LeukoSEQ: Whole Genome Sequencing as a First-Line Diagnostic Tool for Leukodystrophies

Leukodystrophies are a group of approximately 30 genetic diseases that primarily affect the
white matter of the brain, a complex structure composed of axons sheathed in myelin, a glial
cell-derived lipid-rich membrane. Leukodystrophies are frequently characterized by early
onset, spasticity and developmental delay, and are degenerative in nature. As a whole,
leukodystrophies are relatively common (approximately 1 in 7000 births or almost twice as
prevalent as Prader-Willi Syndrome, which has been far more extensively studied) with high
associated health-care costs; however, more than half of the suspected leukodystrophies do
not have a definitive diagnosis, and are generally classified as "leukodystrophies of unknown
etiology". Even when a diagnosis is achieved, the diagnostic process lasts an average of
eight years and results in test expenses in excess of $8,000 on average per patient,
including the majority of patients who never achieve a diagnosis at all. These diagnostic
challenges represent an urgent and unresolved gap in knowledge and disease characterization,
as obtaining a definitive diagnosis is of paramount importance for leukodystrophy patients.
The diagnostic workup begins with findings on cranial Magnetic Resonance Imaging (MRI)
followed by sequential targeted genetic testing, however next generation sequencing
technologies (NGS) offer the promise of rapid and more cost effective approaches.

Despite significant advances in diagnostic efficacy, there are still significant issues with
respect to implementation of NGS in clinical settings. First, sample cohorts demonstrating
diagnostic efficacy are generally small, retrospective, and susceptible to ascertainment
bias, ultimately rendering them poor candidates for utility analyses (to determine how
efficient a test is at producing a diagnosis). Second, historic sample cohorts have not been
examined prospectively for information about impact on clinical management (whether the test
results in different clinical monitoring, a change in medications, or alternate clinical
interventions).

To address these issues, the study team conducted an investigation of patients with suspected
leukodystrophies or other genetic disorders affecting the white matter of the brain at the
time of initial confirmation of MRI abnormalities, with prospective collection of patients
randomly received on a "first come, first served" basis from a network of expert clinical
sites. Subjects were randomized to receive early (1 month) or late (6 months) WGS, with SoC
clinical analyses conducted alongside WGS testing. An interim analysis performed in May 2018
assessed these study outcomes for a cohort of thirty-four (34) enrolled subjects. Two of
these subjects were resolved before complete enrollment and were retained as controls. Nine
subjects were stratified to the Immediate Arm, of which 5 (55.6%) were resolved by WGS and 4
(44.4%) were persistently unresolved. Of the 23 subjects randomized to the Delayed Arm, 14
(60.9%) were resolved by WGS and 5 (21.7%) by SoC, while the remaining 4 (17.4%) remained
undiagnosed. The diagnostic efficacy of WGS in both arms was significant relative to SoC
(p<0.005). The time to diagnosis was significantly shorter in the immediate WGS group
(p<0.05). The overall diagnostic efficacy of the combination of WGS and SoC approaches was
26/34 (76.5%; 95% CI = 58.8% to 89.3%) over <4 months, greater than historical norms of <50%
over more than 5 years.

The study now seeks to determine whether WGS results in changes to clinical management in
subjects affected by undiagnosed genetic disorders of the white matter of the brain relative
to standard diagnostic approaches. We anticipate that WGS will produce measurable downstream
changes in clinical management, as defined by disease-specific screening for complications or
implementation of disease-specific therapeutic approaches.

Inclusion Criteria:

1. Abnormalities of the white matter signal on neuroimaging (MRI) with T2 hyperintensity
which must be diffuse or involve specific anatomical tracts consistent with a genetic
diagnosis;

2. No pre-existing genetic diagnosis;

3. A clinical decision has been made to perform WGS;

4. Less than 18 years of age;

5. Availability of both biologic parents for blood sampling;

6. Availability both biological parents to provide informed consent;

7. Concurrently enrolled in CHOP IRB 14-011236 (New Diagnostic Approaches in
Leukodystrophy - The Myelin Disorders Biorepository Project)

Exclusion Criteria:

1. Candidates with acquired disorders, including infection, acute disseminated
encephalomyelitis (ADEM), multiple sclerosis, vasculitis or toxic
leukoencephalopathies;

2. Patients who have had previous genetic testing*, including WES or WGS;

3. Those with no third-party payer insurance, unable to receive standard of care
diagnosis and therapeutic approaches;

4. Candidates who have already received a diagnosis.

- Note: Karyotype or microarray testing that did not yield a definitive diagnosis
should not be considered as an excluding factor.