A biochemical, genetic, and nutritional characterization of tetrahydrobiopterin responsiveness in patients with phenylketonuria Public

Abstract

In a subset of patients with phenylketonuria (PKU), pharmacological doses of
tetrahydrobiopterin (BH4) work in conjunction with the mutated phenylalanine
hydroxylase (PAH) enzyme to promote disposal of phenylalanine. Current algorithms for
assessing BH4 responsiveness rely on acute change in blood phenylalanine
concentrations as the only criterion for patient classification. This approach inadequately
characterizes responses seen clinically.

We explore the use of a novel set of criteria to classify BH4 responsiveness in 58 patients
with PKU. "Definitive responders" experienced ≥15% decrease in plasma phenylalanine
concentrations after one month of BH4 therapy and had substantial improvements in
dietary phenylalanine tolerance (n=19). "Provisional responders" also experienced an
initial ≥15% decrease in plasma phenylalanine concentrations, but had limited
improvements in dietary phenylalanine tolerance (n=9). Patients with <15% decrease in
plasma phenylalanine concentrations after one month of BH4 therapy were classified as
"non-responders" (n=25), and patients lost to follow-up remained unclassified (n=5).

Next, we explore the clinical utility of assessing PAH genotype severity to classify BH4
response using a previously developed tool (assigned value sum). While the majority of
definitive responders (17/19 patients) had genotypes with molecular basis for
responsiveness, most of the provisional responders (7/9 patients) had severe genotypes
indicative of a false-positive response. Furthermore, the heterogeneity in genotype
severity within the non-responder group suggests that false-negative classification may
have occurred. The simple genotype severity tool which was assessed has the potential to
reveal misclassified patients and may have implication for identifying candidates for BH4
therapy.

The potential response misclassification, however, could not be attributed to overt or
divergent trends in dietary total protein, phenylalanine, and medical food intake during
the first month of BH4 therapy. Pediatric definitive responders reported consuming
significantly more dietary phenylalanine and less medical food than the provisional
responders, further highlighting the phenotypic differences between the two groups.

Thus, dichotomization of patients' acute plasma phenylalanine response to BH4 therapy
is clinically insufficient. As demonstrated by our provisionally responsive group, patients
can experience a marked decrease in plasma phenylalanine concentrations, but not have
the added benefit of diet liberalization. A comprehensive approach is necessary to
sufficiently characterize BH4 responsiveness in patients with PKU.

Table of Contents

CHAPTER 1: INTRODUCTION

CHAPTER 2: THE BIOCHEMICAL, GENETIC, AND CLINICAL PRINCIPLES OF PHENYLKETONURIA

THE AFFECTED BIOCHEMICAL PATHWAY

Normal Phenylalanine Metabolism

Phenylalanine Metabolism in Patients with PKU

GENETIC BASIS OF PKU
DETECTION AND CLINICAL MANIFESTATIONS

PKU Screening and Differential Diagnosis
Clinical Manifestations of PKU

NUTRITION THERAPY

Low-Phenylalanine Diet
Medical Food
Monitoring Nutrition Therapy - Circulating Phenylalanine Concentrations
Issues with Nutrition Therapy

CHAPTER 3: TETRAHYDROBIOPTERIN RESPONSIVENESS IN PATIENTS WITH PHENYLKETONURIA

MODES OF ACTION OF PHARMACOLOGICAL BH4
VARIABILITY OF PROTOCOLS ASSESSING BH4 RESPONSIVENESS

BH4 Doses Used in Responsiveness Protocols
Number of BH4 Doses Administered Prior to Classification
Use of a Phenylalanine Challenge
Dietary Regimen Prior to and During the Protocols
Duration of the Protocols
Timing and Frequency of Sample Collections
Definition of Responsiveness

CLINICAL OUTCOMES

Blood Phenylalanine Concentrations
Dietary Phenylalanine Tolerance and Medical Food Needs

LIMITATIONS OF THE CURRENT BH4 RESPONSE CLASSIFICATION APPROACHES

CHAPTER 4: USING CHANGE IN PLASMA PHENYLALANINE CONCENTRATIONS AND ABILITY TO LIBERALIZE DIET TO CLASSIFY RESPONSIVENESS TO TETRAHYDROBIOPTERIN THERAPY IN PATIENTS WITH PHENYLKETONURIA

ABSTRACT

4.1. INTRODUCTION
4.2. MATERIAL AND METHODS

4.2.1. Patient Eligibility
4.2.2. Responsiveness Classification Algorithm
4.2.3. Plasma and Blood Amino Acid Analysis
4.2.4. Dietary Intake and Diet Prescription
4.2.5. Statistical Analysis

4.3. RESULTS

4.3.1. Baseline Characteristics and Preliminary Responsiveness Classification
4.3.2. BH4 Responsiveness: Diet Liberalization
4.3.3. Summary of Responsiveness Classification

4.4. DISCUSSION

CHAPTER 5: UTILITY OF PHENYLALANINE HYDROXYLASE GENOTYPE FOR TETRAHYDROBIOPTERIN RESPONSIVENESS CLASSIFICATION IN PATIENTS WITH PHENYLKETONURIA

ABSTRACT
5.1. INTRODUCTION

5.2. PATIENTS AND METHODS

5.2.1. Patients and Clinical BH4 Response Classification
5.2.2. PAH Mutation Identification
5.2.3. Assessing PAH Genotype Severity Using Assigned Value (AV) Sum
5.2.4. Assessment of Classification Approaches and Statistics

5.3. RESULTS

5.3.1. Summary of Identified Mutations
5.3.2. PAH Genotype AV Sum by BH4-Response Classification
5.3.3. Discordant BH4 Response Classification of Matching PAH Genotypes
5.3.4. Ability of AV Sum to Predict BH4 Response

5.4. DISCUSSION

CHAPTER 6: DIETARY PROTEIN INTAKE OF PATIENTS WITH PHENYLKETONURIA DURING AN EXTENDED TETRAHYDROBIOPTERIN RESPONSE PROTOCOL

ABSTRACT
6.1. INTRODUCTION
6.2. PATIENTS AND METHODS

6.2.1. Patient Selection
6.2.2. Classification of BH4 Response
6.2.3. Assessment of Dietary Intake
6.2.4. Statistical Analysis

6.3. RESULTS

6.3.1. Analysis of All Patients
6.3.2. Pediatric Analysis

6.4. DISCUSSION

CHAPTER 7: SUMMARY OF RESULTS AND CONCLUSIONS

CAVEATS TO AND LIMITATIONS OF IMPLEMENTED BH4 CLASSIFICATION PROTOCOL
NEED FOR EXTERNAL VALIDATION
CLINICAL IMPLICATIONS FOR THE PKU POPULATION
CLINICAL IMPLICATIONS FOR BH4 BEYOND THE PAH PATHWAY
FUTURE DIRECTIONS
CONCLUSIONS
CITED LITERATURE

APPENDIX A: HETEROGENEITY OF PROTOCOLS CLASSIFYING BH4 RESPONSIVENESS IN PATIENTS WITH PKU
APPENDIX B: SENSITIVITY ANALYSIS USING GENERAL LOGIT MODELS TO EVALUATE PROTEIN INTAKE PRIOR TO BH4 RESPONSE CLASSIFICATION

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Health Sciences, Nutrition
Mot-clé	phenylalanine phenylketonuria genotype nutrition diet tetrahydrobiopterin
Committee Chair / Thesis Advisor	Singh, Rani H, Emory University
Committee Members	Jones, Dean P, Emory University Quyyumi, Arshed Ali, Emory University Ziegler, Thomas R, Emory University Le, Ngoc-Anh, Emory University

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