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 Childrens HospitalHarvard Medical School

Dr. Levy has the following disclosures

Maternal PKU Study, NPKUAMaternal PKU Study, Milton FundEnzyme Therapy for PKU, BioMarin Pharmaceuticals GMP therapy for PKU, FDAUrea Cycle Disorders, NIH

Dr. Charles E. Dent: Richards made a chance observation from a phenylketonuric mother at 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 mothers high blood phenylalanine level that damaged their brains in utero.

From Discussion at the 23rd Ross Pediatric Research Conference 1957

PAHPhenylalanineTyrosine BH4

Phenylpyruvic Acid

Phenyllactic AcidPhenylacetic Acid

MATERNAL PKU: A PROBLEM BORN OF SUCCESS

Aims of the StudyLonger term medical and intellectual outcome of the offspringPsychological-emotional-social functioning of the offspringMedical-nutritional-emotional status of the mothersRelative roles of metabolic control in pregnancy and postnatal maternal stimulation in offspring outcome

The Sample27 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 subjects3-615 subjects7-11 8 subjects12-1812 subjects>18 3 subjects

Measures Used In StudyIQ (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)Labsincluding blood Phe (mothers)Photos to look at dysmorphology (mothers, offspring, and fathers if possible)

Offspring outcome was determined by ABASABAS- Adaptive Behavior Assessment System (questionnaire completed by mother that includes 10 skills areas of offspring)

ABAS correlates with offspring IQn=27, r=0.73, p

Prenatal Metabolic ControlStarted 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 throughoutGood35% (9)On diet prior, blood phe

Maternal Postnatal Diet

Mothers currently on diet: 52% (14/27)On diet = taking medical food and restricting phe

Maternal Blood PheAverage1176 umol/L (19 mg/dL) Range(386-1934)(6-32)

Prenatal Treatment vs. Offspring Outcome MaternalOffspring ABASPre-conception 9816 Post-conception 9222

Current Maternal Dietary Status vs. Offspring Outcome Dietary Status Offspring ABAS On diet 10316 Off diet 9712

Mothers Prenatal and Postnatal Diet vs. Offspring ABAS

Excellent ControlNot Excellent ControlOn Dietn=10

11218

ConclusionsLonger term follow-up of offspring from treated maternal PKU pregnancies confirms that outcome is usually within normal limitsOffspring outcome is not only IQ but also function (social, behavioral) as measured by ABASOptimal offspring outcome requires not only prenatal metabolic control but also postnatal stimulationGood postnatal stimulation requires maternal continuation of diet

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

DisclosureD 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.

Funding: National PKU Alliance & USDA Hatch Grant

Denise Ney Robert Blank Sangita Murali Patrick Solverson UW-Madison PKU Bone Research Team

BackgroundPKU is associated with low bone mass, or osteopenia, and fractures in early adulthood.57% of 28 patients had osteopenia/osteoporosisPerez-Dueas 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 osteoporosisde Groot et al. Mol Genet Metab 101:566, 2012

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.

Objective & DesignTo determine how the PKU genotype and the source of dietary protein affect growth, body composition and bone development.

PKU & WT Mice Housed With Same Sex Littermatesphe GMP phe AA phe Casein

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

GrowthSolverson, P et al Am J Physiol Endocrinol Metab 302:E885-95, 2012

Metabolic Phenotyping PlatformFood & water intakeO2 consumption & CO2 production

PKU mice show increased energy expenditure with casein diet

Values with different letter superscripts are significantly different, p

AA Diet Increases Kidney Workload in both WT and PKU MiceValues with different letter superscripts are significantly different, p

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, 20101Am J Clin Nutr 62:797-804, 1995

Reduced growth occurs in children with PKUMol Genet Metab 101:99-109, 2010J Pediatr 26:1-11, 2002

Dual Energy X-Ray Absorptiometry (DXA)

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

Femur Strength: 3 Point Loading Test Loading force being applied to a mouse femur

Displacement, mmLoad-Displacement Curve

PKU mice show bones that are brittle and break easily

PKU mice show reduced femoral bone mineralization compared to WT mice

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

GMP Increases Bone Size in PKU & WT MiceAACaseinGMP

GMP Improves Bone Strength in PKU and WT Mice Compared with AA Diet

Summary

PKU mice have femora that are brittle and weak. This suggests defects in both collagen synthesis and mineralization.

GMP increases bone size and strength; femora tolerate a higher max load before fracture compared with the AA diet. How does GMP work to improve bone strength in mice?

What causes skeletal fragility in PKU?

ConclusionSkeletal fragility is inherent to the PKU genotype and is attenuated by a GMP compared with an AA diet in mice. Future research is needed to determine if improved low-phe diets containing GMP reduce skeletal fragility in human PKU.

Take Home Message: How can bone health be improved in PKU?

Follow a low-phe diet to improve growth and bone development

Include weight bearing exercise each day

Important that just about half the subjects are over 10 because the previous study only went up to 10****I am pleased to acknowledge our UW Madison PKU Bone Research Team. Funding for 2 yrs from NPKUA and USDA Hatch grant. Support from NPKUA means more than the money itself it sends a message that the research in important to the PKU community. PKU is associated . . . This is referred to as skeletal fragility .

A study published earlier this year from the Netherlands assessed BMD in 53 treated PKU patients. This is the most comprehensive PKU bone study published to date.

*This question cannot be answered in a human PKU study, because it is essential that the low-phe AA-based diet be started shortly after birth to prevent cognitive impairment. *Male and female, WT and PKU mice, were fed high-phe casein or low phe AA or GMP diets from weaning - 23 wk, ~ 25 yr of age for humans. This comprehensive, long term feeding included over 217 mice in 12 treatment groups. *We have our own PKU mouse colony and breed mice to generate homozygous PKU and WT mice. WT means the mice do not carry the gene for PKU; it doesnt mean they are especially mischievious. The diets are color coded and the sole source of protein is provided by GMP, AA or the natural dairy protein casein. *Pure GMP contains no phe. However, in the process of isolating GMP from cheese whey (shown in this vat) it becomes contaminated with trace of phe from other whey proteins 2 mg phe/g GMP protein. Thus, the GMP products that Cambrooke markets contain a small amount of phe. *Here we examine growth based on gain in body wt of male and female mice from weaning to young adulthood.

As expected, the WT mice fed the casein control diet, shown in the blue lines, showed the greatest growth. Point

The low-phe AA and GMP diets supported similar growth in both WT and PKU mice. This is important because bone development is a reflection of overall growth. Reduced growth leads to reduced bone development.

For example, WT male mice fed casein showed the best growth and the strongest bones whereas PKU females fed the high-phe casein diet were the smallest mice and had the weakest bones.

Note optimal conditions to show growth with AA diet in mice: used validated AA based diet developed at MIT in 1965; mice are nibblers during the dark cycle, like infant eat every 2-3 hr. *We used a new sophisticated system for metabolic phenotyping. Mice spend 3 days alone in this special cage unit. This allowed us to accurately determine food and water intake and energy expenditure based on oxygen consumption and carbon dioxide production. *Oxygen consump reflects energy expenditure. Here we look at the relationship between oxygen consumption and lean body mass with the male and female mice combined. Two points: 3 groups of WT mice showed signif lower oxygen consumption vs PKU mice that was independent of diet; largest increase observed in PKU mice fed casein or off diet (15%); 13% inc with AA diet and 3% inc with GMP.

This suggests that PKU mice, esp if they are eating a high-phe casein diet, show increased energy expenditure.

*Food intake provides another way to look at EE because mice eat for energy to maintain their growth. Here we see that PKU mice fed casein and AA diets need to eat more to grow. In contrast, PKU mice fed GMP can eat less to maintain growth similar to WT mice diet is more efficient for growth. Why would energy needs and food intake be increased in PKU mice fed casein or AA diet cost of pro TO, urea syn; kidney work and ion transport costs. Kidney 10% REE and liver 27% REE.

*The AA diet . . . That is, significant increase in kidney mass and 24 hr water intake is observed in both WT and PKU mice fed the AA diet. In contrast, GMP normalizes kidney mass and water intake to levels seen in WT mice. What is the connection with bone development? Increased EE and kidneys workload represent metabolic stress for a growing animal. Making new bone is a costly process that may be impaired with limited energy and metabolic stress. *Studies in children with PKU also suggest increased energy needs and reduced growth.

*Similar to human studies, we use a mouse DXA scanner to assess BMD and body composition. At end of exp mice were put to sleep, euthanized and tissues collected saved both femurs, long thigh bone shown . * We noted that PKU mice . . . as noted in human PKU. In human studies, BMD is used as surrogate for bone strength. In our mouse study we were able to test bone strength directly. *This slide shows how we determined the strength of the mice femora using the 3 pt loading test.Cartoon: When a load or force is applied, the bone bends first (displacement) and then eventually fractures or breaks. Photo: Shown is a loading force being applied to a mouse femur. ***Relationship between load applied to femur and displacement is shown here.When the load is first applied to the bone it behaves like a spring; release load and it springs back (called elastic region). When additional force is applied you reach a critical point, beyond which permanent damage to the bone structure occurs. This is called the yield pointThe Max load represents the greatest load can tolerate before fracture.

Displacement=amount the bone bends before fracture. Strength = load or force applied to the bone before fracture; Displacement = amount the bone bends. AUC=amount of energy needed to cause a fracture (toughness at tissue level). Energy to failure =how much energy has bone absorbed with displacement. Brittle = sustains very little post yield displacement before fracture. This slide shows representative load-displacement curves for PKU and WT mice. What you see is that the yield load is significantly lower in PKU vs WT mice, as shown in histogram. This indicates that the bone from PKU mouse reaches permanent damage with less force compared with WT mice. The post yield displacement (amount the bone bends before fracture) is also lower in PKU mice which indicates a brittle bone. Think of a piece of dry spaghetti and how quickly it breaks, it does not bend very much before breaking.

We conclude that PKU mice show bones that are brittle, which suggests a defect in the protein matrix of bone, and break easily, which suggests a defect in mineralization. *Consistent with reduced bone strength here you see that . .. . So far I have established that the PKU mice show a bone phenotype, which appears to be similar to the skeletal fragility described in human PKU.

*Move from discussion of effect of PKU genotype to how .

*Shown here are representative photographs of cross sections of femora from male PKU mice fed casein, AA or GMP diets. You can see that the AA diet results in a smaller mouse bone compared with casein or GMP diet. This effect of smaller bones with the AA diet occurs in both PKU and WT mice and . . . GMP increases bone size in both PKU and WT mice, as shown in histogram. However, it does not normalize bone size as shown by . . . WT mice fed casein diet.

This is important because bigger bones are stronger and more resistant to fracture.

*Read title .. . The max load, shown here, is the greatest force a bone can tolerate before it fractures. Mice fed GMP diet tolerate a greater force or max load before fracture compared with the AA diet. Occurs in both PKU and WT mice.

There is NSD in BMD or BMC with dietary treatments. Fits with data as calcification is not the only problem, problem is with collagen or bone matrix protein. Sometimes when collagen is weak there is actually over calcification.

Casein diet was most harmful to bone in female PKU, but not male mice. No consistent association between plasma phe concentration and bone but phe levels not different in PKU and WT mice on AA diet.

Humans off diet (like mice on casein diet) perhaps less gross motor activity and less stress on bones which improves bone development; off diet = poor nutritional quality diet overall and dont grow as well; casein diet in mice = high quality. **As shown in this picture, collagen is the primary bone matrix protein that forms the scaffolding upon which mineralization with Ca-P04 occurs. Bone strength is a function of both collagen synthesis and mineralization.

How does GMP work to improve bone in mice? We speculate that GMP improves collagen synthesis and bone mineralization by reducing metabolic stress, reducing the dietary acid load, and improving the dietary AA profile needed for collagen synthesis.

I started my talk by asking . . .. Is it inherent to PKU or secondary to diet. The answer is that both genotype and diet contribute to skeletal fragility in PKU. * This is a hopeful conclusion because it suggests that . .. Future research is needed to understand the pathophysiology of skeletal fragility in PKU and its dietary management.

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