- Email: [email protected]
Genotype-phenotype correlation of patients with tuberous sclerosis complex–associated renal angiomyolipoma: a descriptive study
Human Pathology (2018) 82, 61–67
www.elsevier.com/locate/humpath
Original contribution
Genotype-phenotype correlation of patients with tuberous sclerosis complex–associated renal angiomyolipoma: a descriptive study☆ Shuqiang Li MD a,⁎, Yushi Zhang MD b , Zhiyong Wang MS a , Yanfeng Yang MS a , Wansheng Gao MD a , Dongsheng Li MS a , Jinxing Wei MD a a
Department of Urology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, PR China Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, PR China b
Received 9 May 2018; revised 1 July 2018; accepted 14 July 2018
Keywords: Tuberous sclerosis complex; Renal angiomyolipoma; Genotype; Phenotype; High-throughput sequencing
Summary TSC2 gene mutation was repeatedly reported to be associated with a more severe phenotype in patients with tuberous sclerosis complex (TSC). Our current study aims to further explore whether there is such a correlation in patients with TSC-associated renal angiomyolipoma (TSC-RAML). TSC1/TSC2 gene mutation was screened by high-throughput sequencing in 25 TSC-RAML patients from 2 medical centers. Clinical data were also carefully collected. Linear regression analysis and Student t‐test were conducted by IBM SPSS Statistics Version 21.0 to analyze the genotypic-phenotypic relationship. The results indicated a high level of TSC gene mutation (80%; 20/25) in TSC-RAML patients, with higher frequency of TSC2 mutation (68%; 17/25) than TSC1 mutation (12%; 3/25). Seven novel mutation sites were detected in this study. In general, there were no significant correlations between tumor size and age (r = 0.134, P = .522), hemoglobin (r = 0.255, P = .219), and serum creatinine (r = 0.043, P = .839). Patients with larger tumor size have higher risk of bleeding. Specially, it was higher hemoglobin level in patients with TSC1 mutation than ones with TSC2 mutation and without TSC mutation (P b .05). However, no difference was found in either tumor size or serum creatinine by TSC mutation genes (P N .05). Furthermore, no difference was found in tumor size, hemoglobin, and serum creatinine by TSC mutation types (P N .05). In conclusion, TSCRAML is TSC2 mutation dominant, with the individual differences varying greatly. No definite genotype-phenotype correlation exists in patients with TSC-RAML, and it needs to be further explored. © 2018 Elsevier Inc. All rights reserved.
1. Introduction ☆
Competing interests: None. ⁎ Corresponding author at: Department of Urology, The First Affiliated Hospital of Zhengzhou University, No.1 Jianshe East Road, Zhengzhou, Henan 450052, PR China. E-mail address: [email protected] (S. Li).
https://doi.org/10.1016/j.humpath.2018.07.017 0046-8177/© 2018 Elsevier Inc. All rights reserved.
Tuberous sclerosis complex (TSC), a rare autosomal dominant genetic disorder with a neonatal morbidity of 1/6,000 to 1/10,000 [1,2], seriously affects the health of patients and results in a heavy financial burden on the family and society [3,4]. Apart from the multiple-organ damages of the brain, eyes,
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heart, lung, liver, kidney, skin, teeth, and so on [5], the cerebral, renal, or pulmonary lesions always leave patients at great risk of death or disability. Especially the acute bleeding of renal angiomyolipoma (RAML) caused by spontaneous or traumatic rupture is the leading cause of death for being unable to rescue timely. Published studies have identified the tumor suppressor gene, TSC1 and TSC2. TSC1 gene is located on 9q34 [6], containing 23 exons with a length of 50 kb and coding the hamartin protein (molecular weight, 130 kDa; number of amino acids, 1164). TSC2 gene is located on 16p13.3 [7], containing 42 exons with a length of 45 kb and coding the tuberin protein (molecular weight, 190 kDa; number of amino acids, 1807). The hamartin protein and tuberin protein form the hamartintuberin complex normally [8], and then negatively regulates the mammalian target of rapamycin (mTOR) signaling pathway by acting on Rheb and the downstream transcription factors such as S6K1 and 4E-BP1, resulting in the inhibition of cell metabolism, proliferation, differentiation, and growth [5]. Either TSC1 or TSC2 mutation will lead to the overactivity of the mTOR signaling pathway and result in the development of TSC [9-11]. It has been confirmed that the mTOR signaling pathway is overactive in lymphangioleiomyomatosis, fetal tubers, and subependymal giant cell astrocytomas [12-15]. The discovery of the underlying genetic defects in TSC has furthered our understanding of this complex genetic disorder and made it possible to explore the genotype-phenotype Table
correlation. Previous studies indicated that more severe neurologic phenotype, including an earlier age of seizure onset, lower cognition, and more tubers, in patients with TSC2 mutation as compare with those with TSC1 mutation [16]. Moreover, the results in the study by Sampson et al [17] indicated that the large genomic deletions at TSC2 will cause the development of polycystic kidney disease early. However, information on the correlation of genetic and clinical characteristics in TSC-associated RAML (TSC-RAML) patients is still rare. This study aims to detect the TSC1/2 gene mutation in TSCRAML patients and analyze the relationship between genotype and phenotype.
2. Materials and methods 2.1. Patients and data collection The research was conducted according to the principles expressed in the Declaration of Helsinki and approved by the ethics committee (The reference number: S-714), and all patients or their parents were fully informed and provided the written informed consent. The 25 TSC-RAML patients in this study were all Chinese Han people from Peking Union Medical College Hospital and The First Affiliated Hospital
TSC1/TSC2 mutations in patients with TSC-RAML.
Patients
Age (y)
Sex
Gene
Nucleotide change
Amino acid change
Mutation type
Tumor size (cm)
1 2 3a 4 5 6 7a 8 9 10 a 11 a 12 13 a 14 a 15 a 16 17 18 19 20 21 22 23 24 25
14 38 40 29 24 8 35 25 24 39 35 13 30 37 30 28 30 25 9 28 31 27 28 31 14
Male Male Female Female Female Male Female Female Male Female Female Female Male Female Male Female Female Female Female Female Female Female Female Female Female
TSC1 TSC1 TSC1 TSC2 TSC2 TSC2 TSC2 TSC2 TSC2 TSC2 TSC2 TSC2 TSC2 TSC2 TSC2 TSC2 TSC2 TSC2 TSC2 TSC2 None None None None None
c.733CNT c.2227CNT EX9_12 DEL CAGNTAG c.3235GNT c.3412CNT c.4129CNT c.3412CNT c.3412CNT c.203_204insA c.788_789insC c.788_789 insC c.2738_2739 insT c.3683_3684insG c.4006_4007insC c.4926delC c.3601_3602insGGCCC EX22_24 DEL c.5228GNA c.5126CNT None None None None None
p.Arg245Ter p.Gln743Ter _ p.Gln417Ter G1123W p.Arg1138Ter p.Gln1377Ter p.Arg1138Ter p.Arg1138Ter p.Ala68AlafsX7 p.Leu263LeufsX75 p.Leu263LeufsX75 p.Thr913ThrfsX2 p.Leu1228LeufsX6 p.Ser1336SerfsX78 p.Asn1643ThrfsX29 p.Thr1203GlyfsX9 _ p.Arg1743Gln p.Pro1709Leu None None None None None
NM NM DM NM NM NM NM NM NM FM FM FM FM FM FM FM FM DM MM MM None None None None None
10.3 3.8 8.3 3.5 10 3.2 10 42 11 10.4 16 1 32 8 7.3 12.1 13.6 11.2 4 8.6 6.8 3.2 23 7.4 11
Abbreviations: FM, frameshift mutation; MM, missense mutation; NM, nonsense mutation; DM, deletion mutation. a Novel gene mutation site.
Genotype-phenotype correlation of patients with TSC-RAML
63
Fig. 1 Characteristics of TSC-RAML in computed tomography. A-C, No renal contours can be observed at bilateral renal regions, which are occupied by huge lesions, filling the entire abdominal and pelvic cavity, with multiple aneurysms in the lesions. D, Both of the bilateral renal pelvises are clearly severely deformed with the right renal hydronephrosis.
of Zhengzhou University and were enrolled between October 2012 and May 2016. They were all diagnosed clinically according to the updated diagnostic criteria [18]. Abdominal enhanced computed tomographic scanning, blood routine, and biochemical examination were conducted as outpatient procedures. The patient's demographic data and previous medical records were also collected in detail.
2.2. Mutational screening A total of 3 mL of peripheral venous blood was taken from each patient. Genomic DNA was extracted from the blood samples using the standard process of the QIAamp DNA Blood Midi Kit (Qiagen, Hilden, Germany) and fragmented into 200- to 250-bp fragments using the Covaris LE220 soni-
Fig. 2 Representative histologic staining of TSC-RAML. A, Smooth muscle, adipose tissue, and thin-walled blood vessels were observed in the hematoxylin-eosin staining. B, Positive expression of mTOR was obviously observed in the immunohistochemical staining.
64 cator (Woburn, MA), and then purified in accordance with the operating procedures of the Agencourt AMPure XP kit (Agencourt AMPure XP 60 mL Kit; Beckman, Indianapolis, Indiana). The 3′ and 5′ ends of the purified DNA fragments were modified by T4 DNA polymerase and dNTP, and terminal A was added by incubating with dATP and the Klenow 3′-5′ exo-enzyme in accordance with the standard Illumina protocols, then the ligation-mediated polymerase chain reaction (PCR) and purification were conducted, following the hybridization reaction using the customized gene fragments capturing chips (Roche NimbleGen, Madison, WI). After 72 hours of hybridization, captured samples were obtained from the chips by washing and eluting, and then the ligation-mediated PCRs were conducted to generate a DNA library. The size, concentration, and enrichment of the DNA fragments were detected by Agilent 2100 Bioanalyzer (Agilent, Santa Clara, California) and ABI StepOne (Applied Biosystems, Foster City, California). Amplification with the high-fidelity DNA polymerase and high-throughput
S. Li et al. sequencing of the qualified DNA samples were carried out on the Illumina HiSeq 2500 sequencing platform (Illumina, San Diego, CA) for continuous bidirectional sequencing of 90 cycles. The Illumina base calling software (version 1.7) was used to deal with the original image data according to the Illumina analysis process, and the BWA (BurrowsWheeler Aligner, Sanger Institute, Hinxton, Cambridge, version 0.7.10) software was used for sequence alignments of qualified raw reads, which had been conducted sequencing quality assessment. With the SOAPsnp (BGI, Shenzhen, Guangdong, version 1.03) and Samtools (Sanger Institute, Hinxton, Cambridge, version 1.9) software, the nucleotide polymorphisms of the target region were generated through finding single-nucleotide variant and insertion and deletion, respectively, and then compared with the database (NCBI dbSNP, HapMap, and 1000 Genomes Project) to identify suspicious mutations. Finally, the Sanger sequencing or quantitative PCR method was used to verify the suspicious mutation sites.
Fig. 3 Representative sequencing of TSC1/2 gene mutation in TSC-RAML patients. A, Nonsense mutation in TSC1 gene (c.2227CNT). B, Nonsense mutation in TSC2 gene (c.4129CNT). C, Frameshift mutation in TSC2 gene (c.203_204 insA).
Genotype-phenotype correlation of patients with TSC-RAML
65
Fig. 4 Analysis of clinical characteristics based on TSC gene mutation. A-C, Scatterplots summarizing the correlation of tumor size and age, hemoglobin, and serum creatinine in patients with TSC-RAML. D and E, Differences of tumor size in patients with TSC-RAML by sex and bleeding. F-L, Differences of tumor size, hemoglobin, and serum creatinine by TSC1/TSC2 mutation and by mutation types. Pearson correlation analysis was conducted in panels A to C; one-way analysis followed by Tukey test was conducted in panels D to L.
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2.3. Statistical analysis
4. Discussion
Linear regression analysis and Student t test were conducted by IBM SPSS Statistics Version 21.0 (IBM, Armonk, NY).
TSC is an autosomal dominant genetic disease. Either TSC1 or TSC2 gene mutation can lead to the structural or functional abnormalities of the hamartin-tuberin complex, causing the overactivation of mTOR signaling pathway and resulting in the development of TSC [19,20]. Although TSC1 and TSC2 gene mutations were found in about 15% to 20% and 60% to 70% of TSC patients, respectively, no TSC gene mutation was found in the remaining 10% to 15% [21]. In the present study, 3 (12%) TSC1 and 17 (68%) TSC2 mutations were detected eventually in the 25 TSC-RAML patients. However, neither TSC1 nor TSC2 mutation was found in the remaining 5 (20%). The ratio of TSC1 to TSC2 mutation (TSC1/TSC2 = 1:5.67) in this study was obviously higher than the current records of LOVD database (TSC1, 873; TSC2, 2482; TSC1/ TSC2, 1:2.84) (http://www.LOVD.nl). It may suggest that patients with TSC2 mutation are susceptible to the development of TSC-RAML. The main mutation types of TSC-RAML patients in this study were nonsense and frameshift mutation, each accounting for 40%. In addition, 1 novel TSC1 (EX9_12 DEL) and 6 novel TSC2 mutation sites (c.203_204insA, c.4129CNT, c.788_789insC, c.2738_2739insT, c.3683_3684insG, and c.4006_4007insC) were detected in the present study. The results not only have further enriched the TSC gene mutation database but also indicate that TSC2 mutation is dominant in TSC-RAML patients and mainly nonsense and frameshift mutation. In terms of the relationship between genotype and phenotype, it has been described that TSC patients with TSC2 mutation have much more severe conditions compared with those with TSC1 mutation and without TSC mutation, coexisting large individual differences even within one family with the same mutation [22,23], which maybe resulted from some certain regulatory factors or possible mutations in noncoding regions interfering with the activation of mTOR signaling pathway [24]. However, although the large individual differences of TSCRAML patients are also observed in the present study, we have found some new discoveries that are different from the previous reports. First, the tumor size has no significant correlation with age. It means that the TSC-RAML occurs at different ages and grows at different rates. Second, the TSC-RAML patients with larger tumor size have higher risk of bleeding. However, the influence on anemia degree and renal function in daily life is relatively minor. Third, the tumor size, as well as the hemoglobin and serum creatinine, has no significant correlation with different mutation genes and mutation types. It indicates that TSC-RAML patients with TSC2 mutation do not necessarily have a more serious condition compared with those with TSC1 mutation or without TSC mutation. In view of the characteristics that TSC-RAML occurs multiply and bilaterally in kidneys and will gradually worsen with increasing age, it is rather difficult to manage clinically. Currently, a relatively consistent consensus of most scholars is to retain the renal function as far as possible. Therefore,
3. Results 3.1. Clinical data The 25 TSC-RAML patients (male, 6; female, 19) aged 8 to 40 years (x = 26.88 ± 9.07), with the TSC-RAMLs occurring bilaterally and multiply. The maximum diameter of TSC-RAML ranged from 1 to 42 cm (x = 11.11 ± 9.19) (Table), with 13 patients' tumor diameter of 10 cm or greater (Fig. 1). Sixteen (16/25; 64%) patients had a history of lumbar pain and spontaneous bleeding. One patient underwent the open enucleation of TSC-RAML because of the high risk of repeated spontaneous bleeding. The smooth muscle, adipose tissue, and thin-walled blood vessels were shown in the hematoxylin-eosin staining (Fig. 2A), and the overactivation of mTOR was also indicated in the immunohistochemical staining (Fig. 2B). The level of hemoglobin and serum creatinine ranged from 66 to 158 g/L (x = 106.84 ± 19.60) and from 40 to 90 μmol/L (x = 60.94 ± 12.85), respectively.
3.2. TSC1/2 mutation TSC1 and TSC2 mutations were detected in 3 (12%) and 17 (68%) cases, respectively, including totally 17 different mutation sites and 7 novel TSC mutations (Table). Neither TSC1 nor TSC2 mutation was detected in the remaining 5 (20%) patients. The distribution of mutation sites was very dispersed, with no centralized mutation region. The common mutation types were nonsense and frameshift mutation, each accounting for 40% (8/20), following the missense and deletion mutation, each accounting for 10% (2/20) (Table). All of the detected mutation sites had been verified by Sanger sequencing or quantitative PCR (Fig. 3).
3.3. Genotype-phenotype correlation analysis There was no significant correlation between tumor size and age (r = 0.134, P = .522), hemoglobin (r = 0.255, P = .219), and serum creatinine (r = 0.043, P = .839) (Fig. 4A-C). The statistical difference of tumor size was found between patients with and without a history of spontaneous bleeding (P = .020), but not between patients with different sexes (P = .963) and not among patients with different mutation genes (P N .05) and patients with different mutation types (P N .05; Fig. 4D-G and J). The hemoglobin in patients with TSC1 mutation was higher than that in those with TSC2 mutation (P = .006) and without TSC mutation (P = .017; Fig. 4H). No statistical difference of hemoglobin was found among patients with different mutation types (Fig. 4K). In terms of serum creatinine, no statistical difference was found among patients with different mutation genes (P N .05) and mutation types (P N .05; Fig. 4I and L).
Genotype-phenotype correlation of patients with TSC-RAML selective arterial embolization or surgical excision is only for patients with acute bleeding or with a high risk of bleeding [25,26], and only the mTOR inhibitors will prevent the further development of TSC-RAML to some extent [27-29], being a milestone in the treatment of TSC-RAML and bringing new hopes for TSC-RAML patients.
5. Conclusions As a TSC2 mutation-dominant autosomal genetic disease, TSC-RAML occurs bilaterally and multiply at different ages and grows at different rates. Unlike previous studies, our findings suggest that TSC-RAML patients with TSC2 mutation do not necessarily have a more serious condition than ones with TSC1 mutation or without TSC mutation. The increase in tumor diameter only increases the risk of bleeding, without affecting the anemia degree and renal function in daily life in general. Although the present study included a limited number of patients, it still showed several significant findings and preliminarily revealed the correlation between genotype and phenotype. Further researches and data analysis are needed to confirm these findings.
Acknowledgement The authors would like to thank Dr Zi-Yi Guo (Department of Radiology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, PR China), for his support in analyzing some of the images.
References [1] Osborne JP, Fryer A, Webb D. Epidemiology of tuberous sclerosis. Ann N Y Acad Sci 1991;615:125-7. [2] Sampson JR, Scahill SJ, Stephenson JB, et al. Genetic aspects of tuberous sclerosis in the west of Scotland. J Med Genet 1989;26:28-31. [3] Rentz AM, Skalicky AM, Liu Z, et al. Tuberous sclerosis complex: a survey of health care resource use and health burden. Pediatr Neurol 2015; 52:435-41. [4] Kingswood JC, Nasuti P, Patel K, et al. The economic burden of tuberous sclerosis complex in UK patients with renal manifestations: a retrospective cohort study in the clinical practice research datalink (CPRD). J Med Econ 2016;19:1116-26. [5] Curatolo P, Bombardieri R, Jozwiak S. Tuberous sclerosis. Lancet 2008; 372:657-68. [6] van Slegtenhorst M, de Hoogt R, Hermans C, et al. Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science 1997; 277:805-8. [7] European Chromosome 16 Tuberous Sclerosis Consortium. Identification and characterization of the tuberous sclerosis gene on chromosome 16. Cell 1993;75:1305-15.
67 [8] Tee AR, Manning BD, Roux PP, et al. Tuberous sclerosis complex gene products, tuberin and hamartin, control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb. Curr Biol 2003;13: 1259-68. [9] Orlova KA, Crino PB. The tuberous sclerosis complex. Ann N Y Acad Sci 2010;1184:87-105. [10] Niida Y, Wakisaka A, Tsuji T, et al. Mutational analysis of TSC1 and TSC2 in Japanese patients with tuberous sclerosis complex revealed higher incidence of TSC1 patients than previously reported. J Hum Genet 2013;58:216-25. [11] Jang MA, Hong SB, Lee JH, et al. Identification of TSC1 and TSC2 mutations in Korean patients with tuberous sclerosis complex. Pediatr Neurol 2012;46:222-4. [12] Tsai V, Parker WE, Orlova KA, et al. Fetal brain mTOR signaling activation in tuberous sclerosis complex. Cereb Cortex 2014;24:315-27. [13] Kristof AS. mTOR signaling in lymphangioleiomyomatosis. Lymphat Res Biol 2010;8:33-42. [14] Chan JA, Zhang H, Roberts PS, et al. Pathogenesis of tuberous sclerosis subependymal giant cell astrocytomas: biallelic inactivation of TSC1 or TSC2 leads to mTOR activation. J Neuropathol Exp Neurol 2004;63: 1236-42. [15] Muhlebner A, van Scheppingen J, Hulshof HM, et al. Novel histopathological patterns in cortical tubers of epilepsy surgery patients with tuberous sclerosis complex. PLoS One 2016;11:e0157396. [16] Kothare SV, Singh K, Chalifoux JR, et al. Severity of manifestations in tuberous sclerosis complex in relation to genotype. Epilepsia 2014;55: 1025-9. [17] Sampson JR, Maheshwar MM, Aspinwall R, et al. Renal cystic disease in tuberous sclerosis: role of the polycystic kidney disease 1 gene. Am J Hum Genet 1997;61:843-51. [18] Northrup H, Krueger DA. Tuberous sclerosis complex diagnostic criteria update: recommendations of the 2012 International Tuberous Sclerosis Complex Consensus Conference. Pediatr Neurol 2013;49:243-54. [19] Jozwiak J, Jozwiak S, Wlodarski P. Possible mechanisms of disease development in tuberous sclerosis. Lancet Oncol 2008;9:73-9. [20] Kwiatkowski DJ. Rhebbing up mTOR: new insights on TSC1 and TSC2, and the pathogenesis of tuberous sclerosis. Cancer Biol Ther 2003;2:471-6. [21] Crino PB, Nathanson KL, Henske EP. The tuberous sclerosis complex. N Engl J Med 2006;355:1345-56. [22] Henske EP, Jozwiak S, Kingswood JC, et al. Tuberous sclerosis complex. Nat Rev Dis Primers 2016;2:16035. [23] Li S, Zhang Y, Wei J, et al. Clinical and genetic analysis of tuberous sclerosis complex–associated renal angiomyolipoma in Chinese pedigrees. Oncol Lett 2017;14:7085-90. [24] Dunlop EA, Tee AR. mTOR and autophagy: a dynamic relationship governed by nutrients and energy. Semin Cell Dev Biol 2014;36:121-9. [25] Rouviere O, Nivet H, Grenier N, et al. Kidney damage due to tuberous sclerosis complex: management recommendations. Diagn Interv Imaging 2013;94:225-37. [26] Harabayashi T, Shinohara N, Katano H, et al. Management of renal angiomyolipomas associated with tuberous sclerosis complex. J Urol 2004;171:102-5. [27] Bissler JJ, Kingswood JC, Radzikowska E, et al. Everolimus for angiomyolipoma associated with tuberous sclerosis complex or sporadic lymphangioleiomyomatosis (EXIST-2): a multicentre, randomised, doubleblind, placebo-controlled trial. Lancet 2013;381:817-24. [28] Peng ZF, Yang L, Wang TT, et al. Efficacy and safety of sirolimus for renal angiomyolipoma in patients with tuberous sclerosis complex or sporadic lymphangioleiomyomatosis: a systematic review. J Urol 2014; 192:1424-30. [29] Brakemeier S, Bachmann F, Budde K. Treatment of renal angiomyolipoma in tuberous sclerosis complex (TSC) patients. Pediatr Nephrol 2017;32:1137-44.
www.elsevier.com/locate/humpath
Original contribution
Genotype-phenotype correlation of patients with tuberous sclerosis complex–associated renal angiomyolipoma: a descriptive study☆ Shuqiang Li MD a,⁎, Yushi Zhang MD b , Zhiyong Wang MS a , Yanfeng Yang MS a , Wansheng Gao MD a , Dongsheng Li MS a , Jinxing Wei MD a a
Department of Urology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, PR China Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, PR China b
Received 9 May 2018; revised 1 July 2018; accepted 14 July 2018
Keywords: Tuberous sclerosis complex; Renal angiomyolipoma; Genotype; Phenotype; High-throughput sequencing
Summary TSC2 gene mutation was repeatedly reported to be associated with a more severe phenotype in patients with tuberous sclerosis complex (TSC). Our current study aims to further explore whether there is such a correlation in patients with TSC-associated renal angiomyolipoma (TSC-RAML). TSC1/TSC2 gene mutation was screened by high-throughput sequencing in 25 TSC-RAML patients from 2 medical centers. Clinical data were also carefully collected. Linear regression analysis and Student t‐test were conducted by IBM SPSS Statistics Version 21.0 to analyze the genotypic-phenotypic relationship. The results indicated a high level of TSC gene mutation (80%; 20/25) in TSC-RAML patients, with higher frequency of TSC2 mutation (68%; 17/25) than TSC1 mutation (12%; 3/25). Seven novel mutation sites were detected in this study. In general, there were no significant correlations between tumor size and age (r = 0.134, P = .522), hemoglobin (r = 0.255, P = .219), and serum creatinine (r = 0.043, P = .839). Patients with larger tumor size have higher risk of bleeding. Specially, it was higher hemoglobin level in patients with TSC1 mutation than ones with TSC2 mutation and without TSC mutation (P b .05). However, no difference was found in either tumor size or serum creatinine by TSC mutation genes (P N .05). Furthermore, no difference was found in tumor size, hemoglobin, and serum creatinine by TSC mutation types (P N .05). In conclusion, TSCRAML is TSC2 mutation dominant, with the individual differences varying greatly. No definite genotype-phenotype correlation exists in patients with TSC-RAML, and it needs to be further explored. © 2018 Elsevier Inc. All rights reserved.
1. Introduction ☆
Competing interests: None. ⁎ Corresponding author at: Department of Urology, The First Affiliated Hospital of Zhengzhou University, No.1 Jianshe East Road, Zhengzhou, Henan 450052, PR China. E-mail address: [email protected] (S. Li).
https://doi.org/10.1016/j.humpath.2018.07.017 0046-8177/© 2018 Elsevier Inc. All rights reserved.
Tuberous sclerosis complex (TSC), a rare autosomal dominant genetic disorder with a neonatal morbidity of 1/6,000 to 1/10,000 [1,2], seriously affects the health of patients and results in a heavy financial burden on the family and society [3,4]. Apart from the multiple-organ damages of the brain, eyes,
62
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heart, lung, liver, kidney, skin, teeth, and so on [5], the cerebral, renal, or pulmonary lesions always leave patients at great risk of death or disability. Especially the acute bleeding of renal angiomyolipoma (RAML) caused by spontaneous or traumatic rupture is the leading cause of death for being unable to rescue timely. Published studies have identified the tumor suppressor gene, TSC1 and TSC2. TSC1 gene is located on 9q34 [6], containing 23 exons with a length of 50 kb and coding the hamartin protein (molecular weight, 130 kDa; number of amino acids, 1164). TSC2 gene is located on 16p13.3 [7], containing 42 exons with a length of 45 kb and coding the tuberin protein (molecular weight, 190 kDa; number of amino acids, 1807). The hamartin protein and tuberin protein form the hamartintuberin complex normally [8], and then negatively regulates the mammalian target of rapamycin (mTOR) signaling pathway by acting on Rheb and the downstream transcription factors such as S6K1 and 4E-BP1, resulting in the inhibition of cell metabolism, proliferation, differentiation, and growth [5]. Either TSC1 or TSC2 mutation will lead to the overactivity of the mTOR signaling pathway and result in the development of TSC [9-11]. It has been confirmed that the mTOR signaling pathway is overactive in lymphangioleiomyomatosis, fetal tubers, and subependymal giant cell astrocytomas [12-15]. The discovery of the underlying genetic defects in TSC has furthered our understanding of this complex genetic disorder and made it possible to explore the genotype-phenotype Table
correlation. Previous studies indicated that more severe neurologic phenotype, including an earlier age of seizure onset, lower cognition, and more tubers, in patients with TSC2 mutation as compare with those with TSC1 mutation [16]. Moreover, the results in the study by Sampson et al [17] indicated that the large genomic deletions at TSC2 will cause the development of polycystic kidney disease early. However, information on the correlation of genetic and clinical characteristics in TSC-associated RAML (TSC-RAML) patients is still rare. This study aims to detect the TSC1/2 gene mutation in TSCRAML patients and analyze the relationship between genotype and phenotype.
2. Materials and methods 2.1. Patients and data collection The research was conducted according to the principles expressed in the Declaration of Helsinki and approved by the ethics committee (The reference number: S-714), and all patients or their parents were fully informed and provided the written informed consent. The 25 TSC-RAML patients in this study were all Chinese Han people from Peking Union Medical College Hospital and The First Affiliated Hospital
TSC1/TSC2 mutations in patients with TSC-RAML.
Patients
Age (y)
Sex
Gene
Nucleotide change
Amino acid change
Mutation type
Tumor size (cm)
1 2 3a 4 5 6 7a 8 9 10 a 11 a 12 13 a 14 a 15 a 16 17 18 19 20 21 22 23 24 25
14 38 40 29 24 8 35 25 24 39 35 13 30 37 30 28 30 25 9 28 31 27 28 31 14
Male Male Female Female Female Male Female Female Male Female Female Female Male Female Male Female Female Female Female Female Female Female Female Female Female
TSC1 TSC1 TSC1 TSC2 TSC2 TSC2 TSC2 TSC2 TSC2 TSC2 TSC2 TSC2 TSC2 TSC2 TSC2 TSC2 TSC2 TSC2 TSC2 TSC2 None None None None None
c.733CNT c.2227CNT EX9_12 DEL CAGNTAG c.3235GNT c.3412CNT c.4129CNT c.3412CNT c.3412CNT c.203_204insA c.788_789insC c.788_789 insC c.2738_2739 insT c.3683_3684insG c.4006_4007insC c.4926delC c.3601_3602insGGCCC EX22_24 DEL c.5228GNA c.5126CNT None None None None None
p.Arg245Ter p.Gln743Ter _ p.Gln417Ter G1123W p.Arg1138Ter p.Gln1377Ter p.Arg1138Ter p.Arg1138Ter p.Ala68AlafsX7 p.Leu263LeufsX75 p.Leu263LeufsX75 p.Thr913ThrfsX2 p.Leu1228LeufsX6 p.Ser1336SerfsX78 p.Asn1643ThrfsX29 p.Thr1203GlyfsX9 _ p.Arg1743Gln p.Pro1709Leu None None None None None
NM NM DM NM NM NM NM NM NM FM FM FM FM FM FM FM FM DM MM MM None None None None None
10.3 3.8 8.3 3.5 10 3.2 10 42 11 10.4 16 1 32 8 7.3 12.1 13.6 11.2 4 8.6 6.8 3.2 23 7.4 11
Abbreviations: FM, frameshift mutation; MM, missense mutation; NM, nonsense mutation; DM, deletion mutation. a Novel gene mutation site.
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Fig. 1 Characteristics of TSC-RAML in computed tomography. A-C, No renal contours can be observed at bilateral renal regions, which are occupied by huge lesions, filling the entire abdominal and pelvic cavity, with multiple aneurysms in the lesions. D, Both of the bilateral renal pelvises are clearly severely deformed with the right renal hydronephrosis.
of Zhengzhou University and were enrolled between October 2012 and May 2016. They were all diagnosed clinically according to the updated diagnostic criteria [18]. Abdominal enhanced computed tomographic scanning, blood routine, and biochemical examination were conducted as outpatient procedures. The patient's demographic data and previous medical records were also collected in detail.
2.2. Mutational screening A total of 3 mL of peripheral venous blood was taken from each patient. Genomic DNA was extracted from the blood samples using the standard process of the QIAamp DNA Blood Midi Kit (Qiagen, Hilden, Germany) and fragmented into 200- to 250-bp fragments using the Covaris LE220 soni-
Fig. 2 Representative histologic staining of TSC-RAML. A, Smooth muscle, adipose tissue, and thin-walled blood vessels were observed in the hematoxylin-eosin staining. B, Positive expression of mTOR was obviously observed in the immunohistochemical staining.
64 cator (Woburn, MA), and then purified in accordance with the operating procedures of the Agencourt AMPure XP kit (Agencourt AMPure XP 60 mL Kit; Beckman, Indianapolis, Indiana). The 3′ and 5′ ends of the purified DNA fragments were modified by T4 DNA polymerase and dNTP, and terminal A was added by incubating with dATP and the Klenow 3′-5′ exo-enzyme in accordance with the standard Illumina protocols, then the ligation-mediated polymerase chain reaction (PCR) and purification were conducted, following the hybridization reaction using the customized gene fragments capturing chips (Roche NimbleGen, Madison, WI). After 72 hours of hybridization, captured samples were obtained from the chips by washing and eluting, and then the ligation-mediated PCRs were conducted to generate a DNA library. The size, concentration, and enrichment of the DNA fragments were detected by Agilent 2100 Bioanalyzer (Agilent, Santa Clara, California) and ABI StepOne (Applied Biosystems, Foster City, California). Amplification with the high-fidelity DNA polymerase and high-throughput
S. Li et al. sequencing of the qualified DNA samples were carried out on the Illumina HiSeq 2500 sequencing platform (Illumina, San Diego, CA) for continuous bidirectional sequencing of 90 cycles. The Illumina base calling software (version 1.7) was used to deal with the original image data according to the Illumina analysis process, and the BWA (BurrowsWheeler Aligner, Sanger Institute, Hinxton, Cambridge, version 0.7.10) software was used for sequence alignments of qualified raw reads, which had been conducted sequencing quality assessment. With the SOAPsnp (BGI, Shenzhen, Guangdong, version 1.03) and Samtools (Sanger Institute, Hinxton, Cambridge, version 1.9) software, the nucleotide polymorphisms of the target region were generated through finding single-nucleotide variant and insertion and deletion, respectively, and then compared with the database (NCBI dbSNP, HapMap, and 1000 Genomes Project) to identify suspicious mutations. Finally, the Sanger sequencing or quantitative PCR method was used to verify the suspicious mutation sites.
Fig. 3 Representative sequencing of TSC1/2 gene mutation in TSC-RAML patients. A, Nonsense mutation in TSC1 gene (c.2227CNT). B, Nonsense mutation in TSC2 gene (c.4129CNT). C, Frameshift mutation in TSC2 gene (c.203_204 insA).
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Fig. 4 Analysis of clinical characteristics based on TSC gene mutation. A-C, Scatterplots summarizing the correlation of tumor size and age, hemoglobin, and serum creatinine in patients with TSC-RAML. D and E, Differences of tumor size in patients with TSC-RAML by sex and bleeding. F-L, Differences of tumor size, hemoglobin, and serum creatinine by TSC1/TSC2 mutation and by mutation types. Pearson correlation analysis was conducted in panels A to C; one-way analysis followed by Tukey test was conducted in panels D to L.
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2.3. Statistical analysis
4. Discussion
Linear regression analysis and Student t test were conducted by IBM SPSS Statistics Version 21.0 (IBM, Armonk, NY).
TSC is an autosomal dominant genetic disease. Either TSC1 or TSC2 gene mutation can lead to the structural or functional abnormalities of the hamartin-tuberin complex, causing the overactivation of mTOR signaling pathway and resulting in the development of TSC [19,20]. Although TSC1 and TSC2 gene mutations were found in about 15% to 20% and 60% to 70% of TSC patients, respectively, no TSC gene mutation was found in the remaining 10% to 15% [21]. In the present study, 3 (12%) TSC1 and 17 (68%) TSC2 mutations were detected eventually in the 25 TSC-RAML patients. However, neither TSC1 nor TSC2 mutation was found in the remaining 5 (20%). The ratio of TSC1 to TSC2 mutation (TSC1/TSC2 = 1:5.67) in this study was obviously higher than the current records of LOVD database (TSC1, 873; TSC2, 2482; TSC1/ TSC2, 1:2.84) (http://www.LOVD.nl). It may suggest that patients with TSC2 mutation are susceptible to the development of TSC-RAML. The main mutation types of TSC-RAML patients in this study were nonsense and frameshift mutation, each accounting for 40%. In addition, 1 novel TSC1 (EX9_12 DEL) and 6 novel TSC2 mutation sites (c.203_204insA, c.4129CNT, c.788_789insC, c.2738_2739insT, c.3683_3684insG, and c.4006_4007insC) were detected in the present study. The results not only have further enriched the TSC gene mutation database but also indicate that TSC2 mutation is dominant in TSC-RAML patients and mainly nonsense and frameshift mutation. In terms of the relationship between genotype and phenotype, it has been described that TSC patients with TSC2 mutation have much more severe conditions compared with those with TSC1 mutation and without TSC mutation, coexisting large individual differences even within one family with the same mutation [22,23], which maybe resulted from some certain regulatory factors or possible mutations in noncoding regions interfering with the activation of mTOR signaling pathway [24]. However, although the large individual differences of TSCRAML patients are also observed in the present study, we have found some new discoveries that are different from the previous reports. First, the tumor size has no significant correlation with age. It means that the TSC-RAML occurs at different ages and grows at different rates. Second, the TSC-RAML patients with larger tumor size have higher risk of bleeding. However, the influence on anemia degree and renal function in daily life is relatively minor. Third, the tumor size, as well as the hemoglobin and serum creatinine, has no significant correlation with different mutation genes and mutation types. It indicates that TSC-RAML patients with TSC2 mutation do not necessarily have a more serious condition compared with those with TSC1 mutation or without TSC mutation. In view of the characteristics that TSC-RAML occurs multiply and bilaterally in kidneys and will gradually worsen with increasing age, it is rather difficult to manage clinically. Currently, a relatively consistent consensus of most scholars is to retain the renal function as far as possible. Therefore,
3. Results 3.1. Clinical data The 25 TSC-RAML patients (male, 6; female, 19) aged 8 to 40 years (x = 26.88 ± 9.07), with the TSC-RAMLs occurring bilaterally and multiply. The maximum diameter of TSC-RAML ranged from 1 to 42 cm (x = 11.11 ± 9.19) (Table), with 13 patients' tumor diameter of 10 cm or greater (Fig. 1). Sixteen (16/25; 64%) patients had a history of lumbar pain and spontaneous bleeding. One patient underwent the open enucleation of TSC-RAML because of the high risk of repeated spontaneous bleeding. The smooth muscle, adipose tissue, and thin-walled blood vessels were shown in the hematoxylin-eosin staining (Fig. 2A), and the overactivation of mTOR was also indicated in the immunohistochemical staining (Fig. 2B). The level of hemoglobin and serum creatinine ranged from 66 to 158 g/L (x = 106.84 ± 19.60) and from 40 to 90 μmol/L (x = 60.94 ± 12.85), respectively.
3.2. TSC1/2 mutation TSC1 and TSC2 mutations were detected in 3 (12%) and 17 (68%) cases, respectively, including totally 17 different mutation sites and 7 novel TSC mutations (Table). Neither TSC1 nor TSC2 mutation was detected in the remaining 5 (20%) patients. The distribution of mutation sites was very dispersed, with no centralized mutation region. The common mutation types were nonsense and frameshift mutation, each accounting for 40% (8/20), following the missense and deletion mutation, each accounting for 10% (2/20) (Table). All of the detected mutation sites had been verified by Sanger sequencing or quantitative PCR (Fig. 3).
3.3. Genotype-phenotype correlation analysis There was no significant correlation between tumor size and age (r = 0.134, P = .522), hemoglobin (r = 0.255, P = .219), and serum creatinine (r = 0.043, P = .839) (Fig. 4A-C). The statistical difference of tumor size was found between patients with and without a history of spontaneous bleeding (P = .020), but not between patients with different sexes (P = .963) and not among patients with different mutation genes (P N .05) and patients with different mutation types (P N .05; Fig. 4D-G and J). The hemoglobin in patients with TSC1 mutation was higher than that in those with TSC2 mutation (P = .006) and without TSC mutation (P = .017; Fig. 4H). No statistical difference of hemoglobin was found among patients with different mutation types (Fig. 4K). In terms of serum creatinine, no statistical difference was found among patients with different mutation genes (P N .05) and mutation types (P N .05; Fig. 4I and L).
Genotype-phenotype correlation of patients with TSC-RAML selective arterial embolization or surgical excision is only for patients with acute bleeding or with a high risk of bleeding [25,26], and only the mTOR inhibitors will prevent the further development of TSC-RAML to some extent [27-29], being a milestone in the treatment of TSC-RAML and bringing new hopes for TSC-RAML patients.
5. Conclusions As a TSC2 mutation-dominant autosomal genetic disease, TSC-RAML occurs bilaterally and multiply at different ages and grows at different rates. Unlike previous studies, our findings suggest that TSC-RAML patients with TSC2 mutation do not necessarily have a more serious condition than ones with TSC1 mutation or without TSC mutation. The increase in tumor diameter only increases the risk of bleeding, without affecting the anemia degree and renal function in daily life in general. Although the present study included a limited number of patients, it still showed several significant findings and preliminarily revealed the correlation between genotype and phenotype. Further researches and data analysis are needed to confirm these findings.
Acknowledgement The authors would like to thank Dr Zi-Yi Guo (Department of Radiology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, PR China), for his support in analyzing some of the images.
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