- Email: [email protected]
A novel mutation in xanthine dehydrogenase in a case with xanthinuria in Hunan province of China
Clinica Chimica Acta 504 (2020) 168–171
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/cca
Case report
A novel mutation in xanthine dehydrogenase in a case with xanthinuria in Hunan province of China Tao Xua, Xiaobing Xieb, Zhen Zhangb, Ningzhi Zhaob, Yuanfu Dengb, Ping Lib, a b
T
⁎
Department of Emergency Medicine, First Affiliated Hospital, Hunan University of Chinese Medicine, No. 93 Shaoshan Middle Road, Changsha 410007, China Medical Laboratory Center, First Affiliated Hospital, Hunan University of Chinese Medicine, No, 93 Shaoshan Middle Road, Changsha 410007, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Xanthinuria Xanthinemia Hypouricemia Xanthine dehydrogenase Molybdenum cofactor sulfurase
Xanthinuria is a rare genetic metabolic disorder, the biochemical mechanism of xanthinuria is the disturbance of purine to uric acid metabolism due to the deficiency of xanthine dehydrogenase/xanthine oxidase (XDH/XO) and aldehyde oxidase 1 (AOX1). Xanthinuria has large clinical variability and only about half of all patients have urolithiasis. In this article, we present one xanthinuria case from an unrelated family, which diagnosed by clinical, biochemical and finally confirmed by molecular genetics. One mutation in XDH gene c.2737C > T (p.R913W) and another mutation in SEPT9 gene (c.655C > T (p.R219W)) were identified. To our knowledge, this is the first time that these novel mutations reported in the xanthinuria patients.
1. Introduction Xanthinuria, which was first reported by Dent and Philiport in 1954 [1], is a rare autosomal recessive inherited metabolic disorder, characterized by low concentration of uric acid in serum and high excretion of xanthine and hypoxanthine in blood and urine. According to the enzymes involved, xanthinuria can be classified into type I (OMIM 278300), type II (OMIM 603592), and type III (OMIM 252150). Type I is caused by xanthine dehydrogenase/xanthine oxidase (XDH/XO) deficiency due to mutations in XDH gene, but possesses normal activity of aldehyde, while type II results from molybdenum cofactor sulfurase (MOCOS) deficiency, combined XDH/XO and aldehyde oxidase (AOX1) activities caused by mutations in MOCOS and AOX1 gene. The typeIII, involves the molybdenum cofactor deficiency related with triple deficiency of sulfite oxidase (SO) as well as XDH/XO and AOX1 activities, due to a defect in the synthesis of molybdopterin, which is a precursor of molybdenum cofactor for all three enzymes. Symptoms of typeIII include severe neurological disorder, lens dislocation and dysmorphism, and the outcome is poor [2]. Type I and type II are similar in clinical symptoms, but distinct in biochemical manifestation. Type I patients can metabolize allopurinol, whereas type II patients don’t metabolize allopurinol due to deficiency of aldehyde activities. Generally, patients with xanthinuria are considered to be asymptomatic. Xanthinuria has large clinical variability and only about half of all patients have urolithiasis. Patients with either type I or type II sometimes develop xanthine calculi in the urinary tract, acute renal failure
⁎
and myopathy due to tissue deposition of xanthine. In this article, we present one patient from unrelated family with xanthinuria diagnosed by clinical and biochemical techniques and finally screened related mutation sites by molecular genetics. Besides, family analysis was also performed in the patient. 2. Patients and methods 2.1. Subjects The patient and her parents received information about the study, and signed the informed consents. All tests were performed in accordance with the standards of the institutional ethics committee, which approved the projects. 2.2. Patient A 40-year-old female was born of unrelated parents after an uncomplicated pregnancy. She complained of slight pain in her posterior back and elbow during an outpatient examination. Besides, there were no other obvious abnormal manifestations. 2.3. Determination of uric acid, xanthine, and hypoxanthine The level of uric acid in serum and urine were measured by fully automatic chemistry analyzer (Cobas 8000, Roche Diagnstics
Corresponding author. E-mail address: [email protected] (P. Li).
https://doi.org/10.1016/j.cca.2020.02.012 Received 14 July 2019; Received in revised form 13 February 2020; Accepted 13 February 2020 Available online 15 February 2020 0009-8981/ © 2020 Elsevier B.V. All rights reserved.
Clinica Chimica Acta 504 (2020) 168–171
T. Xu, et al.
Table 1 The screening of gene variations using WES in the patient. Gene
Region
GRCh38.p12
Change
Amino acid [Codon]
Variant
Reference SNP cluster report
HGMD
XDH SEPT9
Exon 25 Exon 2
chr2:31350118 chr17:77402691
c.2737C > T (p.R913W) c.655C > T (p.R219W)
R[CGG] > W[TGG] R(Arg) > W(Trp) R[CGG] > W[TGG] R(Arg) > W(Trp)
Missense Variant Missense Variant
rs776794071 rs562811871
Not included Not included
Abbreviation: SNP, single nucleotide polymorphism; HGMD, the human gene mutation database
3.2. Genetic analyses
Company). The urinary levels of xanthine and hypoxanthine were measured by high performance liquid chromatography (HPLC) [3] (Ultimate 3000 UHPLC Dionex coupled with a TSQ Quantiva).
In order to diagnose and further identify the types of xanthinuria, we performed WES analysis for the patient. The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive [5] in BIG Data Center [6], Beijing Institute of Genomics (BIG), Chinese Academy of Sciences, under accession numbers HRA000109 that are publicly accessible at https://bigd.big.ac.cn/gsa. Quality control was as follow: clean bases were 14.6G, Q20 was 97.4%, and mean depth of target region was 234X. Rare mutations with frequency less than 5% were screened out, and then the mutations affecting amino acid levels were screened out (including frame shift mutation, wholecode mutation, missense mutation, nonsense mutation, and shear region mutation). Finally, the mutated sites were screened and verified by Sanger sequencing. The detailed information of the two mutated sites in the patient was showed in Table 1 and Fig. 1. The same two mutated sites were verified by Sanger sequencing in the parents of patient and showed in Fig. 1.
2.4. Gene analysis Genomic DNA was extracted from peripheral blood by a DNA Quick II kit (Dainippon Pharmaceuticals, Japan) and whole exome sequencing (WES) analysis was executed by Illumina NovaSeq platform. Quality control was as follow: clean bases > 8G, Q20 > 90%, and mean depth of target region > 100X. Data processing: after the original raw data was filtered, the quality control was performed. Only with the quality control passed, the clean data would be obtained. It was carried out the next step of mutation detection if the coverage was high, compared clean data with the reference genome, then annotated and finally carried out visual analysis. Processing of sequencing: Firstly, we got fastq data from illumine sequencing machine (NovaSeq6000/HiSeq2500 etc.) by using Bcl2fastq (v2.0.1), the acquired data could be processed by Trimmomatic (Version 0.36) to remove low quality reads, bases, trimming adaptors, etc. and got the data cleaned. Then, the clean data was processed with Burrows-Wheeler Aligner (BWA) for alignment on a reference sequence of hg19 and Genome Analysis Toolkit (GATK) for variant calling. Finally, the Variant Call Format (VCF) files were analyzed using the ANNOVAR tools, which contains annotation databases, such as the 1000 genome database, dbSNP database (dbSNP; http://www.ncbi. nlm.nih.gov/SNP), ClinVar database (ClinVar; http://www.ncbi.nlm. nih.gov/clinvar), the Polymorphism Phenotyping v2 database (Polyphen-2; http://genetics.bwh.harvard.edu/pph2), and the Sorting Intolerant from Tolerant database (SIFT; http://sift.jcvi.org). The mutated sites screened from WES were validated eventually by Sanger sequencing (ABI 3700 DNA Analyzer, USA).
We performed an allopurinol loading test in order to discriminate the type of xanthinuria for the patient. After administration of allopurinol, the 2 h-serum level of allopurinol and oxypurinol was 1.15 μg/ ml (reference range: 0.767–1.483 μg/ml) and 3.12 μg/ml (reference range: 2.962–3.250 μg/ml), respectively. The 24 h urinary excretion of allopurinol and oxypurinol was 318 µmol (reference range: 220–335 µmol) and 1502 µmol (reference range: 1120–1600 µmol), respectively. These findings showed that oxypurinol was metabolized after the allopurinol loading test in patient. Therefore, the patient possessed the normal activity of AO and was diagnosed type I xanthinuria.
2.5. Allopurinol loading test
4. Discussion
3.3. Alloprinol loading test
Xanthinuriais associated with genetic dysfunction of XDH/XO, also named asxanthinemia or hypouricemia, which used to be thought of no more than 150 cases all over the world in some reports [7]. Many patients who suffer from hypouricemia are unlikely to be overlooked by themselves even by medical staff, just like the patient in our report was accidentally discovered by routine tests. XDH/XO-deficient patients are frequently identified based on measurement of uric acid in blood. Although various diseases or disorders other than xanthinuria may lead to hypouricemia, e.g., renal hypouricemia, which can be caused by decreased re-absorption due to impaired function of urate transporter in the nephrons, is also clinically asymptomatic in most cases. Some drugs like xanthine oxidoreductase inhibitor (e.g., allopurinol, febuxostat), dugs used either as uricosuric agents or to block other aspects of renal tubule excretion (e.g., sulfinpyrazone, probenecid, benzbromarone) may also cause hypouricemia [8]. However, the case in our report didn’t have kidney disease and didn’t use any drugs as described above. Xanthinuria is classified into type I, type II, and typeIII. Whereas type Iand type II xanthinuria are not clinically distinguishable, and type III is a severe disease that usually don’t survive to adulthood. In order to differentiate them, allopurinol loading test and gene analysis are
An allopurinol loading test was performed at the dose of 10 mg/kg body weight according to the procedure described previously [4]. Serum samples were taken 2 h after oral administration, and urine was collected for 24 h. Allopurinol and oxypurinol in the serum and urine were measured by the method of HPLC (Ultimate 3000 UHPLC Dionex coupled with a TSQ Quantiva).
3. Results 3.1. Concentration of compounds in serum and urine The routine laboratory examination was executed in the patient and her parents. The concentration of uric acid in serum and urine was extremely low (below the lower detection limit), and the levels of xanthine and hypoxanthine in urine of patient were markedly elevated with 270 µmol/mmol Crea (normal range: < 18 µmol/mmol Crea) and 186 µmol/mmol Crea (normal range: < 14 µmol/mmol Crea) respectively. While the levels of uric acid, xanthine and hypoxanthine in urine of parents of the patient were all in the reference ranges. 169
Clinica Chimica Acta 504 (2020) 168–171
T. Xu, et al.
Fig. 1. Mutated sites by the WES analysis were validated by Sanger sequencing in the patient and her parents. The red arrow and rectangle indicates the mutated position in chromatogram and alignment of sequence in both of patient and her mother, respectively. The green arrow and rectangle indicates the wild type (nonmutation) in patient’s father.
evidence to support the relationship between SEPT9 and xanthinuria. In order to validate the genetic etiology of the patient, we arranged the patient’s parents to take the routine laboratory examination and genetics analysis. Unexpectedly, the mother’s uric acid in serum, xanthine and hypoxanthine in urine were all normal but possessed the completely same two mutation sites like her daughter. And the routine laboratory examination and genetics analysis for the patient’s father had normal condition. Therefore, it should be noted the relationship between the two mutations and xanthinuria, and we would follow-up of the mother’s serum uric acid level regularly. Previous case reports showed the patients with xanthinuria were accompanied by rheumatoid arthritis [11] and migratory arthritis [12]. Besides, the accumulation of xanthine and hypoxanthine might cause the inflammation of muscle of neck, elbow, or other parts of body. In our report, the patient has the symptoms of muscle soreness in different parts of body. Accumulative evidence is necessary to disclose the complications of xanthinuria. In conclusion, we have identified two novel mutation sites based on gene analysis in the diagnosis of xanthinuria. More importantly, mother and daughter share the same mutation sites, but only daughter has symptoms, and the underlying mechanism needs further in-depth study.
performed, because a measurement method for activity of MOCOS has not yet been established [4,9]. In the allopurinol loading test, oxipurinol is detected in serum and urine of type I xanthinuria patients after administration of allopurinol, as conversion of allopurinol to oxipurinol is catalyzed by AO. While oxipurinol is not detected in the case of type II xanthinuria owing to deficiency of AO. For the patient in this article, after taking allopurinol loading test, serum and urinary oxipurinol were detected, so she was diagnosed as type I xanthinuria. In this patient, we found two heterozygous mutations: c.2737C > T (p.Arg913Trp) in XDH gene and c.655C > T (p.Arg219Trp) in SEPT9 gene. The data from the Exome Aggregation Consortium (ExAC) showed that population frequency of c.2737C > T (p.Arg913Trp) in XDH gene was less than 0.2‰. The relationship between c.2737C > T (p.Arg913Trp) in XDH gene and xanthinuria has not been reported yet. However, the HGMD database contains other variation like c.2738G > A (p.Arg913Lln) in XDH gene, which affects the same amino acid that has been reported related with thiopurine intolerance by Coelho in 2016 [10]. Similarly, the mutation of c.655C > T (p.R219W) in SEPT9 gene has not been reported. Although two missense mutations c.2737C > T (p.R913W) in XDH gene and c.655C > T (p.R219W) in SEPT9 gene were found respectively, there was no 170
Clinica Chimica Acta 504 (2020) 168–171
T. Xu, et al.
Funding information
[6] BIG Data Center Members, Database resources of the BIG Data Center in 2019, Nucleic Acids Res. 47 (D1) (2019) D8–D14. [7] B. Stiburkova, J. Krijt, P. Vyletal, J. Bartl, E. Gerhatova, M. Korinek, I. Sebesta, Novel mutations in xanthine dehydrogenase/oxidase cause severe hypouricemia: biochemical and molecular genetic analysis in two Czech families with xanthinuria type I, Clin. Chim. Acta 413 (1–2) (2012) 93–99. [8] K. Ichida, Y. Amaya, K. Okamoto, T. Nishino, Mutations associated with functional disorder of xanthine oxidoreductase and hereditary xanthinuria in humans, Int. J. Mol. Sci. 13 (11) (2012) 15475–15495. [9] T. Yamamoto, K. Higashino, N. Kono, M. Kawachi, M. Nanahoshi, S. Takahashi, M. Suda, T. Hada, Metabolism of pyrazinamide and allopurinol in hereditary xanthine oxidase deficiency, Clin. Chim. Acta 180 (2) (1989) 169–175. [10] T. Coelho, G. Andreoletti, J.J. Ashton, A. Batra, N.A. Afzal, Y. Gao, A.P. Williams, R.M. Beattie, S. Ennis, Genes implicated in thiopurine-induced toxicity: comparing TPMT enzyme activity with clinical phenotype and exome data in a paediatric IBD cohort, Sci. Rep. 6 (2016) 34658. [11] A. Jurecka, B. Stiburkova, J. Krijt, W. Gradowska, A. Tylki-Szymanska, Xanthine dehydrogenase deficiency with novel sequence variations presenting as rheumatoid arthritis in a 78-year-old patient, J. Inherit. Metab. Dis. Suppi. 3 (2010) S21–S24. [12] M.J. Bradford, V. Hrehorovich, Xanthinuria, psoriasis and arthritis, Am. J. Med. 46 (1) (1969) 137–141.
This work was supported in part by the Youth Program of National Natural Science Foundation of China (81703917). References [1] C.E. Dent, G.R. Philpot, Xanthinuria, an inborn error (or deviation) of metabolism, Lancet 266 (6804) (1954) 182–185. [2] J.L. Johnson, Prenatal diagnosis of molybdenum cofactor deficiency and isolated sulfite oxidase deficiency, Prenat. Diagn. 23 (1) (2003) 6–8. [3] T. Kojima, T. Nishina, M. Kitamura, T. Hosoya, K. Nishioka, Biochemical studies on the purine metabolism of four cases with hereditary xanthinuria, Clin. Chim. Acta 137 (2) (1984) 189–198. [4] K. Ichida, M. Yoshida, R. Sakuma, T. Hosoya, Two siblings with classical xanthinuria type 1: significance of allopurinol loading test, Intern. Med. 37 (1) (1998) 77–82. [5] Y. Wang, F. Song, J. Zhu, et al., GSA: Genome sequence archive < sup/ > , Genomics Proteomics Bioinformatics 15 (1) (2017) 14–18.
171
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/cca
Case report
A novel mutation in xanthine dehydrogenase in a case with xanthinuria in Hunan province of China Tao Xua, Xiaobing Xieb, Zhen Zhangb, Ningzhi Zhaob, Yuanfu Dengb, Ping Lib, a b
T
⁎
Department of Emergency Medicine, First Affiliated Hospital, Hunan University of Chinese Medicine, No. 93 Shaoshan Middle Road, Changsha 410007, China Medical Laboratory Center, First Affiliated Hospital, Hunan University of Chinese Medicine, No, 93 Shaoshan Middle Road, Changsha 410007, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Xanthinuria Xanthinemia Hypouricemia Xanthine dehydrogenase Molybdenum cofactor sulfurase
Xanthinuria is a rare genetic metabolic disorder, the biochemical mechanism of xanthinuria is the disturbance of purine to uric acid metabolism due to the deficiency of xanthine dehydrogenase/xanthine oxidase (XDH/XO) and aldehyde oxidase 1 (AOX1). Xanthinuria has large clinical variability and only about half of all patients have urolithiasis. In this article, we present one xanthinuria case from an unrelated family, which diagnosed by clinical, biochemical and finally confirmed by molecular genetics. One mutation in XDH gene c.2737C > T (p.R913W) and another mutation in SEPT9 gene (c.655C > T (p.R219W)) were identified. To our knowledge, this is the first time that these novel mutations reported in the xanthinuria patients.
1. Introduction Xanthinuria, which was first reported by Dent and Philiport in 1954 [1], is a rare autosomal recessive inherited metabolic disorder, characterized by low concentration of uric acid in serum and high excretion of xanthine and hypoxanthine in blood and urine. According to the enzymes involved, xanthinuria can be classified into type I (OMIM 278300), type II (OMIM 603592), and type III (OMIM 252150). Type I is caused by xanthine dehydrogenase/xanthine oxidase (XDH/XO) deficiency due to mutations in XDH gene, but possesses normal activity of aldehyde, while type II results from molybdenum cofactor sulfurase (MOCOS) deficiency, combined XDH/XO and aldehyde oxidase (AOX1) activities caused by mutations in MOCOS and AOX1 gene. The typeIII, involves the molybdenum cofactor deficiency related with triple deficiency of sulfite oxidase (SO) as well as XDH/XO and AOX1 activities, due to a defect in the synthesis of molybdopterin, which is a precursor of molybdenum cofactor for all three enzymes. Symptoms of typeIII include severe neurological disorder, lens dislocation and dysmorphism, and the outcome is poor [2]. Type I and type II are similar in clinical symptoms, but distinct in biochemical manifestation. Type I patients can metabolize allopurinol, whereas type II patients don’t metabolize allopurinol due to deficiency of aldehyde activities. Generally, patients with xanthinuria are considered to be asymptomatic. Xanthinuria has large clinical variability and only about half of all patients have urolithiasis. Patients with either type I or type II sometimes develop xanthine calculi in the urinary tract, acute renal failure
⁎
and myopathy due to tissue deposition of xanthine. In this article, we present one patient from unrelated family with xanthinuria diagnosed by clinical and biochemical techniques and finally screened related mutation sites by molecular genetics. Besides, family analysis was also performed in the patient. 2. Patients and methods 2.1. Subjects The patient and her parents received information about the study, and signed the informed consents. All tests were performed in accordance with the standards of the institutional ethics committee, which approved the projects. 2.2. Patient A 40-year-old female was born of unrelated parents after an uncomplicated pregnancy. She complained of slight pain in her posterior back and elbow during an outpatient examination. Besides, there were no other obvious abnormal manifestations. 2.3. Determination of uric acid, xanthine, and hypoxanthine The level of uric acid in serum and urine were measured by fully automatic chemistry analyzer (Cobas 8000, Roche Diagnstics
Corresponding author. E-mail address: [email protected] (P. Li).
https://doi.org/10.1016/j.cca.2020.02.012 Received 14 July 2019; Received in revised form 13 February 2020; Accepted 13 February 2020 Available online 15 February 2020 0009-8981/ © 2020 Elsevier B.V. All rights reserved.
Clinica Chimica Acta 504 (2020) 168–171
T. Xu, et al.
Table 1 The screening of gene variations using WES in the patient. Gene
Region
GRCh38.p12
Change
Amino acid [Codon]
Variant
Reference SNP cluster report
HGMD
XDH SEPT9
Exon 25 Exon 2
chr2:31350118 chr17:77402691
c.2737C > T (p.R913W) c.655C > T (p.R219W)
R[CGG] > W[TGG] R(Arg) > W(Trp) R[CGG] > W[TGG] R(Arg) > W(Trp)
Missense Variant Missense Variant
rs776794071 rs562811871
Not included Not included
Abbreviation: SNP, single nucleotide polymorphism; HGMD, the human gene mutation database
3.2. Genetic analyses
Company). The urinary levels of xanthine and hypoxanthine were measured by high performance liquid chromatography (HPLC) [3] (Ultimate 3000 UHPLC Dionex coupled with a TSQ Quantiva).
In order to diagnose and further identify the types of xanthinuria, we performed WES analysis for the patient. The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive [5] in BIG Data Center [6], Beijing Institute of Genomics (BIG), Chinese Academy of Sciences, under accession numbers HRA000109 that are publicly accessible at https://bigd.big.ac.cn/gsa. Quality control was as follow: clean bases were 14.6G, Q20 was 97.4%, and mean depth of target region was 234X. Rare mutations with frequency less than 5% were screened out, and then the mutations affecting amino acid levels were screened out (including frame shift mutation, wholecode mutation, missense mutation, nonsense mutation, and shear region mutation). Finally, the mutated sites were screened and verified by Sanger sequencing. The detailed information of the two mutated sites in the patient was showed in Table 1 and Fig. 1. The same two mutated sites were verified by Sanger sequencing in the parents of patient and showed in Fig. 1.
2.4. Gene analysis Genomic DNA was extracted from peripheral blood by a DNA Quick II kit (Dainippon Pharmaceuticals, Japan) and whole exome sequencing (WES) analysis was executed by Illumina NovaSeq platform. Quality control was as follow: clean bases > 8G, Q20 > 90%, and mean depth of target region > 100X. Data processing: after the original raw data was filtered, the quality control was performed. Only with the quality control passed, the clean data would be obtained. It was carried out the next step of mutation detection if the coverage was high, compared clean data with the reference genome, then annotated and finally carried out visual analysis. Processing of sequencing: Firstly, we got fastq data from illumine sequencing machine (NovaSeq6000/HiSeq2500 etc.) by using Bcl2fastq (v2.0.1), the acquired data could be processed by Trimmomatic (Version 0.36) to remove low quality reads, bases, trimming adaptors, etc. and got the data cleaned. Then, the clean data was processed with Burrows-Wheeler Aligner (BWA) for alignment on a reference sequence of hg19 and Genome Analysis Toolkit (GATK) for variant calling. Finally, the Variant Call Format (VCF) files were analyzed using the ANNOVAR tools, which contains annotation databases, such as the 1000 genome database, dbSNP database (dbSNP; http://www.ncbi. nlm.nih.gov/SNP), ClinVar database (ClinVar; http://www.ncbi.nlm. nih.gov/clinvar), the Polymorphism Phenotyping v2 database (Polyphen-2; http://genetics.bwh.harvard.edu/pph2), and the Sorting Intolerant from Tolerant database (SIFT; http://sift.jcvi.org). The mutated sites screened from WES were validated eventually by Sanger sequencing (ABI 3700 DNA Analyzer, USA).
We performed an allopurinol loading test in order to discriminate the type of xanthinuria for the patient. After administration of allopurinol, the 2 h-serum level of allopurinol and oxypurinol was 1.15 μg/ ml (reference range: 0.767–1.483 μg/ml) and 3.12 μg/ml (reference range: 2.962–3.250 μg/ml), respectively. The 24 h urinary excretion of allopurinol and oxypurinol was 318 µmol (reference range: 220–335 µmol) and 1502 µmol (reference range: 1120–1600 µmol), respectively. These findings showed that oxypurinol was metabolized after the allopurinol loading test in patient. Therefore, the patient possessed the normal activity of AO and was diagnosed type I xanthinuria.
2.5. Allopurinol loading test
4. Discussion
3.3. Alloprinol loading test
Xanthinuriais associated with genetic dysfunction of XDH/XO, also named asxanthinemia or hypouricemia, which used to be thought of no more than 150 cases all over the world in some reports [7]. Many patients who suffer from hypouricemia are unlikely to be overlooked by themselves even by medical staff, just like the patient in our report was accidentally discovered by routine tests. XDH/XO-deficient patients are frequently identified based on measurement of uric acid in blood. Although various diseases or disorders other than xanthinuria may lead to hypouricemia, e.g., renal hypouricemia, which can be caused by decreased re-absorption due to impaired function of urate transporter in the nephrons, is also clinically asymptomatic in most cases. Some drugs like xanthine oxidoreductase inhibitor (e.g., allopurinol, febuxostat), dugs used either as uricosuric agents or to block other aspects of renal tubule excretion (e.g., sulfinpyrazone, probenecid, benzbromarone) may also cause hypouricemia [8]. However, the case in our report didn’t have kidney disease and didn’t use any drugs as described above. Xanthinuria is classified into type I, type II, and typeIII. Whereas type Iand type II xanthinuria are not clinically distinguishable, and type III is a severe disease that usually don’t survive to adulthood. In order to differentiate them, allopurinol loading test and gene analysis are
An allopurinol loading test was performed at the dose of 10 mg/kg body weight according to the procedure described previously [4]. Serum samples were taken 2 h after oral administration, and urine was collected for 24 h. Allopurinol and oxypurinol in the serum and urine were measured by the method of HPLC (Ultimate 3000 UHPLC Dionex coupled with a TSQ Quantiva).
3. Results 3.1. Concentration of compounds in serum and urine The routine laboratory examination was executed in the patient and her parents. The concentration of uric acid in serum and urine was extremely low (below the lower detection limit), and the levels of xanthine and hypoxanthine in urine of patient were markedly elevated with 270 µmol/mmol Crea (normal range: < 18 µmol/mmol Crea) and 186 µmol/mmol Crea (normal range: < 14 µmol/mmol Crea) respectively. While the levels of uric acid, xanthine and hypoxanthine in urine of parents of the patient were all in the reference ranges. 169
Clinica Chimica Acta 504 (2020) 168–171
T. Xu, et al.
Fig. 1. Mutated sites by the WES analysis were validated by Sanger sequencing in the patient and her parents. The red arrow and rectangle indicates the mutated position in chromatogram and alignment of sequence in both of patient and her mother, respectively. The green arrow and rectangle indicates the wild type (nonmutation) in patient’s father.
evidence to support the relationship between SEPT9 and xanthinuria. In order to validate the genetic etiology of the patient, we arranged the patient’s parents to take the routine laboratory examination and genetics analysis. Unexpectedly, the mother’s uric acid in serum, xanthine and hypoxanthine in urine were all normal but possessed the completely same two mutation sites like her daughter. And the routine laboratory examination and genetics analysis for the patient’s father had normal condition. Therefore, it should be noted the relationship between the two mutations and xanthinuria, and we would follow-up of the mother’s serum uric acid level regularly. Previous case reports showed the patients with xanthinuria were accompanied by rheumatoid arthritis [11] and migratory arthritis [12]. Besides, the accumulation of xanthine and hypoxanthine might cause the inflammation of muscle of neck, elbow, or other parts of body. In our report, the patient has the symptoms of muscle soreness in different parts of body. Accumulative evidence is necessary to disclose the complications of xanthinuria. In conclusion, we have identified two novel mutation sites based on gene analysis in the diagnosis of xanthinuria. More importantly, mother and daughter share the same mutation sites, but only daughter has symptoms, and the underlying mechanism needs further in-depth study.
performed, because a measurement method for activity of MOCOS has not yet been established [4,9]. In the allopurinol loading test, oxipurinol is detected in serum and urine of type I xanthinuria patients after administration of allopurinol, as conversion of allopurinol to oxipurinol is catalyzed by AO. While oxipurinol is not detected in the case of type II xanthinuria owing to deficiency of AO. For the patient in this article, after taking allopurinol loading test, serum and urinary oxipurinol were detected, so she was diagnosed as type I xanthinuria. In this patient, we found two heterozygous mutations: c.2737C > T (p.Arg913Trp) in XDH gene and c.655C > T (p.Arg219Trp) in SEPT9 gene. The data from the Exome Aggregation Consortium (ExAC) showed that population frequency of c.2737C > T (p.Arg913Trp) in XDH gene was less than 0.2‰. The relationship between c.2737C > T (p.Arg913Trp) in XDH gene and xanthinuria has not been reported yet. However, the HGMD database contains other variation like c.2738G > A (p.Arg913Lln) in XDH gene, which affects the same amino acid that has been reported related with thiopurine intolerance by Coelho in 2016 [10]. Similarly, the mutation of c.655C > T (p.R219W) in SEPT9 gene has not been reported. Although two missense mutations c.2737C > T (p.R913W) in XDH gene and c.655C > T (p.R219W) in SEPT9 gene were found respectively, there was no 170
Clinica Chimica Acta 504 (2020) 168–171
T. Xu, et al.
Funding information
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