | − | 55 | + | + | + | / | / | Bone+ | / |
| 91 | Female | − | 3.25 | + | − | − | / | + | / | 1) c.343-1G > A, heterozygous, paternal; 2) c.1027 A > C(p.Met343Leu), heterozygous, maternal |
Patient 11 and 12, b Patient 20 and 21, c Patient 22, 23 and 24, d Patient 37 and 38, e Patient 57 and 58, and f Patient 68 and 69 were siblings.
This variant was named c.1100_1101insCAGCAC(p.Ser366_Thr367insThrSer) in the original reference; however, in this study, it was named c.1095_1100dupCAGCAC(p.Ser366_Thr367dup) according to the Human Genome Variation Society (HGVS) guidelines.
Among 91 Chinese AKU patients, 60 were male and 31 were female, with a male-to-female ratio of approximately 2:1. Twenty-two of the patients (24.18 %, 22/91) had a family history of darkened urine and/or arthropathy. Their ages at diagnosis ranged from 1 day to 70 years, with an average age of 36.99 ± 23.05 years and a median age of 45 years.
All patients (100.00 %, 91/91) had darkened urine, 58.24 % of patients (53/91) presented pigmentation of the skin or sclera, and 69.23 % of patients (63/91) suffered from arthropathy. The earliest onset age was 2 years for pigmentation of the skin or sclera and 6.75 years for arthropathy. Among the patients presenting pigmentation of the skin or sclera, 6 (11.32 %, 6/53) were children, whereas the majority of patients (88.68 %, 47/53) were adults older than 38 years of age. Among the patients undergoing arthropathy, 3 (4.76 %, 3/63) were children, whereas the majority of patients (95.24 %, 60/63) were adults older than 38 years of age.
Based on the age at diagnosis, 91 patients were divided into 4 groups: (1) Children group (0–18 years) comprising 29 cases (31.87 %, 29/91); (2) Young adult group (19–45 years) comprising 19 cases (20.88 %, 19/91); (3) Middle-aged group (46–59 years) comprising 27 cases (29.67 %, 27/91); (4) Elderly group (≥60 years) comprising 16 cases (17.58 %, 16/91).
The majority of patients (68.13 %, 62/91) were adults over 19 years old. Darkened urine was present in all patients across all age groups. However, the frequencies of pigmentation of the skin or sclera and of arthropathy significantly differed among age groups (p < 0.0001), being markedly higher in adult patients. Only 20.69 % (6/29) and 10.34 % (3/29) of the children exhibited pigmentation of the skin or sclera, and arthropathy, respectively, whereas the proportions were greater than 63.00 % and 89.00 % in adults. All patients in the middle-aged group and elderly group were affected by arthropathy (Table 2).
Table 2.
Comparison of clinical manifestations among different age groups of Chinese AKU patients.
| Age group | Clinical manifestations |
||
|---|---|---|---|
| Darkened urine | Pigmentation of the skin or sclera | Arthropathy | |
| Children group | 100.00 % (29/29) | 20.69 % (6/29) | 10.34 % (3/29) |
| Young adult group | 100.00 % (19/19) | 63.16 % (12/19) | 89.47 % (17/19) |
| Middle-aged group | 100.00 % (27/27) | 85.19 % (23/27) | 100.00 % (27/27) |
| Elderly group | 100.00 % (16/16) | 75.00 % (12/16) | 100.00 % (16/16) |
| Pearson Chi-square test | / | p < 0.0001 | p < 0.0001 |
Of the 91 Chinese AKU patients, the diagnosis was made in 65 (71.43 %, 65/91) by qualitative/quantitative tests for HGA, in 15 (16.48 %, 15/91) by histopathological examinations, and in the remaining 11 (12.09 %, 11/91) by both criteria. Among 76 patients who received qualitative/quantitative tests for HGA, 17 (22.37 %, 17/76) underwent urinary organic acid analysis using chromatography or gas chromatography–mass spectrometry.
Only 9 of 91 patients (9.89 %, 9/91) were subjected to genetic testing for the HGD gene. Eleven different variants in the HGD gene were identified, including 5 known pathogenic variants: c.15G > A(p.Lys5=), c.124delG(p.Glu42Serfs*69), c.342 + 3 A > C, c.469 + 1G > C, and c.986 T > G(p.Phe329Cys); and 6 variants absent from HGMD (Professional 2025.3): c.34 A > C(p.Asn12His), c.221 A > T(p.Glu74Val), c.343-1G > A, c.910 A > G(p.Lys304Glu), c.1027 A > C(p.Met343Leu), and c.1095_1100dupCAGCAC(p.Ser366_Thr367dup). Based on nucleotide changes, these 11 variants were classified as 9 base substitutions (81.82 %, 9/11), 1 small deletion (9.09 %, 1/11), and 1 small insertion/duplication (9.09 %, 1/11). Based on amino acid changes, they were classified as 4 splice site variants (36.36 %, 4/11), 1 frameshift variant (9.09 %, 1/11), 5 missense variants (45.45 %, 5/11), and 1 amino acid insertion/duplication (9.09 %, 1/11).
4. Discussion
AKU is a very rare disease. Reports of AKU in Chinese population are limited to single cases or small series, whereas large cohort studies are lacking. To better describe clinical and genetic characteristics of AKU patients in China and provide an epidemiological synthesis, we reviewed 112 publications [5,6,[10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24]], and other 95 Chinese publications] and enrolled 91 Chinese AKU patients with complete clinical data, including the present patient, for statistical analyses.
The male-to-female ratio of Chinese AKU patients was 2:1, which fits well with the previous studies [25,26]. A family history of darkened urine and/or arthropathy was shown in 24.18 % of Chinese AKU patients, highlighting the necessity of AKU screening and testing for the family members.
AKU is an insidious disease. The diagnosis of this disease is often delayed or missed. Darkened urine, the first clinical clue for AKU, first appears in infancy. Some infants are diagnosed because their black-stained diapers or clothes are noticed [6,10], whereas most AKU patients are not recognized that early as their fresh urine shows a normal color if it is not exposed to air for a long time. In Chinese AKU patients, the average age at diagnosis was 36.99 ± 23.05 years, which is older than the reports from the USA and Pakistan [1,25]. It had been reported that about 21 % of AKU patients in the USA (12/58) could be diagnosed before 1 year of age [1]. However, in Chinese AKU patients, only 7.69 % (7/91) were diagnosed that early, though most of them recalled a history of darkened urine in infancy or childhood after diagnosis. The poor awareness and limited recognition of this disease in China lead to delayed diagnosis.
The most common clinical feature of Chinese AKU patients was darkened urine (100.00 %), followed by arthropathy (69.23 %) and pigmentation of the skin or sclera (58.24 %), comprising the typical triad of this disease. Although the majority of patients with pigmentation of the skin or sclera (88.68 %) and arthropathy (95.24 %) were older than 38 years of age, the earliest onset ages for these symptoms were 2 years and 6.75 years, respectively, which indicates the high clinical heterogeneity of AKU.
The causative gene of AKU is the HGD gene. Only 9 of 91 Chinese AKU patients (9.89 %) received genetic testing for the HGD gene, revealing that the diagnosis of AKU in the past was mainly based on the clinical phenotypes and urinary biochemical indicators, with a lack of molecular genetic evidence. Actually, the genetic techniques, including Sanger sequencing and next-generation sequencing, have developed rapidly and are used widely in China, especially in recent decades. It is therefore surprising that the rate of genetic testing among Chinese AKU patients remains extremely low. This inadequate use of genetic testing might be due to limited awareness among clinicians and poor adherence among patients. Since the overlooking of darkened urine and the late onset of obvious manifestations result in delayed diagnosis of Chinese AKU patients compared to other populations, the rational utilization of genetic testing will facilitate early diagnosis and improve diagnostic practices.
Currently, 201 different variants in the HGD gene have been documented in HGMD (Professional 2025.3), including 193 disease-causing mutations (DM) and 7 possible disease-causing mutations (DM?). In this study, 11 different variants in the HGD gene were identified in 9 patients, including 5 known pathogenic variants and 6 variants absent from HGMD. Among 6 variants absent from HGMD, the c.1027 A > C(p.Met343Leu) variant was newly suggested with a probably damaging effect in this study. The other 5 variants, despite having been reported before 2023, remain undocumented in HGMD because HGMD does not incorporate non-PubMed-indexed Chinese publications. The same bias is also observed in other global databases, such as abSNP and ClinVar. As China has the largest population in the world and possesses a vast and diverse patient resource pool, this bias creates a substantial knowledge gap. To address this, it is crucial to either develop a Chinese population-specific genetic framework or database, or to advocate for the inclusion of non-PubMed-indexed Chinese publications in these global databases.
The current managements of AKU are generally supportive and palliative to reduce HGA production, delay disease progression, and improve living quality, including a low-protein diet, ascorbic acid supplement, and symptomatic treatments [3,4]. Besides, nitisinone, an inhibitor of 4-hydroxyphenylpyruvate dioxygenase that decreases the formation of HGA [27,28], recently has been approved by the Food and Drug Administration (FDA) for the treatment of AKU; however, its long-term therapeutic effect remains to be observed.
Thus, early diagnosis and intervention are crucial for preventing severe symptoms and improving prognosis of AKU. However, AKU is not currently screened for newborns. Since AKU cannot be identified by a routine tandem mass spectrometry (MS) assay of blood, traditional MS/MS newborn screening for metabolic disorders using heel stick dried blood spot does not include it. In this study, the patient's urine was light yellow when fresh and turned brown-black after being exposed to air for 1 day (Fig. 4A). Therefore, observation of color change for the newborns' urine after exposure to air for 1 day might help achieve early diagnosis for AKU. This simple, inexpensive and non-invasive method represents a potential approach for newborn screening for AKU, being particularly acceptable in low-income settings.
In addition, the present patient first visited because of short stature. However, WES did not identify any definitive pathogenic variants underlying this condition. Furthermore, her height showed a catch-up growth, reaching the normal reference range during follow-up. Therefore, her transient short stature was likely coincidental.
5. Conclusions
This study provides a definite diagnosis of AKU for a new Chinese patient, which will be of benefit to the disease screening and genetic counseling for the family members. This study also summarizes the clinical and molecular characteristics of Chinese AKU patients, which will improve the understanding of this disease and be conducive to early recognition, diagnosis and treatment.
CRediT authorship contribution statement
Xiaomei Qiu: Writing – original draft, Formal analysis, Data curation. Yuqing Liu: Writing – original draft, Formal analysis, Data curation. Yongxian Shao: Investigation, Formal analysis. Xiaojian Mao: Resources, Data curation. Jingqi Zhang: Formal analysis. Xiaodan Chen: Validation. Xi Yin: Investigation. Huiying Sheng: Investigation. Xiuzhen Li: Writing – review & editing, Supervision, Resources, Project administration, Methodology, Investigation, Data curation. Yunting Lin: Writing – review & editing, Writing – original draft, Supervision, Project administration, Methodology, Investigation, Formal analysis.
Consent
Informed consent was obtained from the participants or the guardians of under-aged participants.
Ethics approval
This study was approved by the Institutional Review Board of Guangzhou Women and Children's Medical Center (Guangzhou, China) (No. 2015–112).
Funding
This work was supported by the “Zhuoxing Plan” Young Talent Cultivation Program - Zhuoxue Category from Guangzhou Women and Children's Medical Center (awarded to Yunting Lin).
Declaration of competing interest
The authors declare that they have no conflict of interest.
Acknowledgements
The authors would like to thank the enrolled family for participation in this study.
Contributor Information
Xiuzhen Li, Email: 13725100840@163.com.
Yunting Lin, Email: linyunting1989@foxmail.com.
Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.