Emerging areas of research into the causes of MS

Professor Gavin Giovannoni Professor of Clinical Neurology & Chair of Neurology

Blizard Institute, Barts and The London School of Medicine and Dentistry, London

Complex diseases

Genes

Epigenetics

Environment

Causation theory

Causation

http://www.campusi.com/bookFind/asp/bookFindPriceLoad.asp?prodId=0029266718

Giovannoni & Cutter; Lancet Neurol 2006; 5: 887–94.

Diffusion Curve

Causation - Koch’s Postulates

1. The specific organism should be shown to be present in all cases of animals suffering from a specific disease but should not be found in healthy animals.

2. The specific microorganism should be isolated from the diseased animal and grown in pure culture on artificial laboratory media.

3. This freshly isolated microorganism, when inoculated into a healthy laboratory animal, should cause the same disease seen in the original animal.

4. The microorganism should be reisolated in pure culture from the experimental infection. 1843-1910

Sir Bradford-Hill: Criteria for Causation

Bradford-Hill A. The environment and disease: association or causation? Proc Royal Soc Med 1966; 58:295.

1. CONSISTENCY AND UNBIASEDNESS OF FINDINGS

2. STRENGTH OF ASSOCIATION

3. TEMPORAL SEQUENCE

4. BIOLOGICAL GRADIENT (DOSE-RESPONSE RELATIONSHIP)

5. SPECIFICITY

6. COHERENCE WITH BIOLOGICAL BACKGROUND AND PREVIOUS KNOWLEDGE

7. BIOLOGICAL PLAUSABILITY

8. REASONING BY ANALOGY

9. EXPERIMENTAL EVIDENCE

Causation and Disease:

A Chronological Journey

Alfred S. Evans

Hardcover / Plenum Pub Corp

March 1993 / 0306442833

“Evidence” or “lack of evidence”

99.2% vs. 90.2%

.

Ascherio et al, 2000

Sero-positivity among MS patients compared with controls by study

The risk of developing MS in individuals seronegative for EBV: a meta-analysis

Pakpoor et al., MSJ 2012

Infectious mononucleosis

Handel et al. 2010.

Small OR

Concealed effects of gene-environment interactions in genome-wide association

Handel, Williamson, Lincoln & Ramagopalan, MSARD 2012

EBV antibody titres: anti-EBNA1

.

Levin et al, 2005

Odds ratio of MS in subjects seronegative for EBV

Ascherio et al, 2007

Distribution of MS in England

Ramagopalan et al., JNNP 2011.

Distribution of IM in England

Ramagopalan et al., JNNP 2011.

Correlation Between Distribution of IM and MS in England

Females- Pearson R= 0.70, p=0.000025

Males- Pearson R= 0.55, p=0.003

Correlations don’t imply causation Ramagopalan et al., JNNP 2011.

Primary infection with the EBV and risk of MS

• Nested case-control study including from over 8 million military personnel with serum stored in the Department of Defense Serum Repository.

Levin et al. Ann Neurol 2010.

MS

Controls N = 610

N = 305 10/305 (3.3%) EBV –ve

32/610 (5.2%) EBV –ve 10/28 (35.7%) EBV –ve

10/10 (100%) EBV –ve

• MS risk is extremely low among individuals not infected with EBV, but it increases sharply in the same individuals following EBV infection.

The ugly fact

“The great tragedy of Science; the slaying of a beautiful hypothesis by an ugly fact.” Thomas Henry Huxley

Viral serologies in children with MS

Banwell et al. Lancet Neurology, Sept. 2007

?

Congruency

Compston & Coles, Lancet 2008.

Epidemiology: worldwide distribution & migration studies

Latitude & link with vD & UVB

1Jablonski NG, Chaplin G. J Hum Evol 2000;39:57–106. 2Chaplin G. Am J Phys Anthropol 2004;125:292–302.

45

55

70

47 76

71 78

51 53 51

59

77

62

88

103

98 100

84

82

93

87

95

MS Prevalence by Department Against UVMED Minimum

3–4

4–6

6–7

Department UVMed MIN

7–9

10–11

11–13

14–16

Relationship of MS Prevalence to Ultraviolet exposure

Ramagopalan et al, 2011

Prevalence of MS in Norway

• Prevalence data for counties in Norway (/105):

A Finnmark1 (2003) >83

B Troms1 (2003) >104

C Nordland (1999) 106

D Nord Trøndelag (1999) 164

E Oppland (2002) 190

F Hordaland (2003) 151

G Oslo (2005) 154

• In Norway, MS prevalence does not rise with increasing latitude, unlike other northern European countries and the USA

• As expected, measured UV radiation levels decrease with increasing latitude

1Kampman MT et al. J Neurol 2007;254:471–477.

A

B

C

D

E

F G

Fish consumption?

Kampman et al, 2007

Can serum levels of 25(OH)D3 predict MS risk?

• Nested case-control study including from over 7 million military personnel with serum stored in the Department of Defense Serum Repository.

MS

Controls N = 296

N = 148 25-OH vD3

25-OH vD3

quintiles

• RR of MS significantly decreased with increasing levels of 25-OH-vD

• OR for a 50-nmol/L increase in 25-OH-vD was 0.59 (95%CI = 0.36-0.97)

• RR lowest quintile (<63.3 nmol/L) as the reference, the ORs for each subsequent quintile were 0.57, 0.57, 0.74, and 0.38 (P = .02 for trend across quintiles)

• OR for the highest quintile, corresponding to 25-hydroxyvitamin D levels higher than 99.1 nmol/L, was significantly different from 1.00 (OR = 0.38; 95%CI = 0.19-0.75; P = .006)

Munger KL et al. JAMA 2006;296:2832–2838.

Can vD supplements prevent MS?

• Nurses' Health Study

• NHS I - 92,253 women followed from 1980 to 2000

• NHS II - 95,310 women followed from 1991 to 2001

• Diet assessed at baseline & every 4 years thereafter

Munger KL et al. Neurology. 2004 Jan 13;62(1):60-5.

• Results

• RR highest quintile of total vitamin D intake to the lowest quintile was 0.67 (95% CI = 0.40 to 1.12; p for trend = 0.03)

• RR comparing women with intake of >or=400 IU/day with women with no supplemental vitamin D intake was 0.59 (95% CI = 0.38 to 0.91; p for trend = 0.006)

MS N = 173

1st 2nd 3rd 4th 5th

What dose of vD?

Veith Am J Clin Nutr 1999;69:842–56.

Level of vD supplementation

Genetics of MS: the rate of MS in females is increasing

1Orton SM et al. Lancet Neurol 2006;5:932–936.

Sex- Ratio of IM Admission by Age in England

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Fem

ale

:Mal

e r

atio

Age group at admission

Sreeram Ramagopalan et al., unpublished data.

0.06

0.065

0.07

0.075

0.08

0.085

0.09

0.095

0.1

0.105

1996 1998 2000 2002 2004 2006 2008 2010

L/in

div

idu

al

Ramagopalan & Giovannoni, submitted.

Sunscreen sales per capita in the USA (1997-2008)

A study of childhood environmental factors in Norway

Odds ratio for MS and summer outdoor activities

Kampman MT et al. J Neurol 2007;254:471–477.

Unadjusted odds ratio P- value Adjusted odds ratio* P- value

Summer outdoor activities

OR† 95% CI OR† 95% CI

Age 16–20 0.54 0.38–0.75 0.001 0.55 0.39–0.78 0.001

Age 11–15 0.70 0.52–0.96 0.025 0.74 0.54–1.01 0.055

Age 6–10 0.69 0.51–0.93 0.013 0.71 0.52–0.96 0.025

The regression analysis included 111 patients and 246 controls with complete data for all variables. *Adjusted for use of cod-liver oil supplementation and meals of boiled or fried fish. †Odds ratio per one unit change in summer outdoor activities.

Smoking is a risk factor for multiple sclerosis

Handel et al. PLoS One. 2011 Jan 13;6(1):e16149.

Prevalence of Smoking in England

Source: Office of National Statistics

Compston & Coles, Lancet 2008.

MS Familial Risk

Month of Birth

1Willer CJ et al. BMJ 2005;330:120–125.

Parent-of-origin effects

Ebers et al. Lancet. 2004;363(9423):1773–1774.

IMSGC 2007

GWAS results in MS 110 SNPs / 103 loci

.

IMSGC & WTCCC2, 2011 IMSGC 2013

IMSGC NEJM, 2007 IMSGC & WTCCC2 Nature, 2011

Immune function candidate loci

GWAS results in context

0

100

200

300

400

500

600

% R

isk

Incr

eas

e

Fine-mapping the genetic association of the MHC in MS: HLA and non-HLA effects

Patsopoulos et al. PLoS Genet. 2013 Nov;9(11):e1003926.

Ramagopalan et al. Arch Neurol 2011; 68:469-72.

Gene-environment interactions

Ramagopalan & Giovannoni; Lancet Neurol. 2009

Smoking and two human leukocyte antigen genes interact to increase the risk for multiple sclerosis

Hedstrom et al. Brain 2011:134;653–664.

Biological plausability

EBV & Disease Activity

Farrell et al. Neurology 2009

EBNA-1

NOT VCA or EA

International CIS study: EBNA-1 IgG and conversion to CDMS*

Cox univariate HR 95% CI P value

EBNA-1 nOD 1.01 0.996-1.029 0.137

* similar results in OCB+ve and MRI T2 +ve patients only

Median Survival (days) Log-rank p

< Median 1247

0.216 > Median 1032

Dr Jens Khule, ECTRIMS 2013

Dysregulated EBV infection in the MS brain

Serafini et al. J Exp Med. 2007 Nov 26;204(12):2899-912.

Negative follow-up studies

1. Torkildsen O, et al. Upregulation of Immunoglobulin-related Genes in

Cortical Sections from Multiple Sclerosis Patients. Brain Pathol. 2009 Oct 16.

[Epub ahead of print]

2. Willis SN, et al. Epstein-Barr virus infection is not a characteristic feature of

multiple sclerosis brain. Brain. 2009 Dec;132(Pt 12):3318-28.

3. Peferoen LA, et al. Epstein Barr virus is not a characteristic feature in the

central nervous system in established multiple sclerosis. Brain. 2010

May;133(Pt 5):e137. Epub 2009 Nov 16.

4. Hans Lassman et al, personal communication.

Innate immune activation is a hallmark of the active MS lesions

Tzartos et al., Neurology 2012.

Intrathecal oligoclonal IgG bands (OCBs)

1. Rand KH, et al. (1998) Molecular approach to find target(s) for oligoclonal bands in multiple sclerosis. J Neurol Neurosurg Psychiatry 65:48-55.

2. Bray PF, et al. (1992) Antibodies against Epstein-Barr nuclear antigen (EBNA) in multiple sclerosis CSF, and two pentapeptide sequence identities between EBNA and myelin basic protein. Neurology 42:1798-804.

3. Cepok S, et al. (2005) Identification of Epstein-Barr virus proteins as putative targets of the immune response in multiple sclerosis. J Clin Invest 115:1352-60.

4. Rand KH, et al. (2000) Epstein-Barr virus nuclear antigen-1 (EBNA-1) associated oligoclonal bands in patients with multiple sclerosis. J Neurol Sci 173:32-9.

C+ / S-

Analogy

Japanese macaque encephalomyelitis: spontaneous MS–like disease in a nonhuman primate

Axthelm et al. Ann Neurol 2011.

Experimental evidence

N Engl J Med 2008;358:676-88.

Ocrelizumab Significantly Reduced

ARR by Week 24 (ITT)

Placebo

(n=54)

IFN-β1a

(n=54)

p=0.0019

p=0.0136

OCR 600 mg

arm (n=55)

OCR 1000 mg

arm (n=55)

0.213 0.127

0.557 62% 77%

Ad

jus

ted

AR

R*

(95

% C

I)

*Adjusted for geographical region. CI, confidence interval.

0.364

The Reduction in Gd-Enhancing T1 Lesions

by OCR Is Maintained Through 144 Weeks

Primary endpoint:

OCR vs placebo1

Weeks

* *

1. Kappos L, et al. Lancet. 2011;378(9805):1779–87; 2. Kappos L, et al. Abstract presented (P362) ECTRIMS 2012 , October 12

OCR 600 mg arm (n=55)

OCR 1000 mg arm (n=55)

Placebo (n=54)

IFN-β1a (n=54)

- ‘Core Study' (0–96 weeks)

- ‘Follow-Up' (97–144 weeks)a

*p<0.0001 for both OCR doses vs placebo, N (for primary analysis): Placebo=54, OCR 600 mg=51, OCR 1000 mg=52, IFN-β1a=522

aPatients who withdrew during earlier treatment cycles were also included in the follow-up periods

Patients with baseline MRI

Summimg-up

A causal cascade for multiple sclerosis?

.

Ramagopalan et al, 2010

VS.

Sir Bradford-Hill: Criteria for Causation Is EBV the cause of MS?

Bradford-Hill A. The environment and disease: association or causation? Proc Royal Soc Med 1966; 58:295.

1. CONSISTENCY AND UNBIASEDNESS OF FINDINGS - Yes (not 100%)

2. STRENGTH OF ASSOCIATION – ? / Yes (RR ~ 2 to 3)

3. TEMPORAL SEQUENCE - Yes

4. BIOLOGICAL GRADIENT (DOSE-RESPONSE RELATIONSHIP) - ? (not relevant to infections)

5. SPECIFICITY – No (not 100% other putative autoimmune diseases also associated with EBV)

6. COHERENCE WITH BIOLOGICAL BACKGROUND AND PREVIOUS KNOWLEDGE - Yes

7. BIOLOGICAL PLAUSABILITY - Yes

8. REASONING BY ANALOGY - Yes

9. EXPERIMENTAL EVIDENCE - ?

Kakalacheva et al, 2011

How does EBV increase MS risk?

Conclusions • Genetics

• Increased familial risk proportional to degree of relatedness

• The effect of the HLA dwarfs that of any other region (at least 6 loci)

• 102 imputed genes, of modest effect, now have convincing evidence for involvement in MS

• Genetics support an immunological basis to the aetiology of MS

• Epigenetics • Month of birth effect

• Dizygotic twin vs. sibling concordance rate

• Parent of orgin effect

• vD and EBV biology

• Environment • vD; latitude, migration, outdoor activity, Vd levels, fish consumption, etc.

? epigenetic / immunological mechanisms

• EBV ? Cause; ? biological mechanisms

• Smoking ? Post-translational modification of putative autoantigens

• Is MS a preventable disease? YES!!!!

Acknowledgements

• Ute Meier

• Monica Marta

• Sreeram Ramagopalan

• Ruth Dobson

• George Ebers

• Jo Topping

• Georgina Burrow

• Julian Gold

• Rachel Farrell

• Cosimo Maggiore

• Jaap Middeldorp

• Sandra Amor

• Dorothy Crawford

• Karen McCauley

• Adam Handel

• Giulio DeSanto

• Hadi Amir-Maghzi

• Chris Hawkes

• Klaus Schmierer

• David Baker

• Jens Kuhle

• Arie Gafson

• Julia Pakpoor