MATERNAL PKU: LONGER TERM OFFSPRING OUTCOME RELATED TO PRENATAL AND POSTNATAL FACTORS

2. MATERNAL PKU: LONGER TERM OFFSPRING OUTCOME RELATED TO PRENATAL AND POSTNATAL FACTORS Harvey L. Levy, MD, Susan E. Waisbren, PhD, Fran Rohr, MS, RD, Vera Anastasoaie, Stephanie Petrides Boston Children’s Hospital Harvard Medical School

3. Dr. Levy has the following disclosures Maternal PKU Study, NPKUA Maternal PKU Study, Milton Fund Enzyme Therapy for PKU, BioMarin Pharmaceuticals GMP therapy for PKU, FDA Urea Cycle Disorders, NIH

4. Dr. Charles E. Dent: Richards made a chance observation from a phenylketonuric motherat the mental defective colony in Caterham, near London. She had three children …………….. All three children were mentally retarded from birth but had no abnormal amino acids in their urine. We felt that it might well have been the toxicity of the mother’s high blood phenylalanine level that damaged their brains in utero. • From Discussion at the 23rd Ross Pediatric Research Conference 1957

9. PAH Phenylalanine Tyrosine BH4 Phenylpyruvic Acid Phenyllactic Acid Phenylacetic Acid

12. MATERNAL PKU: A PROBLEM BORN OF SUCCESS

17. Aims of the Study • Longer term medical and intellectual outcome of the offspring • Psychological-emotional-social functioning of the offspring • Medical-nutritional-emotional status of the mothers • Relative roles of metabolic control in pregnancy and postnatal maternal stimulation in offspring outcome

18. The Sample • 27 mothers ranging from 23-48 (average age is 40) • 48 offspring ranging from 2 months-26 years (average age is 9 years) 0-2 10 subjects 3-6 15 subjects 7-11 8 subjects 12-18 12 subjects >18 3 subjects

19. Measures Used In Study • IQ (mothers and offspring) • Executive functioning (mothers and offspring) • Prevalence of Anxiety/Depression/ADHD (mothers and offspring) • Physical exam (mothers and offspring) • Home Scale--measure of home environment (offspring) • Nutrition assessment (mothers) • Labs—including blood Phe (mothers) • Photos to look at dysmorphology (mothers, offspring, and fathers if possible)

20. Offspring outcome was determined by ABAS ABAS- Adaptive Behavior Assessment System (questionnaire completed by mother that includes 10 skills areas of offspring) ABAS correlates with offspring IQ n=27, r=0.73, p<0.0001 ABAS correlates with offspring DQ n=11, r=0.77, p=0.005

21. Prenatal Metabolic Control Started diet prior to pregnancy: 76% In metabolic control prior to pregnancy: 41% Control during pregnancy: Excellent: 41% (11) On diet prior, blood phe < 6 mg/dL throughout Good 35% (9) On diet prior, blood phe <6 mg/dL by 10 weeks Fair 14% (4) Blood phe < 10 mg/dL by second trimester Poor 10% (3) Blood phe in control in third trimester or never

22. Maternal Postnatal Diet Mothers currently “on diet”: 52% (14/27) On diet = taking medical food and restricting phe Maternal Blood Phe Average 1176 umol/L (19 mg/dL) Range (386-1934) (6-32)

23. Prenatal Treatment vs. Offspring Outcome MaternalOffspring ABAS Pre-conception 98±16 Post-conception 92±22

24. Current Maternal Dietary Status vs. Offspring Outcome Dietary StatusOffspring ABAS On diet 103±16 Off diet 97±12

25. Mother’s Prenatal and Postnatal Diet vs. Offspring ABAS

26. Conclusions • Longer term follow-up of offspring from treated maternal PKU pregnancies confirms that outcome is usually within normal limits • Offspring outcome is not only IQ but also function (social, behavioral…) as measured by ABAS • Optimal offspring outcome requires not only prenatal metabolic control but also postnatal stimulation • Good postnatal stimulation requires maternal continuation of diet

29. Effects of Glycomacropeptide, Amino Acid & Casein Diets on Osteopenia in PKU Mice Denise M. Ney, PhD, RD Professor of Nutritional Sciences Waisman Center University of Wisconsin-Madison

30. Disclosure • D Ney is a co-inventor on US Patent Application US-2010-0317597, GMP Medical Foods for Nutritional Management of PKU, which is held by the Wisconsin Alumni Research Foundation and licensed to Cambrooke Foods, LLC. A percentage of all royalty payments is awarded to the inventors. • D Ney has received consulting income from Cambrooke Foods and BioMarin.

31. Funding: National PKU Alliance & USDA Hatch Grant UW-Madison PKU Bone Research Team Denise Ney Robert Blank Sangita Murali Patrick Solverson

32. Background • PKU is associated with low bone mass, or osteopenia, and fractures in early adulthood. • 57% of 28 patients had osteopenia/osteoporosis • Perez-Dueñas et al. Acta Paediatr 91:800, 2002 • Reduced bone mineral density (BMD) in PKU is present from an early age onward and it cannot be predicted by plasma phe levels. • 20% of 53 patients studied had osteoporosis • de Groot et al. Mol Genet Metab 101:566, 2012

33. What causes skeletal fragility in PKU? The fundamental question is whether reduced BMD is inherent to PKU or secondary to its dietary management. In order to isolate the contributions of the PKU genotype itself and dietary treatment of PKU we have conducted a factorial experiment in PKU mice.

34. Objective & Design Casein High Phe ♂ ♀ • WT • PKU AA Low Phe GMP To determine how the PKU genotype and the source of dietary protein affect growth, body composition and bone development.

35. PKU & WT Mice Housed With Same Sex Littermates ↓pheGMP ↓pheAA ↑phe Casein

36. GMP is a natural whey protein produced when making cheese. Pure GMP contains no phe.

37. Growth Solverson, P et al Am J PhysiolEndocrinolMetab302:E885-95, 2012

38. Metabolic Phenotyping Platform ●Food & water intake ●O2 consumption & CO2 production

39. PKU mice show increased energy expenditure with casein diet

40. GMP Diet Normalizes Food Intake in PKU Mice Values with different letter superscripts are significantly different, p<0.05

41. AA Diet Increases Kidney Workload in both WT and PKU Mice Values with different letter superscripts are significantly different, p<0.05

42. Does PKU increase energy needs? • Resting energy expenditure is ~5-10% higher in adolescent females with PKU than that predicted by standard equations. • J Am Diet Assoc 110:922-25, 20101 • Am J Clin Nutr 62:797-804, 1995 • Reduced growth occurs in children with PKU • Mol Genet Metab 101:99-109, 2010 • J Pediatr 26:1-11, 2002

43. Dual Energy X-Ray Absorptiometry(DXA)

44. PKU mice show reduced whole-body bone mineralization compared to WT mice

45. Femur Strength: 3 Point Loading Test Load (F) Loading force being applied to a mouse femur Displacement

46. Yield Point Post-Yield Displacement Fracture Elastic Deformation Load-Displacement Curve Max Load 16 14 12 10 8 Load, N 6 4 2 -0.1 0.1 0.2 0.4 0.5 0.6 0.7 0.8 0.3 -2 Displacement, mm

47. PKU mice show bones that are brittle and break easily

48. PKU mice show reduced femoral bone mineralization compared to WT mice

49. How the casein, AA and GMP diets affect bone size and strength?

50. GMP Increases Bone Size in PKU & WT Mice Casein AA GMP