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

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.