Clinical characteristics of tuberous sclerosis complex in patients with no TSC1 or TSC2 mutations identified

Transcription

1 CHILD NEUROLOGY NEUROLOGIA DZIECIĘCA Vol. 15/2006 Nr 30 PRACA ORYGINALNA/ORIGINAL ARTICLE Clinical characteristics of tuberous sclerosis complex in patients with no TSC1 or TSC2 mutations identified Charakterystyka kliniczna stwardnienia guzowatego u pacjentów bez zidentyfikowanej mutacji w genach TSC1 i TSC2 1 Magdalena Kaczorowska, 1 Julita Borkowska, 1 Dorota Domańska-Pakieła, 1 Jolanta Kasprzyk-Obara, 2 Penelope S. Roberts, 2 Sandra L. Dabora, 2 David J. Kwiatkowski, 1 Sergiusz Jóźwiak 1 Department of Child Neurology and Epileptology, The Children s Memorial Health Institute, Warsaw, Poland Head: prof. dr hab. n. med. S. Jóźwiak 2 Genetics Laboratory, Division of Hematology, Brigham and Women s Hospital, Boston, USA Head: D.J. Kwiatkowski, M.D., Ph.D., professor of medicine (HMS) Abstract Key words: tuberous sclerosis complex, TSC1, TSC2 Streszczenie Słowa kluczowe: stwardnienie guzowate, TSC1, TSC2 Vol. 15/2006, Nr 30 Aim: In the study we reassessed the clinical features of 22 patients diagnosed with tuberous sclerosis complex but with no mutation in TSC1 or TSC2 gene found, and compared them with the phenotypes of TSC1 and TSC2 patients, characterized in our previous study. Material and Methods: In a cohort of over 230 patients with tuberous sclerosis complex (TSC) diagnosed at the Children s Memorial Health Institute mutational analysis of the TSC1 and TSC2 genes has been carried out. In this group we identified 22 subjects (17 sporadic, 5 familial cases) in whom there were no definite mutational findings (the NMI no mutation identified group). Results: Epilepsy, hypomelanotic macules, facial angiofibroma, shagreen patches, ungual fibroma, SEGA, cortical tubers and kidney cysts were less common in the NMI group than in TSC1 or TSC2. Mental retardation was seen at similar frequency in the NMI and TSC2 groups. Frequency of other features: forehead plaque, SENs, kidney AMLs, liver AMLs and cardiac rhabdomyomas remained lower in the NMI than in TSC2, but higher than in TSC1 group. Conclusions: NMI patients had milder clinical features than patients with defined mutations in either TSC1 or TSC2 genes. Mosaicism is a likely explanation for this observation among sporadic TSC patients. In familial cases, however, the possibility of a distinct mutational spectrum or even mutation in a third TSC gene must be considered. Cel: W pracy przeprowadzono analizę objawów klinicznych 22 pacjentów ze stwardnieniem guzowatym (SG), ale bez znalezionej mutacji w genach TSC1 lub TSC2, oraz porównano ich fenotypy do opisanych we wcześniejszym badaniu fenotypów pacjentów ze stwierdzonymi mutacjami w TSC1 i TSC2. Materiał i metoda: W grupie 230 pacjentów ze zdiagnozowanym w Instytucie Pomnik- Centrum Zdrowia Dziecka SG (ang. TSC) wykonano badanie genetyczne poszukując mutacji w genach TSC1 i TSC2. Wśród tych pacjentów u 22 (17 przypadków sporadycznych, 5 rodzinnych) nie znaleziono mutacji w żadnym z genów (grupa NMI no mutation identified). Wyniki: Padaczka, plamy odbarwieniowe, angiofibroma twarzy, skóra szagrynowa, włókniaki okołopaznokciowe, SEGA, guzki podkorowe i torbiele nerek występowały rzadziej w grupie NMI niż w grupach z mutacją w TSC1 lub TSC2. Upośledzenie umysłowe stwierdzano równie często w grupie NMI i u pacjentów z TSC2. Częstość innych cech SG: płaskie włókniaki czoła, guzki okołokomorowe (SEN), angiomiolipoma (AML) nerek i rabdomioma serca pozostawała niższa w grupie NMI niż TSC2, ale wyższa niż w TSC1. Wnioski: Pacjenci z grupy NMI prezentują łagodniejsze objawy kliniczne niż pacjenci ze stwierdzoną mutacją w genach TSC1 lub TSC2. Mozaikowatość może tłumaczyć to zjawisko u pacjentów ze sporadyczną postacią SG. W postaciach rodzinnie występujących można jednak brać pod uwagę obecność mutacji modyfikujących ekspresję TSC1 lub TSC2, a nawet obecność mutacji w trzecim, nieznanym jeszcze genie SG. 15

2 Magdalena Kaczorowska, Julita Borkowska, Dorota Domańska-Pakieła et al. Introduction Tuberous sclerosis complex (TSC) is a genetic disorder characterized by the presence of typical skin abnormalities and hamartoma-like tumors of different organs [1]. The clinical diagnosis of the disease is based on criteria revised in 1998 by Roach et al. [2]. However, since the identification of the TSC2 and TSC1 genes in 1993 and 1997 respectively, genetic verification of the diagnosis screening for mutations in the two known genes is also possible [3,4]. There are few studies in the literature identifying TSC1 and TSC2 phenotypes [5,6,7]. In the current study we analyze clinical features of 22 patients from the Children s Memorial Health Institute with TSC phenotype but no mutation in TSC1/TSC2 genes found. To our knowledge there was only one study in the literature evaluating phenotypes of TSC patients in whom no mutation was found despite careful genetic analysis [6]. Materials and methods Patients The TSC Database of the Children s Memorial Health Institute (over 230 patients) has been screened for the subjects fulfilling the clinical Roach criteria for TSC and having done genetic testing for TSC1 and TSC2 mutation. From this cohort twenty-two patients were chosen whose DNA testing did not reveal either TSC1 or TSC2 mutations. There were seventeen sporadic and five familial cases. All patients were re-evaluated and present clinical status has been recorded. The average age was 8.1 years (ranging from 1 to 19 years). Median age was 7.5 years. The familial cases included 2 with one close relative presenting with some TSC symptoms, 2 with two close relatives presenting with TSC symptoms and 1 where four close relatives presented with TSC symptoms (the total of 10 individuals). Methods Clinical diagnosis of TSC was made according to the Roach criteria [2]. All auxiliary examinations including ultrasonography, echocardiography, brain CT and MR have been done in our Institute by specialists experienced in evaluating TSC features. Mutational analysis of TSC1 and TSC2 genes was carried out as described [5]. All patients provided informed consent. DNA samples were extracted from leukocytes and in some cases from lymphoblastoid cell lines transformed with Epstein Barr virus. The methods used in the analysis included: PCR, multiplex long-range PCR and DHPLC (denaturing high-performance liquid chromatography). A detailed description of the applied methods used is described elsewhere [1]. Statistical analysis was done using chi-square with Yates correction where needed. Results [1] Probands phenotypes Clinical characteristics of our NMI patients are summarized in table I [tabl. I]. In all patients TSC was diagnosed with certainty as they fulfilled at least 2 major Roach s criteria [10]. However we would like to stress that most of the individuals (19/22) had 3 or even more criteria. The average age of the last physical examination was 8.1 years (ranging from 1 to 19). The most frequent TSC trait were multiple (at least 3) hypomelanotic macules, found in over 86% of individuals. Other highly reported TSC features were subependymal nodules (85.7%) and subcortical tubers (81%). The least frequent were kidney cysts (0%), liver angiomyolipoma (4.5%) and ungual fibroma (4.5%). There were two patients in whom we did not find any TSC skin lesions (3DA and 12MK, aged 9 and 3 years respectively). However they had other TSC features: patient 3DA had heart, kidney and liver tumors, patient 12MK subependymal nodules and cortical tubers in the brain. [2] Phenotypes of the family members Phenotypes of the family members are shown in table II [tabl. II]. There were 5 individuals in whom we diagnosed definite TSC (at least 2 major criteria) and 5 in whom the diagnosis was possible (1 major criterion). Most individuals (all except patient 4HK s brother) did not present with any symptoms indicative of involvement of any organ other than the skin, so that few radiographic examinations have been performed and thus the diagnostic status is uncertain. However it should be noted that in 4 out of 5 relatives from the first group the clinical diagnosis of TSC was definite even if only skin lesions were considered. The most common skin feature found in the relatives of our patients was multiple hypomelanotic macules, seen in 8 out of 10 individuals. [3] Comparison of NMI, TSC1 and TSC2 phenotypes The phenotypes of our NMI patients were compared with those of the TSC1 and TSC2 phenotypes characterized in our previous study [1]. The results are shown in table III. Most clinical features were less common in the NMI group than in TSC1, no feature was more frequent in NMI than TSC2 patients. Only a few differences were statistically significant (table III). Epilepsy was found in 81% of the NMI patients versus 86% in TSC1 (the difference not statistically significant) and 97% in TSC2 (p=0.008). 16 Neurologia Dziecięca

3 Charakterystyka kliniczna stwardnienia guzowatego u pacjentów... Table I. Clinical characteristics of the probands. Charakterystyka kliniczna probandów Nr ID Age (years) Seizures Mental handicap Hypome lanotic macules Facial angiof Shagreen patches Ungual fibroma Forehead plaque Subcortical SENs a SEGA b tubers 1 BJ 8 P 0 P 2 P A P P A P A A 6M/0m 0 2 CP 2 P 1 P 2 A A A P A P P A 5M/1m 0 3 DA 9 A 0 A 0 A A A A A A A P 2M/1m 0 4 HK 3 P 1 P 1 A A P P A P 0 1 0?? 6M/0m 2 5 JR 1 P? P 0 A A A P A P P P 4M/1m 0 6 JM 12 P 3 P 2 P A P P A P A P 8M/0m 0 7 KD 4? 3 P 0 A A A P A P P P 5M/1m 0 8 KJ 8 P 1 P 0 A A A P A P A A 3M/0m 0 9 KA 16 P 0 P 0 A A A P A P A A 3M/0m 1 10 KK 6 P 2 P 2 A A P P A P P A 6M/1m 0 11 LM 4 P 2 P 0 A A A P A P A P 4M/0m 0 12 MK 3 P 1 A 0 A A A P A P A A 2M/0m 0 13 OA 8 A 0 P 0 A A A P A P A P 4M/0m 0 14 OM 15 P 2 P 2 A A P P A P A A 6M/0m 0 15 PM 16 P 1 P 3 A P A P A P A A 6M/0m 1 16 PN 6 P 2 P 2 P A P P A P 0 1 0? A 7M/0m 0 17 PA 19 P 3 A 1 A A A A P A A A 3M/0m 0 18 RA 1 A 0 P 0 A A A??? 0 0 0? P 2M/0m 2 19 SI 13 P 2 P 3 P A P A P A A? 6M/0m 0 20 WP 14 P 3 P 3 P A P P A P A A 7M/0m 0 21 ZM 4 P 2 P 0 A A A P A P P P 4M/1m 0 22 OK 7 A 0 P 1 A A A P A A A P 4M/0m 4 cysts AML c Liver AML c Retinal hamart oma Rhabdo myoma Criteria Family Members d Summary 17/21 15/21 19/22 12\22 5\22 1\22 8\22 18\21 2\21 17/21 0\22 12\22 1\22 5\19 9\20 5\22 % a SENs = subependymal nodules; b SEGA = subependymal giant cell astrocytoma; c AML = angiomyolipoma; d number of relatives with TSC features. NOTE: P means the feature is present. A means the feature is absent.? means the presence is unknown. M means major criteria; m means minor criteria. Mental handicap grading: 1 mild; 2 moderate; 3 severe. Facial angiofibroma. kidney and liver AML grading made according to the Dabora et al. criteria [1]. Vol. 15/2006, Nr 30 17

4 Magdalena Kaczorowska, Julita Borkowska, Dorota Domańska-Pakieła et al. Table II. Clinical characteristics of the probands relatives with TSC features. Charakterystyka kliniczna krewnych prezentujących objawy TSC Proband nr (as in table 1) Subcortical SENs a SEGA b tubers cysts AML c Liver Retinal Rhabdo AML c hamart myoma Criteria oma 4 HP 32 P A P 3 P P A???????? 4M/0m Father 4 HK 5 P A P 2 P A P P A P A 1 0?? 7M/0m Brother 15 PT 48 A A A 1 A P A???????? 2M/0m Father 22 OA 8 A A P 1 P A A???????? 3M/0m Brother 22 MK 5 A A P 1 A A A???????? 2M/0m Cousin 9 KR 40 A A A 1 A A A???????? 1M/0m Father 18 RA 28 A A P 0 A A A???????? 1M/0m Mother 18 NN??? P???????????? 1M/0m Aunt 22 OK. 33 A A P 0 A A A???????? 1M/0m Mother 22 MM 28 A A P 0 A A A???????? 1M/0m Aunt Mental Hypome Facial Shagreen pat- fibroad pla- Ungual Forehe- Age Seizures ID handicap lanotic angiof (years) macules ches ma que Relationship to the proband NOTE: abbreviations and symbols as in table I. The first 5 individuals present signs and symptoms enabling to diagnose TSC. In the remaining 5 individuals diagnosis of TSC is possible (1 major criterion). Table III. Comparison of our NMI Patients with the Patients having TSC1 or TCS2 mutations already reported in Dabora et al. study. Porównanie naszej grupy pacjentów NMI z pacjentami ze zidentyfikowanymi mutacjami w TSC1 lub TSC2, przedstawionymi w pracy Dabory i wsp. NMI (Warsaw) (N=22) TSC1 (N=28) TSC2 (N=158) p NMI vs.tsc1 p NMI vs.tsc2 Age (range, average) 1-19 (8.1) 2-51(14.9) 1-44 (11.3) Seizures 17/21 (81 %) 24/28 (86 %) 153/158 (97 %) NS Mental handicap (all grades) 15/21 (71 %) 10/20 (50 %) 80/113 (71 %) NS NS Hypomelanotic macules 19/22 (86 %) 26/27 (96 %) 150/157 (95 %) NS NS Facial angiofibroma 12/22 (54.5 %) 18/28 (64 %) 116/155 (75 %) NS Shagreen patch 5/22 (23 %) 9/25 (36 %) 82/153 (54 %) NS Ungual fibroma 1/22 (4.5 %) 5/25 (20 %) 31/155 (20 %) NS NS (0.071) Forehead plaque 8/22 (36 %) 3/25 (12 %) 64/155 (42 %) NS (0.074) NS SEN s 18/21 (86 %) 20/25 (80 %) 131/134 (98 %) NS SEGA 2/21 (9.5 %) 4/26 (15 %) 17/145 (12 %) NS NS Tubers (any) 17/21 (81 %) 15/18 (83 %) 66/71 (93 %) NS NS cysts 0/22 (0%) 3/24 (12.5 %) 43/149 (29 %) NS (0.086) AMLs 12/22 (54.5 %) 7/24 (29 %) 89/148 (60 %) NS (0.081) NS Liver AMLs 1/22 (4.5 %) 0/20 (0 %) 11/144 (8 %) NS NS Retinal hamartoma 5/19 (26 %) 0/20 (0 %) 41/144 (28 %) NS Rhabdomyoma 9/20 (45 %) 9/21 (43 %) 72/140 (51 %) NS NS 18 Neurologia Dziecięca

5 Charakterystyka kliniczna stwardnienia guzowatego u pacjentów... All skin TSC lesions except forehead plaques were the least common in the NMI group. Hypomelanotic macules were found in 86% of the NMI vs 96% and 95% in TSC1 and TSC2 groups respectively (the differences not statistically significant). Facial angiofibroma were found in 54% subjects from NMI and in 75% from TSC2 group (p<0.05). Shagreen patch was found in 23% of NMI patients, in 36% of TSC1 (the difference not statistically significant) and in 54% of TSC2 group (p=0.004). Ungual fibroma were the least common in the NMI patients 4% vs 20% in TSC1 and TSC2 group, however the differences were not statistically significant. Subependymal giant cell astrocytomas (SEGA) were found in 9% of the NMI patients compared to 15% and 12% in the other two groups (the differences not statistically significant). Cortical tubers were seen in 81% of the NMI subjects. Interestingly in none of our NMI patients kidney cysts were found, whereas in the other two groups the incidence is relatively high (12.5% in TSC1 and 29% in the TSC2 group). Despite the lowest incidence of seizures in the NMI group, mental retardation was seen at similar frequency as in the TSC2 group, more often than in TSC1 subjects (the difference not statistically significant). Among features of higher incidence in NMI than in TSC1 were: forehead plaque (36%, p=0.074), SENs (86%), kidney AMLs (54.5%), liver AMLs (4.5%) (the differences not statistically significant) and retinal hamartoma (26%, p<0.02). Frequency of all these traits was lower in NMI than in the TSC2 patients, however the differences were not statistically significant except for SEN s (p=0.008). Discussion In disorders of defined genetic background, such as TSC where DNA testing is available, all cases of unidentified mutation should be carefully re-evaluated clinically in order to verify the diagnosis. Roach criteria enable to diagnose TSC clinically with certainty if at least 2 major criteria are present. In the study we reevaluated the cases clinically what enabled us to diagnose TSC with certainty in all cases. NMI patients had milder phenotypes than patients with defined mutations in the TSC2 gene. There were no features with higher incidence in NMI than TSC2 group. Some lesions were of similar incidence in NMI and TSC1 patients as cortical tubers or rhabdomyoma. Taking account of a relatively young age of our NMI group as compared to already reported TSC1 and TSC2 (mean 8.1 years vs and 11.3 respectively) one could assume that our NMI patients will develop TSC1 and more probably TSC2 phenotypes when growing older. However, multiple hypomelanotic macules were found in only 86%, Vol. 15/2006, Nr 30 whereas till recently that feature was reported to be present in almost 100% of the TSC cases [8] and appear earliest in life. According to Jozwiak et al. multiple hypomelanotic macules are present in over 83% of TSC patients under 2 years of age [9, 10] so the relatively low median and average age of our NMI group can not sufficiently explain the low incidence of the macules. There are several possible explanations for the low incidence of TSC features in the NMI group and for the differences between the phenotypes of the NMI and both groups with identified mutation. One of the possibilities that must be considered is somatic mosaicism among TSC patients of sporadic occurrence. It was proven that in case of suspected mosaicism DHPLC (denaturing high-performance liquid chromatography) is a better technique than direct DNA sequencing [11, 12]. In several previously published studies somatic mosaicism was found in individuals with clinical features suggestive of TSC [13 15]. In 1995 Verhoef et al. reported on a family where father presenting with 1 major and 1 minor TSC criterion of a certainly affected child was found to have low proportion of blood cells with the TSC2 gene mutation [14]. In another study by Verhoef et al. somatic mosaicism was found among mildly affected parents in 5 out of 6 families [15]. Jones et al. described 3 individuals fulfilling the clinical Roach TSC criteria, in whom mosaicism for TSC2 was detected at levels 13-35% [11]. Kwiatkowska et al. reported on a girl with skin and neurological features enabling to diagnose TSC in whom mutation (in TSC1 gene) was present in only one third of leukocytes and in none of the cells derived from buccal mucosa [16]. The authors even suggested that TSC1 or TSC2 somatic mosaicism may result in total lack of detectable mutant alleles in leukocytes. This latter hypothesis is consistent with the results obtained in our previous studies [5, 13]. In a recent paper Roberts et al. reported screening in 30 families of TSC sporadic cases for somatic mosaicism [13]. None of the parents showed evidence for somatic mosaicism. Therefore it has been suggested that the patients with clinical diagnosis of TSC yet no mutation identified may account for up to 15% of all TSC patients. Other explanations for not detecting any mutation in the two known TSC genes as it has been suggested [5] may be: inability to decide whether the changes found are mutations or polymorphism, presence of mutations remote from coding exons, and presence of other, still unknown loci (third gene), where mutations might lead to TSC phenotype. This latter hypothesis is especially justifiable in familial cases. Further studies of TSC patients from multigenerational families with no mutation identified are needed. 19

6 Magdalena Kaczorowska, Julita Borkowska, Dorota Domańska-Pakieła et al. References [1] Gomez M.R.: Varieties of expression of tuberous sclerosis. Neurofibromatosis 1988:1, [2] Roach E.S., Gomez M.R., Northrup H.: Tuberous Sclerosis Complex Consensus Conference: revised clinical diagnostic criteria. J. Child. Neurol., 1998:13, [3] European Chromosome 16 Tuberous Sclerosis Consortium. Identification and characterization of the tuberous sclerosis gene on chromosome 16. Cell, 1993:75, [4] van Slegtenhorst M.V., Hoogt R.D., Hermans C. et al.: Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science, 1997:277, [5] Dabora S.L., Jozwiak S., Franz D.N. et al.: Mutational analysis in a cohort of 224 tuberous sclerosis patients indicates increased severity of TSC2, compared with TSC1, disease in multiple organs. Am. J. Hum. Genet., 2001:68, [6] Sancak O., Nellist M., Goedbloed M. et al.: Mutational analysis of the TSC1 and TSC2 genes in a diagnostic setting : genotype phenotype correlations and comparison of diagnostic DNA techniques in Tuberous Sclerosis Complex. Eur. J. Hum. Genet., 2005:13, [7] Langkau N., Martin N., Brandt R. et al.: TSC1 and TSC2 mutations in tuberous sclerosis, the associated phenotypes and a model to explain observed TSC1/TSC2 frequency ratios. Eur. J. Pediatr., 2002:161, [8] Webb D.W., Clarke A., Fryer A. et al.: The cutaneous features of tuberous sclerosis: a population study. Br. J. Dermatol., 1996:135, 1 5. [9] Jozwiak S., Schwartz R.A., Krysicka-Janiger C. et al.: Skin lesions in children with tuberous sclerosis complex their incidence, natural course and diagnostic significance. Int. J. Dermatol., 1998:37, [10] Jozwiak S., Schwartz R.A., Krysicka-Janniger C. et al.: Usefulness of diagnostic criteria of tuberous sclerosis complex in pediatric patients. J. Child. Neurol., 2000:15, [11] Jones A.C., Sampson J.R., Cheadle J.P.: Low level mosaicism detectable by DHPLC but not by direct sequencing. Hum. Mutat., 2001:17, [12] Emmerson P., Maynard J., Jones S. et al.: Characterizing mutations in samples with low-level mosaicism by collecting and analysis of DHPLC fractionated heteroduplexes. Hum. Mutat., 2003:21, [13] Roberts P.S., Dabora S., Thiele E.A. et al.: Somatic mosaicism is rare in unaffected parents of patients with sporadic tuberous sclerosis. J. Med. Genet., 2004:41, e69. [14] Verhoef S., Vrtel R., van Essen T. et al.: Somatic mosaicism and clinical variation in tuberous sclerosis complex. Lancet, 1995:345, 202. [15] Verhoef S., Bakker L., Tempelaars A.M. et al.: High rate of mosaicism in tuberous sclerosis complex. Am. J. Hum. Genet., 1999:64, [16] Kwiatkowska J., Wigowska-Sowinska J., Napierala D. et al.: Mosaicism in tuberous sclerosis as a potential cause of the failure of molecular diagnosis. N. Engl. J. Med., 1999:340, Adres do korespondencji: Sergiusz Jóźwiak Department of Child Neurology and Epileptology The Children s Memorial Health Institute Al. Dzieci Polskich Warsaw, Poland s.jozwiak@czd.pl 20 Neurologia Dziecięca