ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. CYSTINURIA: GENETIC ASPECTS AND NOVEL PHARMACOTHERAPEUTICS DIANA STACHULA, AMRIK SAHOTA (FACULTY ADVISOR) ✵ ABSTRACT This review provides an overview of the ge- netic aspects of cystinuria, as well as the novel phar- macotherapeutics that could potentially be used to treat the disease. Cystinuria is an inherited disorder characterized by the formation of painful stones in the kidneys, bladder, and other parts of the renal system. Currently, mutations responsible for cysti- nuria have been identified in two genes (SLC3A1 and SLC7A9 ), and cystinuria patients are catego- rized based on their genotypes - which versions, or alleles, of these genes they have (mutated or wild- type). Regardless of genotype, however, current treatments for all cystinuria patients have significant limitations. This has led researchers to search for more promising therapeutics. One potential treat- ment uses cystine analogs—compounds that are structurally similar to cystine, which is the naturally occurring chemical substance from which the stones are formed. These compounds have demon- strated the ability to inhibit stone formation by stunt- ing cystine crystallization – the process by which cys- tine crystals aggregate to form stones. Gene ther- apy may also be used to treat cystinuria in the future by replacing mutated copies of SLC3A1 and SLC7A9 with healthy ones. Technological advance- ments and an improvement of our understanding of how gene therapy functions in the renal system could reveal even more treatment possibilities. 1 INTRODUCTION Cystinuria generally arises from mutations in the SLC3A1 and SLC7A9 genes. There are likely more genetic factors that contribute to the disease that are yet to be identified, as 5% of cystinuria pa- tients do not have mutations in either of the two genes. (Sahota et al., 2019). SLC3A1 and SLC7A9 encode crucial components of the biochemical pathway responsible for the reabsorption of dibasic amino acids in the renal system. Dibasic amino acids are organic compounds that form proteins and con- tain two basic functional groups, typically amino groups (NH2). One dibasic amino acid is cystine (FIG- URE 2), which is made of two cysteine molecules joined by a disulfide bond (S - S) (FIGURE 1). The de- fective reabsorption of cystine from the kidneys into the bloodstream causes its supersaturation in urine and the formation of cystine stones in the kidneys, bladder, and ureters (Sahota et al., 2019). Cystine stones are jagged in shape and are considered to be the hardest stones formed in the human renal system (Ringdén & Tiselius, 2007). Most cystinuria patients that develop their first stone in adolescence are prone to recurrent stone formation throughout their lifetimes (Rogers et al., 2007). In addition to ab- dominal pain, patients may also experience nausea, hematuria (blood in urine), recurrent urinary tract in- fections, and kidney failure (Mattoo & Goldfarb, 2008). Increased fluid intake, reduced protein con- sumption, and the use of currently available medi- cations have proven to be less-than-ideal treatment methods for the disease (Sahota et al., 2019). Poten- tial novel treatments of cystinuria have been studied using Slc3a1 and Slc7a9 knockout mouse models, which are mice with mutated, nonfunctional ver- sions of the SLC3A1 and SLC7A9 genes (Sahota et al., 2019). Cystine diesters, such as cystine di- methylester (CDME) (FIGURE 3), and cystine diamides, FIGURE 1: Cysteine is an amino acid with a thiol side chain (R-SH). Two cysteine molecules can be oxidized to form cystine (FIGURE 2). ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV FIGURE 2: Cystinuria patients form stones made of cystine, an organic molecule containing a disulfide bridge (S – S) and two amine groups (-NH2). FIGURE 3: CDME is an example of a cystine diester, a type of cystine analog. Like cystine, it contains a disulfide bridge (S – S) and two amine groups (-NH2). FIGURE 4: L-cystine bis(N′-methylpiperazide) is an example of a cystine diamide, a type of cystine analog. Like cys- tine, it contains a disulfide bridge (S – S) and two amine groups (-NH2). such as L-cystine bismorpholide and L-cystine bis(N′-methylpiperazide) (FIGURE 4), all of which are analogs of cystine (FIGURE 2), have demonstrated their effectiveness as potential treatments for cysti- nuria through their abilities to inhibit cystine crystal growth in these mouse models (Yang et al., 2018). Continued study of cystine stone formation inhibi- tors, as well as gene therapy, will likely generate promising new treatments for human cystinuria pa- tients. 2 CYSTINURIA: ETIOLOGY AND EPIDEMIOLOGY TRANSPORT DEFECT Genetic mutations in SLC3A1 and SLC7A9 cause the defective reabsorption of several dibasic amino acids — cystine, ornithine, lysine, and arginine (COLA) — from the kidneys into the bloodstream (FIGURE 5) (Sahota et al., 2019). More specifically, these mutations disrupt the COLA transporter (b0,+), which is a heterodimer, or a molecule made up of two protein components (Sahota et al., 2019). SLC3A1 and SLC7A9 each encode one of these components (Sumorok & Goldfarb, 2013); SLC3A1 encodes the rBAT subunit, while SLC7A9 encodes the b0,+ AT subunit (FIGURE 6) (Sahota et al., 2019). Mutations in either gene will cause a defect in the corresponding subunit, leading to the defective re- absorption of the COLA amino acids (Sumorok & Goldfarb, 2013). Since cystine is the least soluble of the COLA amino acids, it has a greater ability to crys- tallize in the urinary tract and form stones when im- properly reabsorbed (Sahota et al., 2019). STONE FORMATION Cystine stones are thought to form by free solution crystallization, the process by which super- saturated solutions transform into solids (Coe et al., 2010). When cystine is supersaturated in urine, it crystallizes into stones that can be found freely throughout the renal system (Coe et al., 2010), though they are predominantly found in the termi- nal collecting ducts within the kidneys (Khan et al., 2016). These stones are named depending on their specific location (FIGURE 7). Their mobility within the renal system allows them to be easily removed dur- ing surgery; crystals of large size wash away when surgically exposed (Coe et al., 2010). ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV FIGURE 5: Mutations in the SLC3A1 and SLC7A9 genes cause the defective reabsorption of the COLA amino acids from the proximal convoluted tubule into the bloodstream. These dibasic amino acids proceed through the rest of the renal system and are excreted in urine. Created with BioRender.com. FIGURE 6: SLC3A1 encodes the rBAT subunit (green) and SLC7A9 encodes the b0,+ AT subunit (blue) of the COLA trans- porter (b0,+), which is responsible for the reabsorption of the COLA amino acids in the renal system. Cystine is reduced to two cysteine molecules when it is reabsorbed into the bloodstream. EPIDEMIOLOGY Although cystine stones make up only ap- proximately 1% of all kidney stones, cystinuria is still one of the most commonly inherited genetic disor- ders (Mattoo & Goldfarb, 2008). The disease has a global prevalence of approximately 1:7,000, rang- ing from 1:2,500 in Libyan Jews to 1:100,000 in Swedes. In the United States, approximately 1 in 15,000 adults have cystinuria (Mattoo & Goldfarb, 2008). Men are twice as likely as women to develop cystine stones (Leslie, Sajjad & Nazzal, 2020). This may be due to shorter urethral length or factors that inhibit cystine crystal aggregation in females (Sa- hota et al., 2019). Patients typically first present a stone be- tween the ages of 2 and 40, with a median onset age of 12 in males and 15 in females (Rogers et al., 2007). Approximately two thirds of cystinuria pa- tients develop stones in both kidneys, while one ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV third only form stones in a single kidney (Usawachintachit et al., 2018). Among patients who develop stones, over 60% experience recurrent stone formation, with males forming new stones about every 3 years and females forming new stones about every 5 years (Dello Strologo et al., 2002). In addition to higher recurrence rates, males also typically experience more aggressive disease symptoms that may require more surgical interven- tions (Edvardsson et al., 2013). FIGURE 7: Cystine stones are found freely throughout the renal system. Created with BioRender.com. 3 GENETICS INHERITANCE AND GENOTYPES Cystinuria patients are classified depending on which of their genes are mutated. Those with type A, or type I, cystinuria have a mutation in SLC3A1 on chromosome 2. Those with type B, or non-type I, cystinuria have a mutation in SLC7A9 on chromosome 19 (Fazaeli et al., 2017). Every person has two copies of each gene. Mutations in SLC3A1 are inherited through an autosomal recessive pat- tern of inheritance (both copies of the gene must be mutated for disease presentation) (Martell et al., 2017). Meanwhile, mutations in SLC7A9 follow an autosomal dominant pattern of inheritance with in- complete penetrance; typically (only one mutated copy of the gene needs to be present to allow for the formation of cystine stones) (Martell et al., 2017). Rarely, patients have type AB cystinuria; people who fall under this category have two mutated copies of one of the genes as well as one mutated copy of the other (Sumorok & Goldfarb, 2013). Depending on which gene has two mutated copies and which has one mutated copy, patients can be designated as either type AAB or type ABB (Sumorok & Goldfarb, 2013). As aforementioned, both copies of SLC3A1 must be mutated for disease presentation, so SLC3A1 heterozygotes, who only have one mutated copy, should not present stones or any characteris- tics of cystinuria. SLC7A9 heterozygotes, however, may present cystinuria symptoms such as variable urinary levels of COLA (Edvardsson et al., 2013). SLC7A9 heterozygotes are unlikely to develop stones unless urine volumes are low or protein in- take is significantly elevated (Sahota et al., 2019). MUTATIONS Over 400 total mutations have been identi- fied in SLC3A1 and SLC7A9 (Stenson et al., 2003), including missense, nonsense, splicing, regulatory, deletion, insertion, indel, duplication, and rear- rangement mutations (Stenson et al., 2003). Each of these mutation types alters the DNA sequences of SLC3A1 and SLC7A9, resulting in the formation of altered or truncated proteins (the subunits of the COLA transporter). Missense mutations are the larg- est group of mutations that result in cystinuria. Such mutations change a single amino acid in the protein being encoded, which can have a range of effects on the protein — protein function may be unim- pacted, impacted to some degree, or lost com- pletely (Martell et al., 2017). Currently, the impact of missense mutations in SLC3A1 and SLC7A9 on pro- tein function and disease presentation is unclear. (Martell et al., 2017). TABLE 1 presents the mutation type and num- ber of mutations found in SLC3A1. Of the 261 muta- tions identified, data on 210 mutations has been made publicly available by the Human Gene Muta- tion Database (HGMD) from the Institute of Medical Genetics in Cardiff. TABLE 2 presents the mutation type and number of mutations found in SLC7A9. Of the 170 mutations identified, data on 143 mutations has been made publicly available by HGMD (Sten- son et al., 2003). ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV TABLE 1: SLC3A1 mutations listed in the HGMD database. TABLE 2: SLC7A9 mutations listed in the HGMD database. Mutation type Number of mutations Missense/nonsense 128 Splicing 13 Regulatory 1 Small deletions 19 Small insertions 11 Small indels (insertions + deletions) 2 Gross deletions 30 Gross insertions/duplications 5 Complex rearrangements 1 Repeat variations 0 Public total (HGMD Professional 2021.4 total) 210 (261) Mutation type Number of mutations Missense/nonsense 75 Splicing 18 Regulatory 0 Small deletions 29 Small insertions 10 Small indels (insertions + deletions) 1 Gross deletions 9 Gross insertions/duplications 1 Complex rearrangements 0 Repeat variations 0 Public total (HGMD Professional 2021.4 total) 143 (170) ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV 4 MOUSE MODELS KNOCKOUT MOUSE MODELS Several mouse models have been gener- ated to observe the traits associated with types A, B, and AB cystinuria (Sahota et al., 2019). Among these is a knockout Slc3a1 mouse model, Slc3a1-/-, in which both copies of the SLC3A1 gene were mu- tated to become nonfunctional, or “knocked out” (Sahota et al., 2019). Urine analyses have revealed the presence of supersaturated cystine crystals in the Slc3a1-/- mice (FIGURE 8). Computed tomography (CT) scanning was also used to view the cystine stones found in these knockouts (FIGURE 9). A Slc7a9- /- knockout mouse model with deletion mutations in both copies of SLC7A9 was also created (Font- Llitjós et al., 2007). Both type A Slc3a1-/- and type B Slc7a9-/- mice presented higher urinary levels of cys- tine in comparison to wild-type (non-mutated) mice (Beckermann et al., 2020; Font-Llitjós et al., 2007). A mouse model of type AB cystinuria (Slc3a1+/−, Slc7a9+/−) was generated by crossing type A and type B mice (Sahota et al., 2019). These type AB mice also had COLA hyperexcretion; however, they presented more severe stone formation than type A or type B mice (Espino et al., 2015). FIGURE 8: The hexagonal cystine crystals observed in an Slc3a1-/- mouse. Image provided by Amrik Sahota, Ph.D. FIGURE 9: Cystine stones in an Slc3a1 knockout mouse (top), shown to scale (bottom). Figure provided by Amrik Sahota, Ph.D. GENDER DIFFERENCES Males with cystinuria experience more ag- gressive disease symptoms than females, a charac- teristic reflected by Slc3a1-/- mice (Sahota et al., 2019). Knockout males and females presented cys- tine crystals of similar size and distribution; how- ever, bladder stones only formed in a few female mice and with a later onset (>18 months) than their male counterparts (Sahota et al., 2019). Sex differences were not observed in the knockout Slc7a9-/- mice, as both males and females formed stones in a 1:1 ratio with an onset age of one month (Feliubadaló et al., 2003). 5 CURRENT TREATMENTS AND LIMITATIONS Individuals with cystinuria will experience recurrent cystine stone formation throughout their lifetimes, so behavioral management and pharma- cological therapies are often necessary to increase quality of life (Siener et al., 2021). Treatment meth- ods for cystinuria have remained largely unaltered for the past few decades. Currently, most cystinuria patients are advised to increase their fluid intake and reduce their protein and sodium consumption https://orcid.org/0000-0002-3603-673X https://orcid.org/0000-0002-3603-673X https://orcid.org/0000-0002-3603-673X ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV (Siener et al., 2021). In addition to behavioral modi- fications, urinary alkalinization (increasing urine pH) is considered a primary treatment because cystine is more soluble at higher pH values (Pearle et al. 2014). Afflicted individuals may take potassium cit- rate to achieve a urine pH of 7.0-7.5 (the normal av- erage urine pH is 6.0) (Pearle et al. 2014). In more severe cases, patients may be prescribed thiol drugs, which contain a thiol functional group (-SH) that binds to cystine (Pearle et al. 2014). The previously mentioned treatment meth- ods all have limitations. Many cystinuria patients have trouble adhering to behavioral modifications, especially young children who may find it difficult to consume large amounts of water (Sahota et al. 2019). Excess potassium citrate can lead to the for- mation of calcium phosphate stones (another type of kidney stone), and thiol drugs have several dose- dependent adverse effects (Pereira, Schoolwerth & Pais, 2015). Such side effects include, but are not limited to, skin diseases, liver abnormalities, and blood disorders (DeBerardinis et al., 2008). There- fore, there is a clear need for more tolerable, pre- ventative treatment options. Due to the limitations of current treatments, most cystinuria patients require multiple surgical in- terventions throughout their lifetimes. Non-invasive stone-removing procedures include extracorporeal shockwave lithotripsy (ESWL), which directs a shock wave at the stone (Wood et al., 2011). However, cys- tine stones are somewhat resistant to ESWL, so mul- tiple rounds of treatment are necessary (Wood et al., 2011). Furthermore, only 37.5% of cystinuria pa- tients remain stone-free for three months after un- dergoing ESWL (Landau et al., 2009). Several con- current ESWL treatments increase the risk of kidney damage. Renal injuries as the result of ESWL in- clude, but are not limited to, hemorrhages, ruptur- ing of small veins and capillaries, necrosis (prema- ture cell death), hematomas (severe bruises), and complete loss of kidney function (McAteer & Evan, 2008). 6 CYSTINE STONE INHIBITORS CYSTINE ANALOGS Cystine crystallization is a critical step in stone formation; therefore, potential treatments for cystinuria have been evaluated for their ability to in- hibit cystine crystallization (Yang et al., 2018). Atomic force microscopy (AFM), a high-resolution microscopy technique, was used to visualize growth on the surface of cystine crystals in the presence of 31 prospective crystal inhibitors (Poloni et al., 2017). The data showed that the most effective inhibitors of cystine crystal growth were cystine analogs, also known as “molecular imposters.” Cystine diesters and cystine diamides, two types of cystine analogs, demonstrated the greatest inhibitory effects on cys- tine crystal growth (Poloni et al., 2017). In the pres- ence of these inhibitors, cystine crystals were smaller and changed shape from hexagonal to te- tragonal, making them more soluble (Poloni et al., 2017). Maintaining higher levels of cystine in solu- tion is crucial to inhibiting cystine crystallization (Hu et al., 2016). CYSTINE DIAMIDES A series of cystine diamides were designed, synthesized, and then evaluated for their ability to inhibit cystine crystallization (Yang et al., 2018). Of the synthesized cystine diamides, L-cystine bismor- pholide and L-cystine bis (N′-methylpiperazide) were the greatest crystallization inhibitors; they were 7 and 24 times more potent, respectively, as well as more stable than a previously studied cystine diester, L-cystine dimethylester (CDME) (Yang et al., 2018). Additionally, L-cystine bis (N′-methylpiper- azide) has been able to successfully inhibit stone formation in an Slc3a1 knockout mouse model, in- dicating that cystine diamides could potentially be used to prevent the formation of cystine stones in human cystinuria patients (Yang et al., 2018). How- ever, because the knockout mice form bladder stones rather than kidney stones (Woodard et al., 2019), the direct application of these results to hu- man patients may have some limitations. Since cystine diamides have greater chemi- cal stability than cystine diesters (e.g., CDME), they are likely more resistant to proteolytic degradation ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV (the breakdown of peptide bonds in an amino acid) (Hu et al., 2016). L-cystine bismorpholide and L-cys- tine bis (N′-methylpiperazide) are more promising treatments than CDME not only because of their in- creased chemical stability, but also because they are orally bioavailable (they can be easily ingested by mouth and absorbed by the body) (Hu et al., 2016). While CDME has been effective in decreasing cys- tine stone size and mass, its efficacy post oral admin- istration may be reduced due to esterase-mediated hydrolysis, a process causing the degradation of diesters (Hu et al., 2016). Furthermore, AFM has re- vealed that cystine diamides are better than CDME and other cystine diesters at maintaining higher lev- els of cystine in solution, an important factor in pre- venting cystine crystals from aggregating into stones. (Hu et al., 2016). 7 FUTURE DIRECTION As our knowledge and understanding of the pathophysiology of cystinuria expands, new thera- pies and treatments will continue to emerge. Ad- vancements in imaging technology and its interpre- tation will progress the current treatment manage- ment systems toward more effective methods. The application of AFM in identifying crystal growth in- hibitors and the continued use of mouse models will provide greater insight into these alternative thera- pies (Pereira et al., 2015). Aside from cystine analogs, gene therapy appears to be a promising treatment for cystinuria as well. Gene therapy is a disease treatment tech- nique in which a diseased gene copy is replaced with a healthy gene copy in a living organism. CRISPR/Cas9 precision gene editing was recently utilized to create a Slc7a9-/- knockout mouse model of cystinuria (Bai et al., 2019). Research groups are attempting to use gene therapy to repair the Slc7a9 deletions in these knockouts. However, multiple ob- stacles have presented themselves, including im- mune responses against vectors, which are organ- isms, usually bacteria, that deliver foreign DNA to recipient cells (Bai et al., 2019). The location and anatomy of the kidney could be causing difficulties in vector delivery (Bai et al., 2019). More must be learned about applying gene therapy technologies to the renal system to proceed (Bai et al., 2019). If research efforts are successful, gene therapy could become an ideal, one-time treatment for cystinuria as it has been for other rare genetic disorders (e.g., spinal muscular atrophy) (Mendell et al., 2017). Even though there is great diversity in the mutations that can lead to cystinuria (Stenson et al., 2003), gene therapy is versatile in the mutations it can correct with a healthy gene copy (Luther et al., 2018). 8 CONCLUSIONS Cystinuria is a genetic disorder that causes the formation of cystine stones in the renal system as a result of mutations in the SLC3A1 and SLC7A9 genes. Distributions of the disease vary by popula- tion, although males are more likely than females to have severe disease presentation. Because cystine stones have a high recurrence rate, cystinuria pa- tients frequently require several surgical interven- tions throughout their lifetimes. Current treatments, such as increased fluid intake, urine alkalinization, and thiol drugs, are aimed at delaying, but not nec- essarily eliminating, the need for surgical interven- tions. This renders them nonoptimal, especially be- cause they may cause severe side effects. Cystine analogs, particularly cystine diesters (e.g. CDME) and cystine diamides (e.g., L-cystine bismorpholide and L-cystine bis (N′-methylpiperazide)), have demonstrated their ability to effectively inhibit cys- tine crystal growth and, in some cases, stone for- mation. This qualifies them as potential alternatives to the cystinuria treatments currently in use. Gene therapy has also been considered as a potential treatment; however, not enough information about its use in the renal system is presently known. With continued research, a new treatment that will im- prove the quality of life of human cystinuria patients could be made available in the near future∎ 9 ACKNOWLEDGEMENTS Amrik Sahota, Ph.D. (Human Genetics Insti- tute of New Jersey) provided the images used in Figures 6 and 7 of this literature review. I’d like to thank him for his contribution and his guidance as a research mentor. 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HTTPS://DOI.ORG/10.1016/J.BMCL.2018.03.024 https://doi.org/10.1016/j.semnephrol.2008.01.003 https://doi.org/10.1016/j.juro.2014.05.006 https://doi.org/10.1021/acs.cgd.7b00236 https://doi.org/10.1080/00365590601154551 https://doi.org/10.1016/j.ucl.2007.04.006 https://doi.org/10.1007/s00240-018-1101-7 https://doi.org/10.3390/nu13020528 https://doi.org/10.1097/MNH.0b013e3283621c5d https://doi.org/10.1089/end.2017.0335 https://doi.org/10.1186/s12882-019-1417-8 https://doi.org/10.1016/j.bmcl.2018.03.024 ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV Diana Stachula is a Rutgers Presidential Scholar who graduated summa cum laude from the Honors College at Rutgers University - New Brunswick in May 2022, having completed a B.A. in Genetics and a minor in Psychology. From 2020-2022, Diana worked as a research assistant for Dr. Amrik Sahota at the Human Genetics Institute of New Jersey (HGINJ), where she studied cystinuria - a rare genetic disorder that causes the formation of kidney stones. More spe- cifically, she researched novel pharmacological agents as potential crystal growth and stone formation inhibitors. As a lab member, Diana performed gel electrophoresis, polymerase chain reactions (PCR), quantitative PCR (qPCR), Nanopore sequencing, RNA/DNA extractions from tissue, and various other genetic techniques. She has presented her research at several research sym- posiums, including the university-wide Aresty Undergraduate Research Sym- posium. Diana also worked as a Peer Instructor at the Aresty Research Center, mentoring new research assistants on professional development and com- municating their research findings. Currently, Diana is working as an ophthalmic technician and medical assistant in order to gain more experience prior to applying to medical school. As a future physician, Diana hopes that she can blend her interests in science and medicine by performing clinical research.