Genetic and Clinical Characteristics of Japanese Cystinuria with Exon and Exon-Intron Boundary Variants

doi:10.21203/rs.3.rs-4889642/v1

Cystinuria is the most common genetic cause of urinary stones. Defect in SLC3A1/SLC7A9 genes coding cystine transporter proteins rBAT/b^0,+AT will cause Cystinuria. The current work analyzed the clinical and genetics characteristics of Japanese Cystinuria patients. In total, 101 Cystinuria patients were studied. Clinical phenotypes were defined, and genetic analysis of SLC3A1 and SLC7A9 by next-generation sequence was performed. Excretion of urine cystine were determined by twenty-four hours urine analysis. The median age of presentation was 17 years old. In total, 51 different mutant variant alleles were identified (22 and 28 mutant variants in SLC3A1 and SLC7A9, respectively), including 28 novel variants. The p.pro482Leu (c.1445C > T) variant in SCL7A9 was predominantly found in 73 patients. Variants in exon-intron boundaries were identified in 6 cases. The patient with homozygote intron (exon-intron boundary) variant in SCL7A9 presented a severe phenotype with significant loss of mRNA expression. Inclusion of exon and exon-intron boundary variants reduced the number of cases that did not fit autosomal recessive inheritance from 14–9%. Predominance of p.pro482Leu (c.1445C > T) variant in SCL7A9 was a unique characteristic of Japanese Cystinuria. Current data may provide a rationale for the inclusion of exon-intron boundary variants in genomic classification.

Biological sciences/Genetics/Genome/Genetic variation

Health sciences/Nephrology/Kidney diseases/Renal calculi

Cystinuria

Urinary Stone

Genetic variant

Germline variant

Cystinuria (OMIM 220100) is a most common inherited urinary stone disease found in 1–2% of overall kidney stones. In the general population, the prevalence of Cystinuria was reported to be 1/15000 to 1/50000¹. Although the prevalence rate of Cystinuria is low, the affected patients will suffer recurrent urinary stones from childhood and may undergo a series of surgeries, which will damage renal function and quality of life. It is essential to obtain the diagnosis at an early age and early stage of urinary stones.

Cystinuria is caused by a pathogenic variants in two genes; SLC3A1 (OMIM 104614) encodes an rBAT (a single transmembrane protein), and SLC7A9 (OMIM 604144) encodes b^0,+AT (a twelve transmembrane protein)^2–6. rBAT and b^0,+AT form a dimer linked by a disulfide bond. B^0,+AT form the channel which transports dibasic amino acids (cystine, lysine, arginine, and ornithine) into the cell with the exchange of neutral amino acids. rBAT contains the multiple glycosylation sites at the extracellular domain. Interaction of b^0,+AT is required for the rBAT to be fully glycosylated and properly folded and stably expressed at the cell membrane^2–6. We have reported the role of carboxyl-terminal of b^0,+AT in controlling the trafficking of the rBAT- b^0,+AT from the endoplasmic reticulum (ER) to Golgi complex and thus control the full glycosylation of the rBAT protein⁷.

Strologo et al. proposed the genome-based cystinuria classification⁸. The genotypes were classified, such as Type A, due to two variants of SLC3A1; type B, due to two variants of SLC7A9; and type AB, with one variant on each of the above-mentioned genes. However, Rhodes et al. reported that over thirty percent of the Cystinuria patients in the United Kingdom only possessed a single variant or even no variant, who will not fit into the genotypes previously proposed which will fit into the autosomal recessive inheritance ^9,10. Thus, it is of need to establish the genomic classification which will cover the unfit cases of genotype in Cystinuria.

Previously, we have been studied Japanese Cystinuria since 1980’s and in 2006, we identified the presence of a unique variant; P482L is located at the carboxyl terminus end of B^0,+AT among Japanese Cystinuria patients^1,11–15. The P482L variant caused a severe functional defect of the Cystine transporter, which was found in over 80% of Japanese Cystinuria patients¹. The high prevalence of the P482L variant had never been reported elsewhere. The prevalence of P482L variant among Cystinuria patients was reported 1/8 in South Korea¹⁶, 1/73 in the United Kingdom ¹⁰, while no report in China¹⁷. Because of a distinct genotype in Japanese Cystinuria patients, a distinct clinical feature was also expected.

Our previous genetic study of Cystinuria was based on exon lesion with sanger sequence. However, in recent years, a number of effects of the exon intron boundary on mRNA splicing have been reported. And also, there may a variant that could be missed by the sanger sequence. In order to precisely understand the genomic feature of Japanese Cystinuria, here we studied the genomic characteristics of 101 Japanese Cystinuria patients by next generation sequencing including exon-intron boundaries.

Clinical Features

In total, 101 patients diagnosed with Cystinuria were identiﬁed comprising 36 (35.6%) women and 65 (64.4%) men. All patients were Japanese. A positive family history of Cystinuria was documented in 29(28.7%) of patients;19(18.8%) patients had only one generation affected (siblings), 10(9.9%) patients had two generations affected. The median age at ﬁrst presentation of stone symptom was 17 years old (range =0–58 years old) with a median age of 17.0 years old in the male and 16.5 years old in the female. The proportion of onset age for less than ten years, teenage, the 20s and 30s, and over were 38%, 24%, 25%, and 13%, respectively(Table 1).

Genetic Analysis

Genetic analysis was performed in all 101patients. At least two distinct genetic variants were detected in 93 patients, while eight patients only had a single variant. All the patients had at least one variant. Variants in SLC3A1 were identified in 18 patients, and variants in SLC7A9 were identified in 88 patients. Variants in both SLC3A1 and SLC7A9 were identified in 5 patients.

Overall, 50 distinct variants were identified in SLC3A1 and SLC7A9 (Table 2 and 3). Among 22 variants identified in SLC3A1, 14 variants were unreported (novel) variant (Table 2). Among 28 variants identified in SLC7A9, 15 variants were unreported (novel) variants (Table 3). In SLC3A1, 13 missense variants were identified, followed by four frameshifts and two splice-site variants, and one nonsense variant (Figure 1A). The most frequent variant in SLC3A1 was p.Val83Ala (c.5487T>C), which was found in 3 patients (3.0%), followed by Exon 10 deletion and p.Asn442fs. (c.1323dupT), which were found in 2 patients (2.0%) (Figure1A, Table 2). In SLC7A9, 24 missense variants were identified, followed by three splice site and frameshift variants. One nonsense and one initial codon variant were also found (Figure 1B).

The most frequent variant in SLC7A9 was p.pro482Leu (P482L)(c.1445C>T), which was found in 73 patients (72.7%)(43 homozygous and 30 heterozygous), followed by p.Val340fs (c.1017delA), which was found in 9 patients (8.9%)(1 homozygous and eight heterozygous) and p.Asn227Asp (c.679A>G), which was found in 4 patients (4.0%)(4 heterozygous)(Figure 1B, Table 3).

Location of variants

Regarding the location of the variants, all the variants in SLC3A1 were located at the extracellular domain except for p.Ala95Thr (c.313A>G) and p.Ile105Val (c.313A>G), which were located in the transmembrane domain of rBAT (Figure 2A). For the SLC7A9, the most common variant, P482L, was located at the carboxyl terminus, while p.Met1Thr (c.2T>C) was located in the N-terminus of b^0,+AT. Other variants were located at either transmembrane domain (14 variants), cytoplasmic loop (8 variants), or extracellular loop (5 variants) (Figure 2B).

Genome-Phenotype Association

Amount of urine cystine for the patients who have variant in SLC3A1, SCL7A9 and SLC3A1/SLC7A9 were 1357.15, 1815.45 and 1434(µ mol/ day), respectively (Table 4). Regarding genotype, 12 patients (11.9%) were type AA and 76 patients (75.2%) were type BB, while one patient (1.0%) were type AAB and four patients (4.0%) were type ABB (Figure 3A).

Genotype-Phenotype Association

Amount of urine cystine for type AA, BB, AAB/BBA, A and B were 1230.6, 1815.45, 1434, 3034.6　and 1968.8, respectively. No significant difference in the amount of urine cystine was observed between genotypes (Figure 3B, Table 4). Age of onset for type AA, BB, AAB/BBA, and B were 13, 16, 27, and 13.5, respectively. No significant difference in the age of onset was observed between genotypes (Figure 3C).

Genotype-Phenotype association based on P482L variant in SLC7A9.

Regarding P482L variant in SLC7A9, 43 patients (42.6%) had homozygote variants, 26 (25.7%) patients had compound heterozygote variants, four patients (4.0%) had single heterozygote variant and 28 patients (27.7%) had no P482L variant (Figure 4A). Amount of urine cystine for the patients of homozygote P482L variant, compound heterozygote P482L variant, single P482L variant and no P482L variant were 1705.2,2359.3, 1280.8, and 1483.7(µ mol/ day), respectively. No significant difference in the amount of urine cystine was observed between P482L based genotypes (Figure 4B, Table 4). Age of onset for type P482L homo, compound P482L hetero, none P482L were 20,10 and 16, respectively. No significant difference in the age of onset was observed based on the P482L variant classification (Figure 4C).

Variants in exon-intron boundary

Since variants in the exon-intron boundaries are responsible for the splicing error of the mRNA, we also study the variant in the exon-intron boundary. Overall, six exon-intron boundary variants were identified in six patients (Table 5). Among two patients without any variant in exon, one patient had homozygote variant (c.1224+3A>C) (classified as Type BB), and the other patient had heterozygote variant (c.1399+4_1399+7delAGTA) in exon-intron boundary (classified as Type B) (Table 5). All six patients who possessed exon-intron boundary variant resulted in the reclassification of the genotype (Table 5). The patients who had intron-exon boundary variants showed a relatively higher amount of cystine (3214.8 µmol/day) compared to those of patients who had exon variants (1705.4 µmol/day) (Table 6).

In the case with homozygote c.1224+3A>C variant, we have studied the mRNA expression of the SLC3A1 and SLC7A9. The male patient had first symptoms of kidney stone at the age of 3. His father was also affected by Cystinuria. At the age of 28, the patient consulted the hospital due to the left back pain. Computed tomographic scanning showed left staghorn calculus (Figure 5A). Based on the genomic analysis, the location of the variant was three sequences at the boundary of the exon 11(Figure 5B). No variant in exon was identified. The patient had a high amount of urinary cystine (3311.6 µmol/day) along with a high amount of lysine, ornithine, and arginine (Figure 5B). mRNA expression of the renal tissue showed a significant loss of SLC7A9 expression compare to that of SLC3A1 by RNA sequence (Figure 5C). Based on the genotype cover exon and exon-intron boundary, the case that did not fit the genotype of autosomal recessive inheritance reduced to 9 cases from 14 cases out of 101 cases (Figure 5E).

Cases with a single variant

When the Exon Intron boundary was included for the genotype, most of the cases had more than two variants, but eight cases had only one variant. Seven cases had variants in the Exon region and one in the Intron region. The median cystine concentration of the case with a single variant was 2501.7 (µmol/day), which was above the median cystine concentration of whole cohort of 1745.7 (µmol/day) (Table S1).

Roots of Cystinuria patients

We also study the origin of Cystinuria patients. 77% of patients are from the Kanto area (central region), where Tokyo and Chiba prefectures are located. 8% of patients are from the Kansai area (middle west), where Osaka prefectures is located. 7% of patients are from the Tokai area (middle south), where Aichi prefecture is located. No patient from the Tohoku area (north) was identified in this study (Figure S1A). Related to the percent of P482L variant, 100% of patients possessed P482L variant from Hokuriku (middle north), Kyusyu (far west), and Shikoku area (south west island). 76% and 57% of patients possessed P482L variant from Kanto (Tokyo and surrounding area) and Tokai area (middle south), respectively, while 37.5% from the Kansai area (middle west). No patients from Hokkaido area (northern island) possessed P482L variant (Figure S1 6B). The brief location of the region was illustrated in Figure S1C.

Cystinuria is the most common genetic related kidney stones that is responsible for 1% of kidney stones^18,19. Since Japan is geographically isolated by the ocean and even conducted a national isolation policy in the past during the Edo era (in 1636–1854), the development of genetic characteristics can be different from the rest of the countries. Current data described the unique genotype and phenotype characteristics of Japanese Cystinuria patients that are distinct from those of European and even Asian patients, such as presence of P482L variant and predominance of Type BB in genomic subtype. Furthermore, the current data is the first large genomic analysis using NGS covering the sequence of Exon and Exon-Intron boundary in Asian Cystinuria patients.

Among Japanese Cystinuria patients, 73 out of 101 patients possessed P482L variant. Based on the common genome database (genome AD https://gnomad.broadinstitute.org/), the distribution of P482L variant in SLC7A9 among the global population is rare. P482L was not found in African, Latino, Ashkenazi Jewish, and European (Finish). Even for Asians, the allele frequency of P482L for south Asian and East Asian are 0.00006533 and 0.00005012, respectively. Among the previous Asian study of Cystinuria, only 1 out of 8 cystinuria patients possessed P482L(heterozygote) in south Korean²⁰, while no P482L was identified in Chinese¹⁷. The P482L variant is located at the carboxyl terminus (C-terminus) end of b^0,+AT, which causes severe transporter defect comparative to those of frameshift and stop codon variant¹. Our previous study identified the "VPP" motif at the carboxyl terminus of b^0,+AT regulate the ER-Golgi trafficking and thus control glycosylation of rBAT protein⁷. The last P in the "VPP" motif represented the location of P482L variant. Since the location of P482L variant is the almost end of the C-terminus, we are speculating protein-protein interaction may be responsible for this functional defect, similar to the PDZ domain of URAT1²¹.

Related to the genotype classification, the distribution of Type BB was found in 76%, while Type AA was only found in 12% of patients in the current Japanese study. In the UK study, Type AA is dominant (36%), followed by Type BB (26%). In the Chinese Study, 37.5% (3 out of 8) were Type AA, and 25% (2 out of 8) were Type BB. In a Korean study, 50% (4 out of 8) of patients were Type AA, and only one patient (12.5%) was type BB. These data indicated the global trends of SLC3A1 variant in European and even Asian Cystinuria patients. Thus, the predominance of Type BB genotype found in Japanese Cystinuria patients seems to be unique characteristics. Beside SLC3A1 and SLC7A9, novel cystine transporter, AGT1 was identified²². However, so far, no pathogenic variants were reported²³.

In a UK study, patients who have a variant in SLC3A1(Type A or AA) demonstrated a significantly lower level of lysine, arginine, and ornithine, but not cystine compare to those of other patients¹⁰. Another study from the UK presented no difference in the clinical parameter between Type AA and Type BB. Furthermore, patients with a single mutated allele demonstrated similar disease severity compare to those of 2 mutated alleles⁹. In the current study, no difference in the severity of Cystinuria, such as urine cystine and age of onset, were observed based on the gene of variant (between SLC3A1 and SLC7A9), genotype (between type AA and type BB), and the number of mutated alleles(between single and two variant alleles). The difference in the clinical phenotype may be derived from a wide difference in the basal genome variants, in which we found 14 novel variants (total of 28 variants) in SLC3A1 and SLC7A9, respectively.

Recent evidence indicates the variant in the exon-intron boundary sequence resulted in the splicing error of the mRNA ²⁴. About 9% of all variants reported in the Human Gene Mutation Database (HGMD) are splicing variants²⁴. In general, variants in canonical acceptor and donor sites affect the strongly conserved sequences that define exon-intron boundaries. The most typical variants affect + 1 and + 2 residues in the 5′ splice site and − 1 and − 2 residues in the 3′ acceptor splice site²⁴. In the case of Neurofibromatosis type 1, c.1845 + 1G > A resulted in exon 15 and 16 skipping²⁵. In the case of the c.1525-1G > A mutant variant in intron 9 of the CFTR gene resulted in the presence of three different mRNA isoforms²⁶. Actually, it has been a missing piece of Cystinuria that up to 30% of patients did not fit into autosomal recessive inheritance category by exon variants^9,10, which may be partially explained by variants in intron. In the current study, six exon-intron variants were identified, which resulted in the reclassification of the genotype. Among them, two patients previously identified no variant. One had homozygote, and the other had heterozygote exon-intron boundary variant. In the case with homozygote c.1224 + 3A > C variant in SLC7A9, we have studied the mRNA expression of the SLC3A1 and SLC7A9. Almost complete loss of SLC7A9 expression was observed by the exon-intron boundary variant. This data is, we believe, the first direct evidence of the significance of exon-intron boundary variant, which resulted in the loss of the transporter expression. Based on the genotype cover exon and exon-intron boundary, the cases that do not fit into the autosomal recessive inheritance category reduced to 9 cases from 14 cases. Based on the data, we propose genome classification based on the exon and exon-intron boundary.

When looking at the origin of Cystinuria patients among the Japanese population, most of the patients were found from the middle (Kanto and Tokai) and west (Kansai) side of Japan. No patient was found on the northeast side of Japan (Tohoku Area) except one patient in Hokkaido (Northern Island). The current Japanese population is proposed to be the result of admixture between the early migrants (Jomon people) and later migrants (Yayoi people). Jomon people lived in the Northern and Southern parts of Japan, while Yayoi people lived in the central part of Japan. Few Cystinuria patients from the northeast side of Japan may indicate Japanese Cystinuria patients' roots may be derived from Yayoi people, rather than Jomon people²⁷. We are currently studying the association of Japanese Cystinuria patients with Yayoi or Jomon people based on the genome-wide SNP data, especially related to the P482L variant in SLC7A9. Further analysis will lead to the link between P482L variant and origin of Japanese people.

The current manuscript contained several limitations. First, the data was obtained from Chiba university hospital with the collaboration with the hospitals from 15 different prefectures ranged from the northern part (Hokkaido) and the west side (Kyusyu) of Japan. Although data is obtained from a wide range of locations, our data missed some of the location in Japan. Second, the data is obtained in a retrospective manner. In order to improve the quality of the data, we are currently organizing the prospective registry system for kidney stone patients (CRYSTAL-J), including the patients with Cystinuria. Third, presence of splicing error induced by the exon-intron boundary variants has not fully been validated except one case presented. We are currently assessing the presence of splicing error due to the variants in exon-intron boundaries by in vitro transcription. Fourth, based on the previous evidence, about 25% of Cystinuria patients develop non-cystine stone²⁸. It will be next project to assess the risk factors for the development of non-cystine stone among Japanese Cystinuria cohort.

In conclusion, the current manuscript described the genomic and clinical signature of Japanese Cystinuria patients, which is distinct from not only European Cystinuria patients but also from Asian Cystinuria patients. Genotype classification may be updated based on not only exon but also exon-intron boundary variants.

Patient information

Patients recruited to this study all had a clinical diagnosis of Cystinuria on the basis of clinical diagnosis of cystine stones from the southwest to northeast part of Japan between 2000 and 2020. Detailed clinical data were retrospectively collected. Demographics (age, sex and ethnicity) and age at the first stone events, treatment history, and urine biochemistry were collected.

Definition of urine amino acids

Cystine, lysine, arginine, and ornithine were measured by twenty-four hours urine sample. The amount of urine amino acids was determined by high performance liquid chromatography (HPLC) at SRL, Inc. (Hachioji, Tokyo, Japan).

Statistical analysis

Mann-Whitney's U test, ANOVA analysis, and the χ2 test were used for comparisons of two or three groups. Spearman's rank correlation coefficient was used to analyze the relationship between the two groups. Statistical computations were performed using JMP 11.0.0 (SAS Institute, NC, USA). P < 0.05 was considered significant.

Genetic Analysis

Genomic DNA was extracted from whole blood using the Blood & Cell Culture DNA Midi Kit (Qiagen, Hilden, Germany). Genetic variants of SLC3A1 (NM_000341.3) and SLC7A9 (NM_001126335.1) were analyzed by next-generation sequence of the protein-coding exons and their intron boundaries essentially as described previously²⁹. In brief, libraries for the next generation sequence were made by a KAPA Hyper Plus Library Kit. Hybridization capture was made by predesigned hybridization capture probes for SLC3A1 and SLC7A9 purchased from IDT. The libraries were sequenced on an Illumina NextSeq500. Variant calling was done using GATKv4 (https://gatk.broadinstitute.org/hc/en-us). The variants thus detected by the next-generation sequencing were again confirmed by Sanger sequencing. The next-generation sequencing analysis was conducted at the Kazusa DNA Research Institute (Kisarazu, Chiba, Japan). Unreported (novel) variant was defined as the variant not reported on the previous publication. These gene variants are described with reference to the sequence of NM_000341.3

RNA sequencing

The kidney tissue was obtained by biopsy. Total RNA from kidney biopsy sample was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA sequencing libraries were prepared using a SureSelect Strand-Specific RNA Library Prep Kit (Agilent Technologies, Inc., Santa Clara, CA, USA). Sequencing was performed on a HiSeq 2500 system (Illumina, Santiago, CA, USA) in a 50-base single-end mode. TopHat (version 2.1.0; with default parameters) was used to map to the human reference genome (hg38). Then, gene expression levels were quantified using Cufflinks version 2.2.1; with default parameters) and are expressed as the number of fragments per kilobase of exon per million reads mapped (FPKM).

Ethical Approval and Consent to Participate

The present study was conducted in accordance with ethical standards that promote and ensure respect and integrity for all human subjects and the Declaration of Helsinki. This study was approved by the institutional review board of Chiba University Hospital (approval number 1050), Nagoya City University Hospital (approval number of 70-19-0018) and other participated hospitals and written informed consent was obtained from all patients.

Conflict of interest

The authors declare no competing financial interests.

Funding information

The present work was supported by 5th research grant from Japanese Society on Urolithiasis Research to S. Sakamoto, the Japan Agency for Medical Research and Development, the Initiative on Rare and Undiagnosed Diseases (IRUD) to T. Ichikawa and the Japan Agency for Medical Research and Development (AMED) (JP21ek0109549)to T. Ichikawa.

Author Contribution

YN and SS conceived and designed the experiments. JR, MM, SS, ST, NS, CI and YI analyzed the data. AA, AC, KW, MN, MF, YS, TU, TY, YK, YN, YY, KA, YI, MS, NA and TI supervised the study. AT, SH and KT provided the samples.

Acknowledgement

The authors thank Osamu Ohara (Kazusa DNA Research Institute, Kisarazu, Japan) for the NGS analysis and RNA sequence. The authors also thank Chiaki Hosaka for organizing the data. The author also thanks, Natsuko Kusama, Masako Takahashi, Hisayo Karahi, Keiko Yamazaki (Experimental assistant, Chiba University) for their support of this study.

Data Availability

The data was generated based on the funding from the Japanese Society on Urolithiasis Research (JSUR), the Initiative on Rare and Undiagnosed Diseases (IRUD) and the Japan Agency for Medical Research and Development (AMED). The data are available from the authors upon reasonable request and with the permission of the JSUR. The contact should be made to a corresponding author: Shinichi Sakamoto, E-mail: [email protected], Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuou-ku, Chiba, Japan.

Shigeta, Y. et al. A novel missense mutation of SLC7A9 frequent in Japanese cystinuria cases affecting the C-terminus of the transporter. Kidney Int. 69, 1198–1206. 10.1038/sj.ki.5000241 (2006).
Bertran, J. et al. Expression cloning of a human renal cDNA that induces high affinity transport of L-cystine shared with dibasic amino acids in Xenopus oocytes. J. Biol. Chem. 268, 14842–14849 (1993).
Chairoungdua, A. et al. Identification of an amino acid transporter associated with the cystinuria-related type II membrane glycoprotein. J. Biol. Chem. 274, 28845–28848. 10.1074/jbc.274.41.28845 (1999).
Feliubadalo, L. et al. Non-type I cystinuria caused by mutations in SLC7A9, encoding a subunit (bo,+AT) of rBAT. Nat. Genet. 23, 52–57. 10.1038/12652 (1999).
Segawa, H. et al. Identification and functional characterization of a Na+-independent neutral amino acid transporter with broad substrate selectivity. J. Biol. Chem. 274, 19745–19751. 10.1074/jbc.274.28.19745 (1999).
Egoshi, K. I., Akakura, K., Kodama, T. & Ito, H. Identification of five novel SLC3A1 (rBAT) gene mutations in Japanese cystinuria. Kidney Int. 57, 25–32. 10.1046/j.1523-1755.2000.00821.x (2000).
Sakamoto, S. et al. A novel role of the C-terminus of b 0,+ AT in the ER-Golgi trafficking of the rBAT-b 0,+ AT heterodimeric amino acid transporter. Biochem. J. 417, 441–448. 10.1042/BJ20081798 (2009).
Dello Strologo, L. et al. Comparison between SLC3A1 and SLC7A9 cystinuria patients and carriers: a need for a new classification. J. Am. Soc. Nephrol. 13, 2547–2553. 10.1097/01.asn.0000029586.17680.e5 (2002).
Rhodes, H. L. et al. Clinical and genetic analysis of patients with cystinuria in the United Kingdom. Clin. J. Am. Soc. Nephrol. 10, 1235–1245. 10.2215/CJN.10981114 (2015).
Wong, K. A. et al. The genetic diversity of cystinuria in a UK population of patients. BJU Int. 116, 109–116. 10.1111/bju.12894 (2015).
Watanabe, Y. et al. Pediatric Cystinuria Patient With Novel Mutation in SLC3A1. Glob Pediatr. Health. 6, 2333794X19862441. 10.1177/2333794X19862441 (2019).
Okada, T. et al. Efficacy of transurethral cystolithotripsy assisted by percutaneous evacuation and the benefit of genetic analysis in a pediatric cystinuria patient with a large bladder stone. Urol. Case Rep. 34, 101473. 10.1016/j.eucr.2020.101473 (2021).
Abe, Y. et al. Persistent Leukocyturia Was a Clue to Diagnosis of Cystinuria in a Female Patient. Glob Pediatr. Health. 1 10.1177/2333794X14551275 (2014).
Akakura, K. et al. The long-term outcome of cystinuria in Japan. Urol. Int. 61, 86–89. 10.1159/000030294 (1998).
Ito, H. et al. The incidence of cystinuria in Japan. J. Urol. 129, 1012–1014. 10.1016/s0022-5347(17)52514-8 (1983).
Lee, E. H., Kim, Y. H., Hwang, J. S. & Kim, S. H. Non-type I cystinuria associated with mental retardation and ataxia in a Korean boy with a new missence mutation(G173R) in the SLC7A9 gene. J. Korean Med. Sci. 25, 172–175. 10.3346/jkms.2010.25.1.172 (2010).
Yuen, Y. P. et al. Heterogeneous mutations in the SLC3A1 and SLC7A9 genes in Chinese patients with cystinuria. Kidney Int. 69, 123–128. 10.1038/sj.ki.5000003 (2006).
Sakamoto, S. et al. Chronological changes in epidemiological characteristics of lower urinary tract urolithiasis in Japan. Int. J. Urol. 26, 96–101. 10.1111/iju.13817 (2019).
Sakamoto, S. et al. Chronological changes in the epidemiological characteristics of upper urinary tract urolithiasis in Japan. Int. J. Urol. 25, 373–378. 10.1111/iju.13552 (2018).
Kim, J. H. et al. Genotype and Phenotype Analysis in Pediatric Patients with Cystinuria. J. Korean Med. Sci. 32, 310–314. 10.3346/jkms.2017.32.2.310 (2017).
Anzai, N. et al. The multivalent PDZ domain-containing protein PDZK1 regulates transport activity of renal urate-anion exchanger URAT1 via its C terminus. J. Biol. Chem. 279, 45942–45950. 10.1074/jbc.M406724200 (2004).
Nagamori, S. et al. Novel cystine transporter in renal proximal tubule identified as a missing partner of cystinuria-related plasma membrane protein rBAT/SLC3A1. Proc. Natl. Acad. Sci. U S A. 113, 775–780. 10.1073/pnas.1519959113 (2016).
Olschok, K., Vester, U., Lahme, S., Kurth, I. & Eggermann, T. No evidence for point mutations in the novel renal cystine transporter AGT1/SLC7A13 contributing to the etiology of cystinuria. BMC Nephrol. 19, 278. 10.1186/s12882-018-1080-5 (2018).
Abramowicz, A. & Gos, M. Splicing mutations in human genetic disorders: examples, detection, and confirmation (vol 59, pg 253, 2018). J Appl Genet 60, 231–231, doi: (2019). 10.1007/s13353-019-00493-z
Fang, L. J. et al. A novel mutation in the neurofibromatosis type 1 (NF1) gene promotes skipping of two exons by preventing exon definition. J. Mol. Biol. 307, 1261–1270. 10.1006/jmbi.2001.4561 (2001).
Ramalho, A. S. et al. Transcript analysis of the cystic fibrosis splicing mutation 1525-1G > A shows use of multiple alternative splicing sites and suggests a putative role of exonic splicing enhancers. J. Med. Genet. 40, e88. 10.1136/jmg.40.7.e88 (2003).
Jinam, T. A. et al. Unique characteristics of the Ainu population in Northern Japan. J. Hum. Genet. 60, 565–571. 10.1038/jhg.2015.79 (2015).
Reinstatler, L. et al. Conversion from Cystine to Noncystine Stones: Incidence and Associated Factors. J. Urol. 200, 1285–1289. 10.1016/j.juro.2018.07.047 (2018).
Fujiki, R. et al. Assessing the Accuracy of Variant Detection in Cost-Effective Gene Panel Testing by Next-Generation Sequencing. J. Mol. Diagn. 20, 572–582. 10.1016/j.jmoldx.2018.04.004 (2018).

Table 1. Demographic and clinical data
Characteristic	value
number of patients (N)	101
Median age at presentation (years) (range)	17.0 (0-58)
Less than 10 years/teen age/20s/30s and over	38/24/25/13(%)
Male	17.0 (0-48)
Female	16.5 (0-58)
Sex: male (N=65)/ female (N=36)	64.4%
Family Histroy (1st sibling/2nd sibling)	29% (19%/10%)
Ethnicity: Japanese	100%
Median age at follow-up (years)	33.0 (0.3-78.0)
Median time from initial symptoms to follow-up (years)	14.0 (0.0-56.0)
Cystine (μmol/day)	1745.7 (184.6 - 5303.4 )
Ornithine (μmol/day)	2044.3 (205.2 - 7471.7)
Lysine (μmol/day)	8646.7 (536.9 - 36961.6)
Arginine (μmol/day)	3334.2 (96.3 - 12096.0)

Table 2. List of variants in SLC3A1
SLC3A1	Variant Type	Variants	Protein Sequence	Homo/Hetero	PT Number	Exon	Location
1	Missense	c.283G>A*	p.Ala95Thr	Hetero	1	1	TM1
2	Missense	c.313A>G*	p.Ile105Val	Hetero	1	1	TM1
3	Missense	c.470T>C*	p.Ile157Thr	Hetero	1	2	Extra
4	Missense	c.548T>C*	p.Val183Ala	Hetero	3	2	Extra
5	Missense	c.659A>G*	p.His220Arg	Hetero	1	3	Extra
6	Missense	c.763T>A *	p.Trp255Arg	Hetero	1	3	Extra
7	Missense	c.989A>C*	p.Gln330Pro	Hetero	1	5	Extra
8	Missense	c.1037T>C	p.Leu346Pro	Hetero	1	6	Extra
9	Missense	c.1323C>G*	p.Pro441Arg	Hetero	1	7	Extra
10	Missense	c.1334T>C	p.Ile445Thr	Hetero	1	8	Extra
11	Missense	c.1742T>G*	p.Val581Gly	Hetero	1	10	Extra
12	Missense	c.1772G>A *	p.Arg591Lys	Hetero	1	10	Extra
13	Missense	c.2017T>C	p.Cys673Arg	Hetero	1	10	Extra
14	Nonsense	c.1113C>A	p.Tyr371X	Hetero	1	6	Extra
15	Nonsense	c.1328G>A*	p.Trp443X	Homo	1	7	Extra
16	Frameshift	c.1323dupT*	p.Asn442fs	Hetero	2	7	Extra
17	Frameshift	c.1820delT	p.Leu607fs	Hetero	1	10	Extra
18	Frameshift	c.1820delT	p.Leu607fs	Homo	1	10	Extra
19	Frameshift	c.1898_1899insAT*	p.Asp634fs	Homo	1	10	Extra
20	Splice site	c.1500+1G>A		Hetero	1	8+1 intro	Extra
21	Splice site	c.1617+1_1617+2dupGT*		Hetero	1	9+1+2 intron	Extra
22	Deletions	Ex10 deletion		Homo	2	10	Extra
Missense: missense variant, Frameshift: frame shift variant, Nonsense: nonsense variant, Frameshift: frameshift variant, Splice site: splice site variant (exon-intron boundary variant), TM: transmembrane domain, Extra: extracellular domain, new: novel variant not reported previously. These gene variants are described with reference to the sequence of NM_000341.3

Table 3. List of variants in SLC7A9
SLC7A9	Variant Type	Variants	Protein Sequence	Homo/Hetero	PT Number	Exon	Location
1	Missense	c.2T>C new	p.Met1Thr	Hetero	1	2	Cytoplasmic
2	Missense	c.206G>A	p.Trp69X	Hetero	2	3	TM2
3	Missense	c.209C>T	p.Ala70Val	Hetero	1	3	TM2
4	Missense	c.217G>A	p.Gly73Arg	Hetero	1	3	TM2
5	Missense	c.308C>T new	p.Ala103Val	Hetero	1	4	TM3
6	Missense	c.511C>T	p.Arg171Trp	Hetero	1	5	Cytoplasmic Loop2
7	Missense	c.583G>A	p.Gly195Arg	Hetero	2	5	TM5
8	Missense	c.650C>G new	p.Ser217Cys	Hetero	1	6	TM6
9	Missense	c.671C>T	p.Ala224Val	Hetero	1	6	TM6
10	Missense	c.679A>G	p.Asn227Asp	Hetero	4	6	TM6
11	Missense	c.698A>G new	p.Asp233Gly	Homo/Hetero	1/1	6	Cytoplasmic Loop3
12	Missense	c.829G>A new	p.Val277Met	Hetero	1	7	Extra Loop4
13	Missense	c.886C>G new	p.Arg296Gly	Hetero	1	9	Extra Loop4
14	Missense	c.968C>T new	p.Thr323Ile	Hetero	1	9	Cytoplasmic Loop4
15	Missense	c.974G>T new	p.Gly325Val	Hetero	1	9	Cytoplasmic Loop4
16	Missense	c.992C>T	p.Ala331Val	Homo	1	10	Cytoplasmic Loop4
17	Missense	c.998G>A	p.Arg333Gln	Hetero	2	10	Cytoplasmic Loop4
18	Missense	c.1061C>A	p.Ala354Asp	Hetero	5	10	TM9
19	Missense	c.1124T>C new	p.Val375Ala	Homo	1	11	TM10
20	Missense	c.1166C>T	p.Thr389Met	Hetero	1	11	TM10
21	Missense	c.1336A>C new	p.Ser446Arg	Hetero	1	12	TM12
22	Missense	c.1445C>T	p.pro482Leu	Homo/Hetero	43/30	13	Cytoplasmic
23	Frameshift	c.611_612delCA new	p.thr204fs	Hetero	1	6	Extra Loop3
24	Frameshift	c.1017delA	p.Val340fs	Homo/Hetero	1	10	Cytoplasmic Loop4
25	Splice site	c.873+2dupT new		Hetero	1	Intron(Exon 8+2)	Extra Loop4
26	Splice site	c.1074+1G>A new		Hetero	1	Intron(Exon 10+1)	Extra Loop5
27	Splice site	c.1224+3A>C new		Homo	1	Intron(Exon 11+3)	Cytoplasmic Loop5
28	Splice site	c.1399+4_1399+7delAGTA		Hetero	1	Intron(Exon 12+4-7)	TM12
Missense: missense variant, Frameshift: frame shift variant, Nonsense: nonsense variant, Frameshift: frameshift variant, Splice site: splice site variant (exon-intron boundary variant), TM: transmembrane domain, Extra: extracellular domain, new: novel variant not reported previously. These gene variants are described with reference to the sequence of NM_000341.3

Table 4. Aminoacid concentration based on Genes, Genotype and P482L variant
*Urine Aminoacids: : µmol/day*
	Cystine		Ornithine		Lysine		Arginine
	Median	Range	Median	Range	Median	Range	Median	Range
*Based on Genes*
*SLC3A1*	1357.2	(186.9-3117.9)	1692.6	(861.3-3848.1)	7051.8	(5307.9-12456.4)	2682.7	(323.4-4590.5)
*SLC7A9*	1832.2	(184.6-5303.4)	2020.15	(205.2-7471.7)	8787.8	(536.9-36961.6)	3334.2	(96.3-12096)
*SLC3A1/SLC7A9*	1434	(1037.3-3722.9)	3174.4	(2302.1-4316.8)	8144	(1300.2-13826.2)	3495.9	(2269-5996.8)
*Based on Genotype*
AA	1230.6	(186.9-3117.9)	1355.8	(861.3-3848.1)	5631.6	(5307.9-9223.1)	1002.9	(323.4-4590.5)
BB	1815.45	(184.6-5303.4)	2036.8	(205.2-7417.7)	8858.5	(536.9-36961.6)	3334.2	(150.4-12096)
AAB/BBA	1434	(1037.3-3722.9)	3174.4	(2302.1-4316.8)	8144	(1300.2-13826.2)	3495.9	(2269-5996.8)
A	3034.6	(3034.6-3034.6)	2029.4	(2029.4-2029.4)	12456.4	(12456.5-12456.5)	4425.4	(4425.4-4425.4)
B	1968.8	(592.8-4094.2)	1616.4	(278.3-2843)	8576.3	(3038.4-10211.1)	555.9	(96.3-5037.6)
*Based on P482L*
P482L Homo	1705.15	(184.6-4368)	2195.6	(205.2-4316.8)	8858.5	(536.9-17412)	3423.4	(150.4-6531)
P482L compound hetero	2359.3	(851-5303.4)	2183.1	(716-7471.7)	8970.1	(2968.4-36961.6)	3875.3	(1416.6-12096)
P482L single Hetero	1280.8	(592.8-1968.8)	1560.65	(278.3-2843)	5807.4	(3038.4-8576.3)	2567	(96.3-5037.6)
None P482L	1483.7	(186.9-4094.2)	1822.9	(845.4-5497.2)	8594.5	(2411.5-16476.1)	1729.4	(323.4-9512.9)

Table 5. List of cases with Exon-Intron boundary variants
	*SLC3A1*		*SLC7A9*		Genotype
	Variants		Variants		Exon only	Exon/Exon-Intron boundary
1			T204fs c.611_612delCA (hetero)	c.873+2dupT (hetero)*	Type B	Type BB
2			c.1224+3A>C (homo)*		No mutation	Type BB
3			c.1399+4_1399+7delAGTA (hetero)*		No mutation	Type B
4	Y371Ter(hetero)C1113A	c.1500+1G>A (hetero)*			Type A	Type AA
5	P441R(hetero)C1322G	c.1617+1_1617+2dupGT (hetero)*			Type A	Type AA
6			P482L(hetero)C1445T	c.1074+1G>A (hetero)*	Type B	Type BB
	* Exon-Intron boundary (Splice site) variant

Table 6. Urine aminoacid concentration based on Exon/Exon-intron boundary variants
	*Urine Aminoacids: : µmol/day*
	Cystine		Ornithine		Lysine		Arginine
	Median	Range	Median	Range	Median	Range	Median	Range
*Exon variant*	1705.4	(184.6-5303.4)	2044.3	(205.2-7471.7)	8538.6	(536.9-36961.6)	3512.6	(96.3-12096)
*Exon-intron boundary variant*	3214.8	(1025.6-4094.2)	2732.3	(845.4-3922.7)	9134.9	(2411.5-10211.1)	1169.5	(555.9-4362.4)

No competing interests reported.

20240807SupportingInfo.docx

Genetic and Clinical Characteristics of Japanese Cystinuria with Exon and Exon-Intron Boundary Variants

Status:

Version 1

Abstract

Figures

Introduction

Results

Discussion

Methods