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

Vol. 15/2006 Nr 30

NEUROLOGIA

DZIECIĘCA

NEUROLOGIA

DZIECIĘCA

PRACA ORYGINALNA/ORIGINAL ARTICLE

Aim: In the study we reassessed the clinical features of 22 patients diagnosed with tuberous scle-rosis complex but with no mutation in TSC1 or TSC2 gene found, and compared them with the phe-notypes 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 mac-ules, 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 de-fined 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 guzo-watym (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: Padacz-ka, 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), angiomio-lipoma (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.

Abstract

Key words: tuberous sclerosis complex, TSC1, TSC2

Streszczenie

Słowa kluczowe: stward-nienie guzowate, TSC1, TSC2

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_{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}

Introduction

Tuberous sclerosis complex (TSC) is a genetic di-sorder characterized by the presence of typical skin abnormalities and hamartoma-like tumors of different organs [1]. The clinical diagnosis of the disease is ba-sed on criteria reviba-sed in 1998 by Roach et al. [2]. Ho-wever, since the identification of the TSC2 and TSC1 genes in 1993 and 1997 respectively, genetic verifica-tion of the diagnosis – screening for mutaverifica-tions 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 stu-dy we analyze clinical features of 22 patients from the Children’s Memorial Health Institute with TSC pheno-type but no mutation in TSC1/TSC2 genes found. To our knowledge there was only one study in the litera-ture 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 He-alth 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 inclu-ding ultrasonography, echocardiography, brain CT and MR have been done in our Institute by specialists expe-rienced in evaluating TSC features. Mutational analy-sis of TSC1 and TSC2 genes was carried out as descri-bed [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 ma-jor 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 fre-quent TSC trait were multiple (at least 3) hypomelano-tic macules, found in over 86% of individuals. Other highly reported TSC features were subependymal no-dules (85.7%) and subcortical tubers (81%). The least frequent were kidney cysts (0%), liver angiomyolipo-ma (4.5%) and ungual fibroangiomyolipo-ma (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 fea-tures: patient 3DA had heart, kidney and liver tumors, patient 12MK – subependymal nodules and cortical tu-bers 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 involve-ment of any organ other than the skin, so that few ra-diographic 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 relati-ves of our patients was multiple hypomelanotic macu-les, 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 charac-terized 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 diffe-rences were statistically significant (table III).

Epilepsy was found in 81% of the NMI patients ver-sus 86% in TSC1 (the difference not statistically signi-ficant) and 97% in TSC2 (p=0.008).

T

able I.

Clinical characteristics of the probands.

Charakterystyka kliniczna probandów

Nr ID Age (years) Seizu -res Mental handi -cap (grade)

Hypome lanotic macules Facial angiof _(grade)

Sha -gr een p a tc h es Ungual fibr oma For e-head _plaque SENs a SEGA b Subcortical tubers Kidney cysts Kidney AML c (grade) Liver AML c (grade) Retinal ha m a rt oma Rhab -do m y o m a Crite -ria Family Mem -bers d 1 BJ 8 P 0 P 2 P A P P A P 0 0 0 A A 6M/0m 0 2 CP 2 P 1 P 2 A A A P A P 0 1 0 P A 5M/1m 0 3 DA 9 A 0 A 0 A A A A A A 0 2 1 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 0 0 0 P P 4M/1m 0 6 JM 12 P 3 P 2 P A P P A P 0 1 0 A P 8M/0m 0 7 KD 4 ? 3 P 0 A A A P A P 0 1 0 P P 5M/1m 0 8 KJ 8 P 1 P 0 A A A P A P 0 0 0 A A 3M/0m 0 9 KA 16 P 0 P 0 A A A P A P 0 0 0 A A 3M/0m 1 10 KK 6 P 2 P 2 A A P P A P 0 1 0 P A 6M/1m 0 1 1 LM 4 P 2 P 0 A A A P A P 0 0 0 A P 4M/0m 0 12 MK 3 P 1 A 0 A A A P A P 0 0 0 A A 2M/0m 0 13 OA 8 A 0 P 0 A A A P A P 0 0 0 A P 4M/0m 0 14 OM 15 P 2 P 2 A A P P A P 0 1 0 A A 6M/0m 0 15 PM 16 P 1 P 3 A P A P A P 0 1 0 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 P A 19 P 3 A 1 A A A A P A 0 1 0 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 0 1 0 A ? 6M/0m 0 20 WP 14 P 3 P 3 P A P P A P 0 1 0 A A 7M/0m 0 21 ZM 4 P 2 P 0 A A A P A P 0 0 0 P P 4M/1m 0 22 OK 7 A 0 P 1 A A A P A A 0 0 0 A P 4M/0m 4 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 % 81 71.4 86.36 54.54 22.72 4.54 36.36 85.71 9.52 80.95 0 54.54 4.54 26.31 45 22.72

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 cri teria; “m” means minor crite

-ria. Mental handicap grading: 1 – mild; 2 – moderate; 3 – severe. Facial angiofibroma. kidney and liver

AML

T

able II.

C

linical characteristics of the probands’

relatives with

TSC features.

Charakterystyka kliniczna krewnych prezentujących objawy TSC

Pr oband nr (as in table 1) ID Age (years) Seizu -res Mental handicap (grade) Hypome lanotic macules Facial angiof (grade) Shagr e-en pat -ches Ungual fibr o -ma For ehe -ad pla -que SENs a SEGA b Subcortical tubers Kidney cysts Kidney AML c (grade) Liver AML c (grade) Retinal hamart oma Rhabdo myoma Criteria Relation -ship to the pr oband 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 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).

T able III. Comparison of our NMI Patients with the Patients having TSC1 or TCS2 mutations already reported in Dabora et al. study . Porównanie naszej grupy pa

-cjentów NMI z pacjentami ze zidentyfikowanymi mutacjami w TSC1 lub TSC2, przedstawionymi w pracy Dabory i wsp.

NMI (W arsaw) (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 (1 1.3) Seizures 17/21 (81 %) 24/28 (86 %) 153/158 (97 %) NS 0.008

Mental handicap (all grades)

15/21 (71 %) 10/20 (50 %) 80/1 13 (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 %) 1 16/155 (75 %) NS 0.046 Shagreen patch 5/22 (23 %) 9/25 (36 %) 82/153 (54 %) NS 0.004 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 SE N s 18/21 (86 %) 20/25 (80 %) 131/134 (98 %) NS 0.008 SEGA 2/21 (9.5 %) 4/26 (15 %) 17/145 (12 %) NS NS T ubers (any) 17/21 (81 %) 15/18 (83 %) 66/71 (93 %) NS NS Kidney cysts 0/22 (0%) 3/24 (12.5 %) 43/149 (29 %) NS (0.086) 0.003 Kidney AMLs 12/22 (54.5 %) 7/24 (29 %) 89/148 (60 %) NS (0.081) NS Liver AMLs 1/22 (4.5 %) 0/20 (0 %) 1 1/144 (8 %) NS NS Retinal hamartoma 5/19 (26 %) 0/20 (0 %) 41/144 (28 %) 0.014 NS Rhabdomyoma 9/20 (45 %) 9/21 (43 %) 72/140 (51 %) NS NS

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 dif-ferences not statistically significant). Facial angiofi-broma 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 statisti-cally 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). Despi-te 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 ha-martoma (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 crite-ria enable to diagnose TSC clinically with certainty if at least 2 major criteria are present. In the study we re-evaluated the cases clinically what enabled us to diag-nose 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. 14.9 and 11.3 respectively) one could assume that our NMI patients will develop TSC1 and more probably TSC2 phenotypes when growing older. However, mul-tiple hypomelanotic macules were found in only 86%,

whereas till recently that feature was reported to be pre-sent in almost 100% of the TSC cases [8] and appear earliest in life. According to Jozwiak et al. multiple hy-pomelanotic 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 mo-saicism 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 propor-tion of blood cells with the TSC2 gene mutapropor-tion [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 ful-filling the clinical Roach TSC criteria, in whom mo-saicism for TSC2 was detected at levels 13-35% [11]. Kwiatkowska et al. reported on a girl with skin and neu-rological 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 buc-cal 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 hy-pothesis 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 diag-nosis 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 fa-milial cases. Further studies of TSC patients from mul-tigenerational families with no mutation identified are needed.

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Adres do korespondencji: Sergiusz Jóźwiak

Department of Child Neurology and Epileptology The Children’s Memorial Health Institute Al. Dzieci Polskich 20

04-730 Warsaw, Poland E-mail: s.jozwiak@czd.pl