Tuberous sclerosis complex without tubers and subependymal nodules: a phenotype genotype study

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1 Clin Genet 2014: 86: Printed in Singapore. All rights reserved Short Report 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: /cge Tuberous sclerosis complex without tubers and subependymal nodules: a phenotype genotype study Boronat S., Shaaya E.A., Doherty C.M., Caruso P., Thiele E.A. Tuberous sclerosis complex without tubers and subependymal nodules: a phenotype genotype study. Clin Genet 2014: 86: John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2013 Tuberous sclerosis complex (TSC) is caused by a mutation in the TSC1 or TSC2 genes. However, 15% of patients have no mutation identified. Tubers and subependymal nodules (SENs) are the typical brain lesions in TSC and are present in 90 95% of patients. The objective of this study is to characterize the specific genotype phenotype of patients without these lesions. We analyzed the of 11 patients without typical TSC neuroanatomic. Ten had TSC1/TSC2 mutational analysis, which was negative. Clinically they had lesions thought to be of neural crest (NC) origin, such as hypomelanotic macules, facial, cardiac rhabdomyomas, angiomyolipomas, and lymphangioleiomyomatosis. We hypothesize that patients without tubers and SENs reflect mosaicism caused by a mutation in TSC1 or TSC2 in a NC cell during embryonic development. This may explain the negative results in TSC1 and TSC2 testing in DNA from peripheral leukocytes. Conflictofinterest The authors declare no conflict of interest. S. Boronat a,c, E.A. Shaaya a, C.M. Doherty a,p.caruso b and E.A. Thiele a a Department of Neurology, Herscot Center for TSC, Massachusetts General Hospital, Boston, MA, USA, b Department of Neuroradiology, Massachusetts General Hospital, Boston, MA, USA, and c Department of Pediatric Neurology, Vall d Hebron Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain Key words: mosaicism neural crest no mutation identified (NMI) tuberous sclerosis complex (TSC) Corresponding author: Elizabeth A. Thiele, MD, PhD, Pediatric Epilepsy Program, Massachusetts General Hospital, 175 Cambridge Street, Suite 340, Boston, MA 02114, USA. Tel.: ; fax: ; ethiele@partners.org Received 14 May 2013, revised and accepted for publication 29 July 2013 Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by an inactivating mutation in the TSC1 or TSC2 genes with the result of hyperactivation of the mtor pathway (1). Many tissues can be affected including the brain, skin, eye, heart, kidney, lung, and bone. Tubers and subependymal nodules (SENs) are the typical TSC lesions and are present in 90 95% of patients. This study analyzes the genotype phenotype correlation in TSC patients without these typical brain lesions. Materials and methods We performed a retrospective review of the charts of 404 patients (182 males and 222 females; mean ± SD age: 24.3 ± 16.4 years; range: 6 months 81 years) with definite clinical diagnosis of TSC (2) and identified 11 patients (2.7%) without tubers and SENs who had brain MRIs available for review. We also performed a search for patients with typical TSC brain lesions and without skin involvement; a single patient was identified. The following information was noted for all patients: age, gender, type of mutation, any clinical manifestation related to TSC in family members, clinical phenotype specifying which major and minor were present, and any other related to TSC but not currently considered as diagnostic criteria. For comparison, gender and type of mutation was recorded for the 385 (174 males and 211 females) TSC patients with typical brain findings. Genetic analysis consisted in sequencing of TSC1 and TSC2 and study 149

2 Boronat et al. of TSC2 deletion, performed by Athena Diagnostics, in all patients. Mutation results were available for 280 patients: 78 had a TSC1 mutation (28%), 160 had a TSC2 mutation (57%) and 42 had no mutation identified (NMI) (15%). Statistical analysis was performed using spss version 11.5 for Windows. An alpha level of 0.05 was used for all statistical calculations. Contingency tables were analyzed using chi-squared or Fisher tests. Results Ten of the 11 TSC patients without tubers and SENs (Table 1) had mutational studies, which resulted in NMI in all patients (100%). This percentage is significantly higher than the 15% of patients with NMI in the group of TSC patients with tubers and/or SENs (p< ). No difference in gender was noted (six females and five males), and none of the patients had a family history of definite TSC nor any clinical manifestation related to TSC in family members. Fundoscopic examination was performed in 7 of the 11 patients with normal results. In 5 of the 11 cases, the brain MRI was completely normal. In one patient, a hemispherectomy had been performed for refractory epilepsy. Pre-surgical brain MRI scans were normal as were subsequent scans of the remaining brain tissue. One patient had a meningioma. Four patients had dilated perivascular spaces. Seven patients had some degree of brain dysfunction, usually mild, such as global development delay, autistic spectrum disorder, attention deficit and hyperactivity disorder, depression, or epilepsy. Discussion Our study shows that TSC patients without tubers and SENs have a high probability of NMI (100% in our study) in mutational studies performed in DNA from peripheral leukocytes. Previous published data support the notion that the lesions in our patients may have in common a neural crest (NC) origin (3, 4). NC dysfunction has been implicated in some of the clinical manifestations of TSC including hypomelanotic macules and facial as well as other neurocutaneous syndromes, such as neurofibromatosis type 1, which also involves an upregulation of the mtor pathway (3). The NC is a multipotent cell population located between the neural and non-neural ectoderms that migrates and colonizes nearly all tissues of the embryo. It gives rise to a wide range of derivatives, including melanocytes, neurons and glia of the peripheral nervous system, cranial meninges, bone, cartilage and connective tissues of the head and face and smooth muscle cells of intracranial vessels (5). The patients in this study represent a phenotype of multiorgan involvement that appears to be restricted to NC cells. We hypothesize that mutations in TSC1 or TSC2 in a NC progenitor, explaining the NMI results from mutation analysis in peripheral lymphocytes in our patients, results in many of the clinical of TSC but not the typical brain TSC involvement. However, not all individuals with TSC and NMI have this phenotype, as it was observed only in 11 of the 53 patients (21%) with NMI in our cohort. In our patients, skin is the most frequently involved organ. The most common lesions, hypopigmented macules and, involve cells of neural cest origin, i.e. melanocytes and facial fibroblasts. If cells of NC origin are involved in other skin lesions in our patients, such as shagreen patches or ungual fibromas, is unknown. However, this is certainly a possibility, as the skin, including the nail bed, is colonized by melanocytes and Merkel cells, which are of NC origin (6). None of our patients have a forehead plaque; one study has closely correlated this manifestation with typical TSC brain lesions (7). Retinal hamartomas were also not detected in our cohort, and this may be explained by the sparing of the eye, similarly to the brain since the retinal epithelium originates from the neural ectoderm (6). Cardiac rhabdomyomas, lymphangioleiomyomatosis and angiomyolipomas are lesions frequently found in our cohort and all stain with the monoclonal antibody HMB-45, which reacts positively against fetal melanocytes (4), while other lesions such as the subependymal giant cell tumor do not (8). The NC play a role in cardiac development, such as forming the septum, dividing the truncus into the aorta and pulmonary arteries and giving rise to the connective tissue insulation of the His-Purkinje conduction system (9). A mouse model of loss of TSC1 in ventricular myocytes failed to induce proliferative cardiac tumors (10). A possible explanation could be that cardiac rhabdomyomas arise from NC cells instead of myocytes. Sporadic lymphangioleiomyomatosis is frequently associated with renal angiomyolipomas and also occasionally with extrarenal tumors in the retroperitoneal area, pancreas, adrenals, uterus, liver, thyroid and parathyroid glands (11), and with sclerotic bone lesions (12). Mutations in both alleles of the TSC2 gene have been found in the lung and kidney lesions but not in normal lung, kidney, or circulating lymphocytes (13). This has led to the current hypothesis that pulmonary lymphangioleiomyomatosis cells reach the lungs via a metastatic process. This is also supported by one case of recurrent lymphangioleiomyomatosis with the same TSC2 mutation after lung transplantation (14) as well as the finding that TSC2-deficient smooth muscle cells have higher migration potential than normal cells in vitro (13). These data argue for different pathomechanisms in brain and extracerebral lesions and suggest a major role for the NC in the TSC pathophysiology of extracranial lesions. We hypothesize that, as adult tissues contain NC stem cells (5), an abnormal proliferation following a second hit may be responsible for many of the manifestations of TSC occurring outside the brain. There is little information about the possible role of the NC in other lesions present in our patients, 150

3 Table 1. Patients with definite TSC and no tubers and SENs Tuberous sclerosis complex without tubers and subependymal nodules P G, A Mut Major and minor criteria Other Brain MRI Brain function TSC in family members 1 M, 4 NMI Major: Four hypomelanotic macules: one midline thorax, 2 L (abdomen), 1 R (calf) Multiple cardiac rhabdomyomas 2 F, 4 ND Major: Four hypomelanotic macules: 2 L (flank, shin), 2 R (chest, upper arm) One cardiac rhabdomyoma 3 M, 6 NMI Major: Four hypomelanotic macules: 4 L (three flanks, one leg) Two cardiac rhabdomyomas 4 M, 7 NMI Major: Five hypomelanotic macules: chest, both scapulas, occiput, L posterior thigh Multiple cardiac rhabdomyomas 5 M, 10 NMI Major: One shagreen patch in chest Multiple cardiac rhabdomyomas 6 M, 30 NMI Major: Facial Ungual fibromas 1 CALS (L back) Normal Normal Parents: no TSC Siblings (one older): no TSC Nevus (L flank) Frontal developmental venous anomaly 1 CALS (scrotum) R choroidal fissure cyst spaces in right convexity Three hypomelanotic macules: 2 R (buttock and posterior knee) 1 L (posterior thigh) 2 CALS (L hip and neck) Normal Parents: no TSC Siblings (one younger): no TSC Normal GDD, ASD Parents: no TSC Siblings: no Normal GDD, febrile seizures, anxiety Febrile seizures ADHD Parents: no TSC Siblings: no Parents: no TSC Siblings: no Normal Normal Parents: NA Siblings: NA 7 F, 40 NMI Major: Facial Ungual fibromas One hypomelanotic macule on back Subtotal L hemispherectomy and wallerian degeneration of L cerebral peduncle and pons Severe GDD, epilepsy Parents: mother: no TSC, father: NA Siblings (1 sister): no TSC 8 F, 47 NMI Major: Facial L Renal AML and LAM Minor: Renal cysts Hepatic AML SBLs MMPH spaces in subcortical WM Normal Parents: NA Siblings: NA Offspring [daughter (14 years) and son (11 years)]: no TSC 9 F, 50 NMI Major: Facial Bilateral renal AMLs and LAM Minor: Confetti lesions Renal cysts Hepatic cysts One hypomelanotic macule on upper back spaces in WM of the L frontal and R parietal lobes Anxiety, depression Parents: NA Siblings (three brothers and two sisters): no TSC Offspring [daughter (27 years)]: no TSC 10 F, 50 NMI Major: Multiple hypomelanotic macules (biggest 2 on R) Bilateral renal AMLs Uterine leiomyoma MMPH Normal Depression Parents: NA Siblings (one sister, one brother): no TSC 151

4 Boronat et al. Table 1. Continued P G, A Mut Major and minor criteria Other Brain MRI Brain function TSC in family members 11 F, 58 NMI Major: Facial Bilateral renal AMLs Uterine leiomyomas spaces in periventricular and subcortical WM. Partial R temporal resection (epilepsy surgery) R parafalcine meningioma Epilepsy from 9 to 30 years. Exploratory surgery at 10 years. Intellectual disability Parents: NA Siblings (one brother): no TSC A, age in years; ADHD, attention deficit and hyperactivity disorder; AMLs, angiomyolipomas; ASD, autistic spectrum disorder; CALS, café-au-lait spot; F, female; G, gender; GDD, global developmental delay; L, left; LAM, lymphangioleiomyomatosis; M, male; MMPH, micronodular multifocal pneumocyte hyperplasia; Mut, mutation; NA, non-available information; ND, not determined; NMI, no mutation identified; P, patient; R, right; SBLs, sclerotic bone lesions; TSC, Tuberous Sclerosis Complex; WM, white-matter; y, years; SEN, subependymal nodules. including sclerotic bone lesions, uterine leiomyomas or micronodular multifocal pneumocyte hyperplasia, so additional characterization of this clinical phenotype is needed. Our patients do not exhibit typical TSC brain lesions but may have involvement of intracranial NC derivatives, such as meninges, smooth muscle cells of the forebrain vessels and pericytes (5, 15). These lesions include an arachnoid cyst, a meningioma, a developmental venous anomaly and dilated perivascular spaces. Although some of these lesions have occasionally been reported in TSC (16, 17), additional characterization is needed about their prevalence in TSC and the role of NC in their pathophysiology. Figure 1 shows some of the intracranial manifestations in our patients. Although none of our patients have cortical tubers or white matter migration lines, it is noteworthy that seven have some manifestation of brain dysfunction, such as global developmental delay, autistic spectrum disorder or depression. As this is a retrospective study from a TSC and neurology clinic, a bias of overrepresentation of a population with neurological problems may be present. However, microscopic anomalies may be widespread in TSC brains (18) and that may be responsible in part for the dysfunction. Two of our patients have focal refractory epilepsy without evidence of tuber or cortical dysplasia. Epilepsy in TSC has been related classically to the presence of tubers although microscopic structural abnormalities, such as focal dyslamination, heterotopic neurons or isolated giant cells, have been reported in non-tuber cortices (18). The cranial NC contributes to the proper patterning of genes that are expressed in the prosencephalon (19) and, therefore, NC dysfunction may exert a critical effect on forebrain development. Also, dysfunction of the blood-brain barrier, which has been implicated in mechanisms of epileptogenesis (20), may contribute to epilepsy in our patients. NC derived pericytes are involved in the maintenance of proper blood-brain barrier properties and architecture such as the expression and distribution of tight junction proteins, vascular stability, diameter, and blood flow, so pericyte dysfunction in TSC may also be involved in epilepsy (21). It is noticeable that the phenotype with typical TSC brain lesions and without skin involvement is extremely rare in our cohort. The only identified patient was first seen in our program at 53 years of age and wood lamp examination was not performed. As hypomelanotic macules can disappear with aging (1), we cannot rule out their presence in our patient during childhood. In our cohort, the phenotype of TSC skin involvement without typical brain lesions (2.7%; 95% CI: 1.4 5%) is more frequent than the phenotype with brain lesions without skin involvement (0.25%; 95% CI: 0.01% 1.6%). Tubers are hallmarks of the presence of a TSC mutation in neuroectoderm. As the mutation would be also present in NC cells that derive from these neural progenitors, it is expected that in the presence of tubers, lesions related to NC derivatives would be present in almost all the cases. Our study has several limitations, including its retrospective nature and the lack of TSC1/TSC2 mutational study in cells of NC origin in our patients. Nonetheless, as above, previous reports support a NC origin of the lesions in our patients, including histological markers (4), embryological studies (5) and previous data involving the NC in the pathophysiology of TSC (3, 22). The existence of 15% of TSC patients with NMI in TSC1 or TSC2 has led to speculation that a third gene (i.e. TSC3) might exist, but, although considerable efforts and the use of new sequencing technologies, it has not been found (23, 24). A third subunit of the TSC1 TSC2 complex, TBC1D7, has recently been discovered, but it does not appear to be TSC3, as no mutations of this subunit were identified in 12 patients with NMI in TSC1 or TSC2 after extensive genetic analyses (24). Mosaicism is another hypothesis for explaining patients with NMI (25), which is supported by our study. In our patients, a mosaicism restricted to cells of NC origin would explain the sparing of the neuroectoderm and the negative mutational studies in peripheral leukocytes. Conclusion Our study suggests that TSC patients without tubers and SENs will probably result in NMI in TSC1/TSC2 152

5 Tuberous sclerosis complex without tubers and subependymal nodules (a) testing in DNA from peripheral leukocytes. We hypothesize that this phenotype is due to a TSC1 or TSC2 mutation in a NC progenitor during embryonic development and the persistence of mutated NC stem cells in the adult. To support this hypothesis, mutational studies of TSC1 and TSC2 in lesions of TSC patients without tubers and SENs are warranted. Acknowledgements This work was supported by the Herscot Center for Tuberous Sclerosis Complex. S. B. is supported by a BAE (Beca de ampliación de estudios) grant from the Carlos III Institute, Spain. (b) (c) Fig. 1. (a) In this coronal spoiled gradient recalled (SPGR) image of patient 4, two prominent perivascular spaces (arrow) in the right frontal white matter lie ipsilateral to a prominent choroidal fissure cyst (arrowhead) seen as a hyperintense lesion in the accompanying axial T2 weighted image (b). Perivascular spaces may be confluent as seen on (c) an axial T2 weighted image of patient 11 (arrowheads), that shows as well a right parafalcine meningioma (arrow). References 1. Crino PB, Nathanson KL, Henske EP. The tuberous sclerosis complex. N Engl J Med 2006: 355: Roach ES, Gomez MR, Northrup H. Tuberous sclerosis complex consensus conference: revised clinical diagnostic criteria. J Child Neurol 1998: 13: Sarnat HB, Flores-Sarnat L. Embryology of the neural crest: its inductive role in the neurocutaneous syndromes. J Child Neurol 2005: 20: Weeks DA, Chase DR, Malott RL. HMB-45 staining in angiomyolipoma, cardiac rhabdomyoma, other mesenchymal processes and tuberous sclerosis associated brain lesions. Int J Surg Pathol 1994: 1: Dupin E, Sommer L. Neural crest progenitors and stem cells: from early development to adulthood. Dev Biol 2012: 366: Schoenwolf GC, Bleyl SB, Brauer PR et al. Larsen s Human Embriology, 4 edn. Edinburgh: Churchill Livingstone, Rama Rao GR, Krishna Rao PV, Gopal KV, Kumar YH, Ramachandra BV. Forehead plaque: a cutaneous marker of CNS involvement in tuberous sclerosis. Indian J Dermatol Venereol Leprol 2008: 74: Gyure KA, Prayson RA. Subependymal giant cell astrocytoma: a clinicopathologic study with HMB45 and MIB-1 immunohistochemical analysis. Mod Pathol 1997: 10: Hutson MR, Kirby ML. Model systems for the study of heart development and disease. Cardiac neural crest and conotruncal malformations. Semin Cell Dev Biol 2007: 18: Meikle L, McMullen JR, Sherwood MC et al. A mouse model of cardiac rhabdomyoma generated by loss of Tsc1 in ventricular myocytes. Hum Mol Genet 2005: 14: Maziak DE, Kesten S, Rappaport DC, Maurer J. Extrathoracic angiomyolipomas in lymphangioleiomyomatosis. Eur Respir J 1996: 9: Avila NA, Dwyer AJ, Rabel A, Darling T, Hong CH, Moss J. CT of sclerotic bone lesions: imaging differentiating tuberous sclerosis complex with lymphangioleiomyomatosis from sporadic lymphangioleiomymatosis. Radiology 2010: 254: Astrinidis A, Henske EP. Aberrant cellular differentiation and migration in renal and pulmonary tuberous sclerosis complex. J Child Neurol 2004: 19: Karbowniczek M, Astrinidis A, Balsara BR et al. Recurrent lymphangiomyomatosis after transplantation: genetic analyses reveal a metastatic mechanism. Am J Respir Crit Care Med 2003: 167: Etchevers HC, Vincent C, Le Douarin NM, Couly GF. The cephalic neural crest provides pericytes and smooth muscle cells to all blood vessels of the face and forebrain. Development 2001: 128: Tatli M, Guzel A. Bilateral temporal arachnoid cysts associated with tuberous sclerosis complex. J Child Neurol 2007: 22: Irving RM, Ford GR, Jones NS. Tuberous sclerosis with primary meningioma of the maxillary antrum. J Laryngol Otol 1991: 105: Marcotte L, Aronica E, Baybis M, Crino PB. Cytoarchitectural alterations are widespread in cerebral cortex in tuberous sclerosis complex. Acta Neuropathol 2012: 123:

6 Boronat et al. 19. Creuzet SE, Martinez S, Le Douarin NM. The cephalic neural crest exerts a critical effect on forebrain and midbrain development. Proc Natl Acad Sci USA 2006: 103: Abbott NJ, Patabendige AA, Dolman DE, Yusof SR, Begley DJ. Structure and function of the blood-brain barrier. Neurobiol Dis 2010: 37: Marchi N, Lerner-Natoli M. Cerebrovascular remodeling and epilepsy. Neuroscientist 2013: 19: Curatolo P. MRI appearance of Sturge-Weber syndrome in tuberous sclerosis complex: is the neural crest the culprit? J Child Neurol 2009: 24: Qin W, Kozlowski P, Taillon BE et al. Ultra deep sequencing detects a low rate of mosaic mutations in tuberous sclerosis complex. Hum Genet 2010: 127: Dibble CC, Elis W, Menon S et al. TBC1D7 is a third subunit of the TSC1-TSC2 complex upstream of mtorc1. Mol Cell 2012: 47: Kwiatkowski D. TSC1, TSC2, TSC3? Or mosaicism? Eur J Hum Genet 2005: 13: