Neurodevelopmental Outcome of Early Dietary Lysine Restriction in Pyridoxine Dependent Epilepsy Patients

I Purpose: The purpose of this multi-centre study is to further assess the safety and
efficacy of the proposed lysine restricted diet in confirmed ATQ deficient PDE patients.

II Hypothesis: Restricting dietary lysine intake in infants from age 3 months or less with
confirmed diagnosis of pyridoxine-dependent epilepsy due to Antiquitin (ATQ) deficiency
will: reduce the accumulation of neurotoxic substratesα-aminoadipicsemialdehydeandits cyclic
equivalent 1-piperideine-6-carboxylate;and will improve overall neurodevelopmental outcome
at 3 years of age by acting as an effective intervention into the complex pathophysiology of
the condition.

III Justification: We hope that participants have a much improved chance to avoid the same
degree of developmental delay and associated problems. If this study demonstrates a
significant improvement of normal development and proves to be safe to use this would
provide a positive benefit to the child, the families involved as well as decreasing the
burden on the health care system which currently must provide for life long care of these
affected children.

IV Objectives: To determine the safety and efficacy of the lysine restricted diet on
numerous primary and secondary outcome measures as listed in the protocol. The primary
objective of this study is to evaluate neurodevelopmental outcome based on
neuropsychological assessments using Bayley-III.

V Research Method:

1. Recruitement - Infants and/or children ranging from less than 3 months up to 3 years of
age who have been diagnosed with PDE and with confirmed ATQ deficiency will be
recruited within the 3 participating TIDE clinics (Medical Genetics, Biochemical
Diseases, Neurology). Families may choose to participate in the TEST group or CONTROL
group if they do not wish to undergo the lysine restricted diet regimen or decline to
participate altogether.

2. Study visits - For participants in the TEST group, this will include a baseline visit
and follow up visits at ages 3 months, 6 months then every 6 months until 3 years of
age. For participants in the CONTROL group study visits include a baseline visit and 1
follow up visit at age 3 years. Routine clinical care visits can be scheduled as per
treating physician's discretion at shorter intervals if needed.

3. Procedures: The following safety and efficacy measures will be performed at the
specified study visits (Refer to table1 protocol or consent): medical history, vital
signs, anthropometric measurements, Physical and Neurological exams, vision & hearing
assessments, blood and urine samples tested for biomarkers, electroencephalogram,
cognitive function test, ages and stages questionnaire, quality of life questionnaire,
MRI, Cerebral Spinal fluid request only if lumbar puncture is being performed for
clinical purposes.

4. Study Outcomes: The following outcomes will be measured.

Efficacy:

Primary Outcomes

- Neurocognitive development at age 3 years defined by total developmental index measured
using the Bayley Scales for Infant and Toddler Development, 3rd Edition (Bayley-III)

- Level of biochemical marker-α- aminoadipic semialdehyde (AASA) its cyclic equivalent
P6C in plasma and urine Secondary Outcomes

- Seizure frequency: clinical and electrical (EEG)

- Quality of life through a HR-QOL questionnaire

- Neurological deficits through neuro exam

Safety:

Primary Outcomes

- Anthropometric measures

- Plasma lysine and branched chain amino acid levels Secondary Outcomes

- Global nutritional assessment with plasma levels for albumin, prealbumin, total
protein, iron parameters, zinc, selenium, CBC, folic acid, vitamin B12

- Peripheral sensory neuropathy (relevant because the lysine restriction is expected to
reduce chemical inactivation of pyridoxine, thus potentially increasing the risk of
toxicity)

VI Statistical Analysis:

I. OVERALL STUDY DESIGN Structure: We will conduct a multicenter, open label,
negative-controlled observational cohort study to assess the safety and the efficacy on
neurodevelopmental outcome of early dietary lysine restriction as an adjunct to pyridoxine
therapy for infants with pyridoxine-dependent epilepsy resulting from ATQ deficiency.

Treatment Exposure: Patients receiving a lysine restricted diet adjunct to pyridoxine
therapy will be considered as participants in the 'exposure'/test group and patients on
pyridoxine mono-therapy will be participants in the 'control' group. The ratio of test
participants to controls in the study will be 1:2.

Duration: All participants will be monitored for safety and neurodevelopmental outcome until
the age of 3 years.

Efficacy & Safety Analysis: Data will be summarized with descriptive statistics, frequency
tabulations, and data listings. Analyses include comparison of treatment effect before and
after diet restriction within each participant, and comparison between test and control
groups. Analysis will be performed on intent-to-treat population including all enrolled
participants. The primary objective of this study is to evaluate neurodevelopmental outcome
based on neuropsychological assessments using Bayley-III. The evaluation comprises the
following scales: Cognitive Scale, Language Composite Scale with Receptive and Expressive
Language subscales, and Motor Composite Scale with Fine- and Gross-Motor subscales. A
composite score of all the scales gives the total developmental index. For the composite
score and each scale, a hypothesis test can be performed to compare test group and the
control group. A 2-sided 95% confidence interval for the mean change in Treatment minus mean
change in control groups will be calculated for the total developmental index and each
individual scale of Bayley-III to evaluate any particular nonnegative quantity of clinical
interest. Similar analysis using appropriate parametric (ANOVA, ANCOVA, Fishers exact test)
and nonparametric tests (Wilcoxon rank sum test) will be done for all efficacy and safety
outcomes. Severity and relationship to treatment of the adverse events will be summarized.

Inclusion Criteria:

- Diagnosis of pyridoxine-dependent epilepsy based on clinical symptoms and elevated
levels of plasma or urine AASA. Confirmation by at least one known disease causing
mutation in the ALDH7A1 gene to be obtained within one month of enrollment.

- Participant is male or female <3 years of age.

- Participants in the test arm have to be less than 3 months of age when the
dietary restriction was started.

- Participants in the control arm may be older than 3 months of age but must not
be older than 3 years of age when they are enrolled into the study and must have
been on pyridoxine treatment prior to age 3 months and not treated with dietary
lysine restriction at any time during their life.

- Participant is managed with a vitamin B6 dose of 15-30 mg/kg/day continuously
beginning at < 3 months age, and willing to maintain this dose for the study
duration.

- Participants must have been offered dietary lysine restriction as adjunct therapy as
part of standard clinical care.

- Parent(s) or guardian(s) is willing and able to provide written informed consent
after the nature of the study has been explained, and prior to any research-related
procedures.

Exclusion Criteria:

- Diagnosis is not confirmed: Participant does not have a mutation in the ALDH7A1 gene.

- Participant was treated prenatally for PDE with pyridoxine (i.e. mother was on
pyridoxine)

- Timing of dietary restriction: Participant is on a lysine-restricted diet from an age
> 3months.

- Confounding factors:

- Participant is a pre-term with a gestational age < 32 weeks

- The participant has a birth weight less than the 2nd percentile or weighs less
than 2nd percentile at study entrance (on age appropriate growth chart).

- Participant shows an intracranial malformation or abnormality unrelated to ATQ
deficiency, as diagnosed on the cranial ultrasound and/or MRI brain scan

- Participant has any other disorder identified that can affect the cognitive
function in the opinion of the coordinating principal investigators.

- A known allergy or sensitivity to any component of the products commonly used in a
lysine-restricted diet or to other products associated with lysine restriction or any
other products associated with general study procedures.

- Participant is on oral folinic acid and/or pyridoxal phosphate treatment at study
entrance.

- Participant has any condition or situation which, in the investigator's opinion,
places the patient at significant risk of adverse events, or may interfere
significantly with their participation and compliance in the study.