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2nd Principal Investigators Meeting

Mutant Analysis of TSC1 & TSC2 in TSC Related Disorders
Sandra L. Dabora, Sergiusz Jozwiak, David Neal Franz, Penelope S. Roberts, Andres Nieto, Joon Chung, Yew-Sing Choy, Mary Pat Reeve, Elizabeth Thiele, John C. Egelhoff, Jolanta Kasprzyk-Obara, Dorota Domanska-Pakiela, and David J. Kwiatkowski


Table of Contents:

Am. J. Hum. Genet. 68:6480, 2001

Mutational Analysis in a Cohort of 224 Tuberous Sclerosis Patients Indicates Increased Severity of TSC2, Compared with TSC1, Disease in Multiple Organs


Sandra L. Dabora, Sergiusz Jozwiak, David Neal Franz, Penelope S. Roberts, Andres Nieto, Joon Chung, Yew-Sing Choy, Mary Pat Reeve, Elizabeth Thiele, John C. Egelhoff, Jolanta Kasprzyk-Obara, Dorota Domanska-Pakiela, and David J. Kwiatkowski

Genetics Laboratory, Division of Hematology, Brigham and Womens Hospital, Boston Divisions of Genetics and Neurology, Childrens Hospital, Boston Division of Neurology and Dept. of Radiology, Childrens Hosp. Med. Center, Cincinnati Department of Child Neurology, Childrens Memorial Hospital, Warsaw




COMPREHENSIVE MUTATION ANALYSIS FOR TSC

224 TSC Patients

Patient origin: #
US/mail 36 (16%)
Warsaw 105 (47%)
Cincinnati 65 (29%)
Boston 18 (8%)

Family:         #
Sporadic 183 (83%)
Familial 38 (17%)
Unknown 3 (1%)


SCREENING METHODS

DHPLC for all exons in TSC2
DHPLC for all exons in TSC1
Long Range PCR
Quantitative PCR







MUTATION SPECTRUM IN 224 TSC PATIENTS

Mutation Type         TSC1         TSC2         TOTAL         DETECTION RATE        
All 28 (15%) 158 (85%) 186 186/224 (83%)
In/Del 15 (8%) 43 (23%)    
Missense 0 31 (17%)    
Splice 2 (1%) 27 (14%)    
Nonsense 11 (6%) 37 (20%)    
Large del/dup 0 20 (11%)    

48% of small mutations never seen before




Distribution of small mutations within TSC1 and TSC2

Distribution of small mutations within TSC1 and TSC2



TSC clinical features grading form


Clinical features - please circle appropriate term
date of last exam:
1. seizures chronic - ever present - never
2. mental retardation none - mild - moderate - severe
Key: mental retardation Grade
3 Severe: no speech intelligible to a stranger
2 Moderate: some speech, no reading
1 Mild: reading at less than 3rd grade level
0 None: reading above 3rd grade level
3. hypomelanotic macules (>2) present - absent
4. facial angiofibroma grade - 0 - 1 - 2 - 3
Key: facial angiofibroma
grade 0: none
grade 1: macular lesions only on cheek
grade 2: papular lesions, < 3 mm diam.
grade 3: papular lesions, > 3 mm diam.;
    and/or extending to below mouth
5. shagreen patch present - absent
6. ungual fibromas present - absent - #____
7. forehead plaque present - absent
Radiographic studies or other evaluation - please circle appropriate term or give number; please give dates of all studies
8. SENs ____# periventricular lesions seen on CT/MRI, date of exam:
9. SEGA present - absent
10. cortical tubers _____# seen on MRI (do not use CT); date of exam:
11. kidney cysts grade - 0 - 1 - 2 - 3 - 4
date of exam:
12. kidney angiomyolipoma grade - 0 - 1 - 2 - 3
13. liver angiomyolipoma grade - 0 - 1 - 2 - 3
14. retinal hamartomas present - absent
15. cardiac rhabdomyomas ever present - never - date of exam
16. lymphangiomatosis severe (clinical symptoms) - mild (abn. chest X-ray) - absent
17. other
Demographic
18. Ethnic background:
Family hx - draw a family tree and/or provide a list of all immediate relatives of all members of family with TS.
Name   relationship   age   medical condition potentially related to TS



(slide 8)

Characteristic         1-5 years         6-10 years         11-15 years         16-25 years         >25 years        
All 67 (30%) 54 (24%) 45 (20%) 42 (19%) 16 (7%)
Sporadic 56 (31%) 44 (24%) 36 (20%) 35 (19%) 12 (7%)
Familial 10 (26%) 10 (26%) 9 (23%) 6 (16%) 3 (8%)
TSC1 7 (25%) 8 (32%) 3 (11%) 7 (25%) 3 (11%)
TSC2 45 (28%) 36 (23%) 35 (22%) 33 (21%) 9 (6%)



(slides 9,10,11,12)

Clinical features with comparisons of sporadic vs familial and TSC1 vs TSC2 mutation subsets
  All (n=224) P,
sporadic (n=183)
vs.
familial (n=38)
TSC1 sporadic
cases (n=22)
TSC2 sporadic
cases (n=129)
P, Sporadic TSC1
vs. TSC2
Age range (Ave. age) 1-51y (11.5y) NS 2-51y (13.4y) 1-44y (11.2y) NS
Median Age 10   9 10  
NEUROLOGIC
Seizures 202/223 (93%) 0.055 19/22 (86%) 127/128 (99%) 0.02
Age range (Ave. age) 1-50y (11.5y)   2-50y (12.3y) 1-44y (11.4y) NS
Mental Handicap (age>6)
Mi+mod+severe 102/155 (66%) NS 7/14 (50%) 66/90 (73%) NS
Mod+Severe 65/155 (42%) NS 2/14 (14%) 41/90 (46%) 0.04
Age range (Ave. Age) 6-44y (12.0y)   9-24y (13.1y) 6-44y (11.7y) NS
Mean MR gr (scale 0-3) 1.25   0.67 1.4 0.007
Subependymal nodules 177/193 (92%) NS 15/20 (75%) 127/136 (93%) 0.02
Age range (Ave. age) 1-51y (11.6y) 2-51y (13.4y) 1-44y (11.6y) 0.055
Mean SEN number 4.7   1.7 6.7 0.0002
SEGA 23/207 (11%) NS 2/21 (9%) 13/118 (12%) NS
Age range (Ave. age) 3-44y (14.7y)   9-11y (10y) 4-44y (15.9y)  
Tubers (any) 100/113 (88%) NS 13/15 (87%) 55/60 (92%) NS
>10 tubers 40/78 (51%) NS 1/9 (11%) 29/42 (69%) 0.002
Age range (Ave. age) 1-50y (11.4y)   2-50y (11.7y) 1-44y (11y) NS
Mean tuber number 10.2   4.4 12.9 0.002
         
   
RENAL  
Kidney cysts (grade 1-4) 52/207 (25%) NS 3/19 (16%) 30/122 (25%) NS
grade 2-4 29/207 (14%)   0/19 (0%) 19/122 (16%) NS (0.08)
grade 4 6/207 (3%)   0/19 (0%) *5/122 (4%) NS
Age range (Ave. age) 1-50y (13.6y)   9-50y (26.3y) 1-44y (13.8y) NS (0.07)
Mean grade (scale 0-4) 0.46   0.16 0.52 NS (0.14)
Kidney AMLs 113/207 (55%) NS 6/19 (31%) 72/121 (60%) 0.03
Age range (Ave. age) 1.5-44y (13.2y)   9-21y (11.8y) 1.5-44y (13.8y) NS
Mean grade (scale 0-3) 0.83   0.32 0.97 0.006
SKIN  
Hypomelanotic macules 202/219 (92%) 0.02 20/21 (95%) 124/128 (97%) NS
Age range (Ave. age) 1-51y (11.2y)  
Facial Angiofibromas (age>2) 155/208 (75%) NS 13/22 (59%) 95/121 (78%) NS
Age range (Ave. age) 2-51y (14.3y)   4-51y (18.5y) 2-44y (13.6y) NS (0.09)
Mean grade (scale 0-3) 1.4   0.9 1.5 0.02
Shagreen patch 100/210 (48%) NS 7/20 (35%) 68/130 (52%) NS
Age range (Ave. age) 2-51y (13.1y)   4-51y (18.9y) 2-42y (12.7y) NS (.06)
Ungual fibroma 39/215 (18%) NS 5/20 (25%) 26/128 (20%) NS
Age range (Ave. age) 4-51y (21y)   6-51y (29.2y) 4-42y (18.7y) NS (0.07)
Forhead Plaque 74/217 (34%) NS 2/20 (10%) 51/128 (40%) 0.01
Age range (Ave. age) 2-42y (13.3y)   6-20y (13y) 2-42y (13.5y) NS
       
       
OTHER  
Liver AMLs 12/195 (6%) NS 0/15 9/117 (8%) NS
Age range (Ave. age) 3-38y (16.7y)   10-38y (17.4y)  
Retinal Hamartomas 46/192 (24%) NS 0/16 32/117 (27%) 0.01
Age range (Ave. age) 1-44y (12.8y)   1.5-44y (13.2y)  
Cardiac Rhabdomyoma 100/195 (51%) NS 8/17 (47%) 58/117 (50%) NS
Age range (Ave. age) 1-33y (9.6y)   3-20y (7.6y) 1-23y (8.5y) NS
LAM (females, age >16) 5/48 (11%) NS 0/5 3/17 (18%) NS
Age range (Ave. age) 18-37y (27.4y)     18-37y (25.3y)  



(slides 13, 14)

Clinical features of NMI patients and comparison to TSC1 and TSC2 patients.
 
  P value P value P value
  NMI (all)           TSC1 (all)           TSC2 (all)           T1 vs. T2           T1 vs. NMI           T2 vs. NMI          
Number of patients 38 28 158  
Age range (Ave. age) 1-33y (9.8y) 2-51y (14.9y) 1-44y (11.3y) NS NS NS
NEUROLOGIC  
Seizures 25/37 (68%) 24/28 (86%) 153/158 (97%) 0.03 NS (.15) <0.0001
Mental Handicap (age>6)  
Mi+mod+severe 12/22 (54%) 10/20 (50%) 80/113 (71%) NS (.08) NS NS (.14)
Mod+Severe 9/22 (41%) 4/19 (21%) 52/113 (46%) 0.05 NS NS
Mean MR gr (scale 0-3) 1.1 0.79 1.49 0.02 NS NS
Subependymal nodules 26/34 (76%) 20/25 (80%) 131/134 (98%) 0.003 NS 0.0001
Mean SEN number 2.9 2 6.3 0.0005 NS 0.0008
SEGA 2/37 (5%) 4/26 (15%) 17/145 (12%) NS NS NS
Tubers (any) 19/24 (79%) 15/18 (83%) 66/71 (93%) NS NS NS (.12)
>10 tubers 6/20 (30%) 1/11 (9%) 34/47 (72%) 0.0002 NS 0.002
Mean tuber number 6.9 4.3 12.9 0.0004 NS 0.002
RENAL  
Kidney cysts (grade 1-4) 6/34 (18%) 3/24 (12.5%) 43/149 (29%) NS NS NS
grade 2-4 4/34 (12%) 0/24 (0%) 25/149 (17%) 0.03 NS NS
grade 4 0/34 (0%) 0/24 (0%) 6/149 (4%) NS NS NS
Mean grade 0.32 0.125 0.55 0.05 NS NS
Kidney AMLs 16/35 (46%) 7/24 (29%) 89/148 (60%) 0.007 NS NS
Mean grade 0.66 0.29 0.96 0.001 0.05 NS
SKIN  
Hypomelanotic macules 26/35 (74%) 26/27 (96%) 150/157 (95%) NS 0.03 0.0004
Facial Angiofibromas (age>2) 21/36 (58%) 18/28 (64%) 116/155 (75%) NS NS NS (.06)
Mean grade 1.2 1 1.5 0.01 NS NS (.06)
Shagreen patch 9/35 (26%) 9/25 (36%) 82/153 (54%) NS NS 0.004
Ungual fibroma 3/35 (9%) 5/25 (20%) 31/155 (20%) NS NS NS
Forhead Plaque 8/35 (23%) 3/25 (12%) 64/155 (42%) 0.006 NS 0.05
OTHER  
Liver AMLs 1/31 (3%) 0/20 (0%) 11/144 (8%) NS NS NS
Retinal Hamartomas 5/28 (18%) 0/20 (0%) 41/144 (28%) 0.004 NS (.07) NS
Cardiac Rhabdomyoma 19/34 (56%) 9/21 (43%) 72/140 (51%) NS NS NS
LAM ( females, age >16) 0/7 (0%) 0/6 (0%) 5/36 (14%) NS NS NS



Conclusions
  • TSC1 mutations (13%) are less common than TSC2 mutations (70%) in both sporadic patients and families, implying a lower rate of germline mutation in TSC1 than TSC2
  • TSC1 disease is correspondingly milder than TSC2 disease (SENs 1.7 vs. 6.7; tubers 4.4 vs. 12.9), consistent with a lower rate of somatic mutation events in TSC1 than TSC2
  • These observations have implications for genetic counseling of TSC patients with defined mutations in TSC1 or TSC2
  • 38 (17%) of all patients had no mutation identified (NMI)
  • 7 of 16 clinical measures substantially less severe in NMI than TSC2 disease
  • Falls to 27 (12%) if potential missense mutations, possible mosaic deletions, and depleted DNA samples excluded
  • Multiple potential causes - missed mutations in TSC1 or TSC2, mosaicism, other genes, etc.
  • Given distinct clinical phenotype, hypothesize that either mosaicism or novel TSC genes are major contributors.