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NEW APPROACHES TO STUDYING THE CAUSES OF CEREBRAL PALSY

NEW APPROACHES TO STUDYING THE CAUSES OF CEREBRAL PALSY . Nigel Paneth MD MPH West Michigan Neurological Society Grand Rapids, Nov 9, 2010. NEWEST DEFINITION OF CEREBRAL PALSY .

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NEW APPROACHES TO STUDYING THE CAUSES OF CEREBRAL PALSY

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  1. NEW APPROACHES TO STUDYING THE CAUSES OF CEREBRAL PALSY Nigel Paneth MD MPH West Michigan Neurological Society Grand Rapids, Nov 9, 2010

  2. NEWEST DEFINITION OF CEREBRAL PALSY “Cerebral palsy describes a group of disorders of the development of movement and posture that are attributed to non-progressive disturbances that occurred in the developing fetal or infant brain. The motor disorders of cerebral palsy are often accompanied by disturbances of sensation, cognition, communication, perception and/or behavior, and/or by a seizure disorder.” Rosenbaum P et al: Dev Med Child Neurol 2005;47:572-4

  3. Sigmund Freud was the leading European authority on CP of the late 19th Century. He authored three Monographs on CP in the 1890’s

  4. William Osler wrote the only 19th century monograph on CP published in the US

  5. A NEW ISSUE FOR NEUROLOGY THE ADULT WITH CEREBRAL PALSY

  6. LIFE EXPECTANCY FOR CP IN GREAT BRITAIN • Survival to age 30 • no severe disabilities – 99% • one severe disability – 95% • two severe disabilities – 78% • three severe disabilities – 59% • four severe disabilities – 33% Hutton JL; Clin Perinatol 2006; 33:545-555

  7. DECLINE IN MOTOR FUNCTION IN CP California CP sample (n = 7,550 at age 10; 5,721 at age 25; 904 at age 60) • About a quarter of children and young adults who could walk and climb stairs without difficulty could not do so 15 years later. • Between ages 60–75, a high fraction lost ambulatory ability; speech and self-feeding less affected.

  8. OVERVIEW OF ADULT CP • Life expectancy in CP is about the same as for the general population for individuals without severe cognitive impairment or multiple disabilities. For the severely impaired, survival rates are improving • Decline in motor abilities can occur in adults with CP. This is found occasionally in early adulthood, but commonly over age 60. • Chronic pain and fatigue are important features of adult CP

  9. FREQUENCY OF CP Population studies from many parts of the world find that CP prevalence at school age is between 1.5 – 2.5 cases per thousand live births This means that about one in 500 children has CP, or about 6,000 – 10,000 new cases each year in the US. Some recent US studies suggest prevalence might be as high as 3.0/1,000

  10. CP PREVALENCE IN 12 NATIONAL REGISTRIES N of observations 1950 - 1; 1955 - 3 1960 - 4; 1965 - 3 1970 - 6;1975 - 5 1980 - 8; 1985 -14 1990 - 7; 1995 - 2

  11. COSTS OF DISABILITIES IN CHILDHOOD • Because of the long duration and severity of many childhood disabilities, including cerebral palsy their economic burden is very high • Estimate in 2003 of annual lifetime costs for CP • $11.5 billion for cerebral palsy • This makes prevention of CP and other severe childhood disabilities a high public health priority

  12. QUICK SUMMARY OF CP EPIDEMIOLOGY • PREMATURE BIRTH • 50 - 100-fold higher risk in infants < 28 wks • BIRTH ASPHYXIA • CP is not as closely linked to “birth asphyxia” as we once thought • INFECTION/INFLAMMATION • Increasing evidence for a role of antepartum infection in both preterm and term births • PREVENTIVE POSSIBILITIES • Head and body cooling for encephalopathic newborns now established • MgSO4 in premature labor nearly standard of care.

  13. HOW DO EPIDEMIOLOGISTS INVESTIGATE CAUSES OF DISEASE? • The best way to study the causes of disease is to measure factors you think might cause disease before the onset of disease • To do this, you mount a cohort study, defined as a study that starts with exposure (risk factors) and then identifies disease as it emerges in the cohort. • The best example of this is the Framingham Heart Study that established that smoking, lipids, BP, etc were modifiable risk factors for heart disease. • Since 1960, ischemic heart disease death rates have halved.

  14. IS A COHORT STUDY OF CP FEASIBLE? • Framingham enrolled 5,000 people to study a disease with a prevalence then of 15-20% by age 60. • To mount a cohort study for CP (prevalence of 0.2%) with the statistical power of Framingham would require enrolling 500,000 births • There is an effort now to pool the Norwegian and Danish birth cohort studies, each with 100,000 subjects and try to look at CP in cohort fashion.

  15. AN ALTERNATIVE: LOOKING BACK AT PREGNANCY AND ASKING WHAT WAS DIFFERENT FOR CHILDREN WITH CP • We can match CP cases to controls (case-control study) and look at medical records in pregnancy, socio-demographic factors, and interview mothers about their pregnancies. • However, the biggest limitation of this approach is that you do not have access to biological information at the time of exposure (during pregnancy and the perinatal period). • To really get at the underlying causes of many diseases, we will have to have information on what was happening to the individual at the molecular level at the time of exposure.

  16. THE DILEMMA On the one hand, CP is too rare to efficiently mount a cohort study On the other hand, retrospective case-control studies cannot identify molecular exposures or markers of exposure in real time.

  17. THE SOLUTION NESTED CASE-CONTROL STUDIES

  18. WHAT IS A NESTED CASE-CONTROL STUDY? • In a case-control study, cases of disease are matched, on factors known to be important to the risk of disease, to people without disease. • Both cases and controls are assessed for antecedent risk factors, generally be interview or medical record review • But if there is a universal record of biological information, it may be possible to search for that information for both cases and controls.

  19. MORE ON THE NESTED CASE-CONTROL STUDY In a nested case-control study, the cases and controls must have been part of some earlier data collection process, that is to say, they are nested in a studied population. Especially valuable is when that earlier data collection process includes an archive of biological material The newborn blood spot collection left over after genetic screening represents such an archive

  20. THE OWL STUDY(ORIGINS, WELLNESS AND LIFE-HISTORY IN CP) A NESTED CASE-CONTROL STUDY OF CEREBRAL PALSY, FUNDED BY NIH, CONDUCTED IN COLLABORATION WITH VARI

  21. MURPHY’S LAW Everything that can go wrong, will go wrong.

  22. PANETH’S COROLLARY TO MURPHY’S LAW Everything that can go wrong in research will go wrong……. and most of the things that can’t go wrong, will also go wrong.

  23. IN WHICH COHORT IS THIS CASE-CONTROL STUDY NESTED? ALL BIRTHS IN MICHIGAN SINCE 1986. NEONATAL BLOOD SPOTS OF THESE BIRTHS, LEFTOVER FROM NEWBORN SCREENING, ARE ARCHIVED BY THE MICHIGAN DEPARTMENT OF COMMUNITY HEALTH

  24. OWL PROTOCOL • Cases of CP are recruited in child neurology and CP clinics in Ann Arbor, Lansing and Grand Rapids • Matched controls are obtained from the primary care practices in the three regions of MI; controls are matched on year of birth, gender, and gestational age (4 categories) • Cases and controls provide • maternal interview • permission to review medical records • Permission to study newborn blood spots. • We also save saliva-derived DNA from family

  25. WHAT CAN BE STUDIED ON THESE BLOOD SPOTS? • The spots have been stored at ambient temperatures • Proteins do not seem to be maintained • Human DNA can be retrieved • Viral and bacterial DNA can be retrieved • Surprisingly, mRNA can also be retrieved, though probably not the full signal

  26. WHAT ARE WE LOOKING FOR IN THE BLOOD SPOTS? • Evidence of viral (CMV, HSV) DNA • Evidence of mRNA expression profiles reflecting activation of • Hypoxic/ischemic signals • Inflammatory signals • Coagulation signals (perinatal ischemic stroke is part of CP) • Thyroid hormone signals (low thyroid hormone prominent CP risk factor in prematures, maybe in terms too) • Anything else that looks different between cases and controls

  27. CAN WE RETRIEVE MRNA FROM THESE NEWBORN BLOOD SPOTS? PILOT WORK at VARI (laboratory of Jim Resau) We obtained, from MDCH, 85 randomly selected MI blood spots from 1996 – 2004 We obtained freshly prepared blood spots from adults in a different study We obtained newborn blood spots from 15 children with CP, including from two sets of twins discordant for CP.

  28. PILOT MICROARRAY FINDINGS1. NUMBER OF GENES EXPRESSED Fresh blood from adults Buffy coat 9,223 genes Whole blood 8,404 genes Filter paper spot 7,067 genes Random Newborn spots 1998 archived spot 3,017 genes 2000 archived spot 3,211 genes 2003 archived spot 3,644 genes 2004 archived spot 4,067 genes

  29. PILOT MICROARRAY FINDINGS 2. RELIABILITY OF GENE EXPRESSION Gene expression in archived newborn blood spots: Reliability in three runs and N of genes detected Sample Year Average Correlation Standard Deviation Average N of genes 1998 .947 .017 3,017 2000 .894 .067 3,211 2003 . .887 .047 3,624 2004 .888 .048 4,067 All years .904 .042 3,480

  30. PILOT MICROARRAY FINDINGS3. QUANTITATIVE PCR (qPCR) FINDINGS

  31. PILOT MICROARRAY FINDINGS 4. qPCR CONFIRMATION (TRIPLICATE) OF GENES IDENTIFIED BY MICROARRAY Amplification plots and dissociation curves for five genes 3 Housekeeping genes CYC = cyclophyllin MRPL 11 = mitochondrial ribosomal protein L 11 HK1 = Hexokinase 1 2 Inflammatory genes ITGAX = alpha X integrin NFKB-1 = nuclear factor kappa B-1

  32. SUMMARY OF THIS PILOT WORK Haak PT, Busik JV, Kort AJ, Tikhonenko M, Paneth N, Resau JH: Archived unfrozen neonatal blood spots are amenable to microarray gene expression analysis. Neonatology 2009;95:210-216. Khoo SK, Dykema K, Vadlapatla NM, LaHaie E, Valle S, Satterthwaite D, Ramirez SA, Carrutthers JA, Haak PT, Resau JR: Acquiring genome-wide gene expression profiles in Guthrie card blood spots using microarrays. Pathology International 2010 (in press)

  33. SUMMARY OF WHAT WE DO WITH BLOOD SPOTS IN OWL

  34. RECRUITMENT SO FAR

  35. LABORATORIES INVOLVED Gene expression Microarray – Van Andel Research Institute, Grand Rapids, MI. James Resau PhD, Kyle Furge PhD, Pete Haak qPCR – Julia Busik PhD, Physiology, MSU Viral DNA – Mark Schleiss MD, Yeon Choi PhD Pediatrics, U Minnesota

  36. OTHER COLLABORATORS • Recruiting physicians • Ann Arbor – Ed Hurvitz MD • Lansing – Zach Dyme MD, David VanDyke MD • Grand Rapids – Nancy Dodge MD, Steve DeRoos MD, Mary Free Bed

  37. THANKS FOR LISTENINGI’M HAPPY TO TAKE QUESTIONS (This presentation is posted at http://www.epi.msu.edu/faculty/paneth.htm)

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