Evaluation of Behavior, Executive Function, Neurotransmitter Function and Genomic Expression Kuvan Nonresponders
Condition: Phenylketonuria; Behavior and Behavior Mechanisms; PAH Gene Expression
MeSH terms: Phenylketonurias
Study type: Observational

Summary: 
      This observational study seeks to establish evidence:

        1. that physiologic changes, unrelated to effect on the Phenylalanine Hydroxylase (PAH)
           enzyme, occur in Phenylketonuria (PKU) patients who are treated with sapropterin
           (Kuvan®) therapy,

        2. that these changes may be caused by enhanced neurotransmitter synthesis in the brain or
           an upregulation of gene expression (increasing the ability of genes to produce
           functional enzymes),

        3. and that beneficial changes in behavior and cognition, especially executive functioning
           skills may result.

      The objective of this study is to correlate any change in behavior and executive function
      skills of PKU patients who are non-responsive to sapropterin effect on the PAH enzyme, as
      defined by lowered blood PHE levels, with urine neurotransmitter levels and broad gene
      expression prior to and after sapropterin administration.

      Expected outcomes would include evidence of sapropterin effects on upregulation of enzymes
      other than PAH that control neurotransmitter synthesis, and any resulting correlation with
      behavioral and cognitive changes.

      The investigators hope this study will inform further detailed investigations into the
      biochemical and molecular actions of sapropterin (Kuvan®) that lead to increased
      understanding of possible treatment effects beyond a lowered blood PHE response.
    

Detailed description: 
      The anticipated study participant population was approximately 30 established PKU patients
      receiving care from Hayward Genetics Center, who were found previously to exhibit no decrease
      in blood phenylalanine (PHE) levels (nonresponders) with administration of sapropterin.
      Subjects acted as their own controls. An additional number of patients naive to sapropterin
      were subsequently added to the study, and the age exclusion of over 21 years of age was
      omitted per IRB approval. Primary endpoints were designed to be measurement of behavioral and
      cognitive function, neurotransmitter levels, and gene expression of enzyme activity after 4
      weeks on treatment compared to baseline levels.

      At study baseline each patient attended an approximately 1 hour clinic visit at their usual
      genetics clinic location. Study purpose, design, and requirements were discussed, and
      consents/assents reviewed and signed.

      Rating inventories of executive function performance and behavior (BASC-2 and BRIEF tools)
      were administered to patients and parents by the Principal Investigator (PI) and/or the Study
      Coordinator.

      Urine samples were collected non-invasively for measurement of neurotransmitter levels. Blood
      as collected by venipuncture (3-5 ml) for microarray expression analysis and analysis of
      plasma amino acids. 3-day food records previously provided to participants for completion
      were collected.

      Participants were provided with a 4 week supply of Kuvan® and instructions on how to take the
      medication during the study period. The importance of maintaining usual dietary intake (food
      choices and metabolic formula) to minimize any research effect not attributable to
      sapropterin administration was emphasized. Sapropterin was discontinued at the end of the 4
      week study period. The exception to this was for the naive patients who were found to be
      responsive to sapropterin.

      All of these measures were repeated at the same sites with study participants at the end of
      week 4 of the study period.

      At the ends of weeks 1 and 2 additional blood samples were sent to Hayward Genetics Center
      for measurement of PHE and tyrosine (TYR) levels to ascertain no significant changes have
      occurred in a patient's usual dietary intake. These samples were drawn at each patient's
      local state health unit, as is done for usual monitoring. Nutrient analysis of the 3-day food
      diaries was conducted at Hayward Genetics Center.

        1. Behavior and executive function were assessed using published validated inventories,
           completed as patient self-reports and as parent (or guardian) reports when appropriate.
           Instruments used were the Behavioral Assessment System for Children (BASC-2) parental
           Rating Scale and Self-Reporting Personality Rating Scale, and the Behavioral Rating
           Inventory of Executive Function (BRIEF) Parent Form Instruments of Executive Function.
           Completed inventories were scored using electronic evaluation instruments by the Study
           Coordinator and PI, with consultation from Harvard Medical Center experts as needed.

        2. Urine samples were non-invasively collected and sent for analysis of catechols and
           neurotransmitters to an NIH laboratory specializing in this technique. Samples were
           blinded to this laboratory to prevent bias.

        3. Microarray analysis of blood samples was conducted at Hayward Genetics Molecular
           Laboratory to determine any effect on gene expression, and thus enzyme activity, as a
           result of sapropterin administration.

        4. Plasma amino acids were analyzed at Hayward Genetics Biochemical Laboratory to document
           that patients are "nonresponsive" to sapropterin (no resultant lowering of blood PHE);
           and to monitor any changes in plasma amino acids that could indicate a patient's failure
           to maintain usual dietary restrictions. Patients naive to Kuvan who responded were noted
           and function as comparators.

        5. 3-day food diaries completed by patients (or parent/guardians) at home documented any
           substantive changes in usual dietary intake during the study period. These were analyzed
           at Hayward Genetics Center using the MetabolicPro web-based analysis program.
    

Intervention name(s): sapropterin dihydrochloride
Intervention type(s): Drug
Intervention description(s): In 30 PKU patients found previously to exhibit no decrease in blood PHE levels behavioral and cognitive function, neurotransmitter levels, and gene expression of enzyme activity will be measured at baseline and after 4 weeks of Kuvan administration. Rating inventories of executive function performance and behavior will be administered to patients and parents.
Urine neurotransmitters, blood microarray expression, and plasma amino acids will be measured (plasma PHE and TYR levels will also be measured at weeks 1 and 2). Nutrient analysis of 3 day food diaries will be conducted.
Other intervention name(s): Kuvan
Intervention MeSH terms: Verapamil
Observational model: Other
Time perspective: Prospective
Sampling method: Probability Sample
Study population: 
        Study participants were solicited from treated patients diagnosed with PKU who are followed
        by our clinic. Participants were initially limited to those who had undergone a trial with
        sapropterin (FDA approved drug available for treatment) as part of their clinical care and
        been found to be non-responsive. Subsequently, with IRB approval, some patients diagnosed
        with PKU from our clinic population who were naive to sapropterin were added to the study.
      
Keywords: phenylketonuria; PKU; phenylalanine; neurotransmitters; gene; Kuvan; sapropterin