Intervention and Outcomes in Duarte

Angela Wittenauer*, MSN, FNP-C, RN Follow Up Director Emory University, Department of Human Genetics

* No conflicts of interest to report.

Published as: Carlock G, Fischer ST, Lynch ME, Potter NL, Coles CD, Epstein MP, Mulle JG, Kable JA, Barrett CE, Edwards SM, Wilson E, and Fridovich-Keil JL. Developmental Outcomes in Duarte Galactosemia. . 2019;143(1):e20182516. In the next 15 minutes....

• What is Duarte galactosemia (DG)?

• What were the goals of our study?

• What did we do?

• What did we find?

• What does it mean for NBS? Duarte galactosemia (DG) results from PARTIAL deficiency of GALT. DG versus classic galactosemia

RBC GALT activity (μmol/hr/g Hb) 10 20 30 40 50 60 0 (109) NN (93) GN Imputed (44) DN (15) GALT DD (127) DG status (22) CG other (7)

Pyhtila et al JIMD Reports 2014 15:79 • November 2015-November 2017 (2 years) States Tested: California • 17 testing blocks in 13 states Texas • 3-5 days per testing block, up to 6 children tested per day Alabama, • Testing team: Study coordinator, 2 Psych testers, 2 Georgia, Speech/Motor testers South Carolina • Some states had low enrollment and so we did not test there, Tennessee but eligible families were invited for testing in adjacent states Iowa Missouri Indiana Illinois Michigan Ohio Virginia

States recruited but not tested: North Carolina New Jersey Pennsylvania Maryland/DC* Newborn screening (NBS) detection rates for CG and DG

False positives Duarte galactosemia cases Classic galactosemia cases per 100,000 births C per 100,000 births B per 100,000 births A (16) AK (16) (16) (15) (13) (11) AL * AR (3) (3) (3) (3) (2) AZ (2) (10) (10) (10) CA (11) (1) (2) FL GA (5) (5) (5) (22) (22) (22) IL (18) (8) IN (16) (11) (14) KS (10) (10) (10) ME (7) MI (7) (7) MO (5) (5) (5) (9) MS (9) (14) (14) (14) NE (6) (6) NJ (6) (18) (18) (18) NY (12) (12) (12) SC UT (6) (6) (6) (22) (22) (22) WI * *(Yearsdata included) of Positives False DG CG

Pyhtila et al JIMD Reports 2014 15:79 Should babies with DG drink milk or low-galactose formula?

• Infants with DG • Newborns with generally thrive DG accumulate while drinking milk elevated galactose • No strong direct metabolites while evidence of drinking milk long-term complications • We know from CG that long-term • Breastfeeding complications can has many known occur even in benefits… do we children who really want to experienced no interfere with that? acute symptoms What were the goals of our study?

Aim 1: Test whether children with Duarte galactosemia, ages 6-12 years old, demonstrate increased prevalence of developmental difficulties relative to controls, and

Aim 2: Test whether dietary exposure to galactose (milk) in infancy associates with developmental outcomes of children with Duarte galactosemia. An observational case-control study

Aim 1 Aim 2 Study design:

Initial recruitment (17 states + DC)

Informed consent

Part 1 (online parent/guardian survey)

Part 2 (direct child assessment + more parent/guardian surveys)

Data analysis What information did we gather from enrolled volunteers? • demographics, SES by parent/guardian • other covariates response surveys • child’s diet in infancy and beyond

by direct assessments

• Thinking and Problem • Speech and Language Solving Skills Development • Socioemotional • Physical Development Development (Movement/ Coordination) • Physical measures (height, weight, head circumference) • Auditory Processing What did our data look like?

• 350 subjects (206 cases, 144 controls)

• Case/control status confirmed by GALT genotyping of DNA from saliva samples

• 73 outcome variables quantified per child (representing 5 developmental domains)

• Covariates collected (age, sex, race/ethnicity, annual household income, breast milk exposure, highest parent education, US region, WASI II IQ) How did we analyze our data?

• All statistical analyses performed using R (version 3.4.0) • Descriptive statistics to compare demographics between groups and identify covariates relevant to different outcomes • Test for “missingness” between cases and controls • Split data set (25:75) into independent discovery (n=87) and validation (n=263) sets • Tested all 73 outcomes for association with DG status in the discovery set; identified top 10 (smallest p values) • Tested these top 10 for association with DG status in the validation set • Tested same top 10 for association with diet among cases in the validation set Top 10 outcomes from the discovery set also tested for possible association with DG in the validation set.

** By chance alone we would have expected to find 1 outcome with p<0.05; we found 2. Bonferroni-adjusted cut-off for significance= 0.0025. A closer look at the NEPSY II Route Finding Score %

Validation set

Discovery set

Diet (dairy exposed)

Children with DG scored BETTER than controls! A closer look at head circumference

Validation set

Discovery set Diet (dairy exposed)

Tiny difference; not significant after multiple test correction Top 10 outcomes from the discovery set also tested for possible association with milk exposure of DGs in the validation set.

** None reached even nominal significance. So what did we find?

1. Did children with Duarte galactosemia, ages 6-12 years old, demonstrate increased prevalence of developmental difficulties relative to controls? NO

2. Did dietary exposure to galactose (milk) in infancy associate with developmental outcomes of children with Duarte galactosemia? NO Example outcome tested: WASI IQ WASI IQ WASI IQ WASI

Controls Children Children Children (n=144) with DG with DG: with DG: (n=206) Soy Milk (n=123) (n=83) Example outcome tested: Height Z Score Differential Differential Differential Height Z Score Z Score Height Height Z Score Z Score Height

Controls Children Children Children (n=144) with DG with DG: with DG: (n=206) Soy Milk (n=123) (n=83) Example outcome tested: Auditory Processing ABER Wave 3 Latency ABER Wave 3 Latency ABER Wave

Controls Children Children Children (n=132) with DG with DG: with DG: (n=191) Soy Milk (n=113) (n=78) Example outcome tested: Speech/ Motor TETRAS Score TETRAS TETRAS Score TETRAS

Controls Children Children Children (n=144) with DG with DG: with DG: (n=206) Soy Milk (n=123) (n=83) Recognizing our limitations:

• This is only one study. • 350 is a big number for DG, but it is not huge. • Age range 6-12 years; we did not test younger or older volunteers. • Diet information gathered from family by retrospective survey. • All volunteers from US; almost all white. • We tested 73 outcomes; there are outcomes we did not test. • Other Mindful of the limitations, what do our results mean? Our results suggest that children with DG are not at significantly increased risk of developmental disorders regardless of milk exposure. IF THIS IS TRUE…

• DG families: can breastfeed with confidence • Clinicians: can give evidence-based prognostic and diet recommendations to DG families • : evidence for removing DG from the NBS panel, adjusting NBS GALT cut-off level to lower the false-positive rate Breast milk consumption in infancy by 324 cases and 243 controls

Controls

Children with DG

0-1 month 2-6 months 7-12 months CG and DG detection rates in Utah before and after changing the GALT NBS cutoff et al JIMD Reports 2014 15:79 Reports JIMD al et

*may include carriers and other variants. Pyhtila Thank you! • Amazing families and wonderful colleagues who made this study possible • Funding from the Patient-Centered Outcomes Research Institute (http://www.pcori.org/research-results/2015/intervention-and- outcomes-duarte-galactosemia) • The late Dr. Paul Fernhoff who encouraged us to start this project

Questions? References and web sites: • Carney AE, Sanders RD, Garza KR, McGaha LA, Bean LJ, Coffee BW, Thomas JW, Cutler DJ, Kurtkaya NL, Fridovich-Keil JL. Origins, distribution and expression of the Duarte-2 (D2) allele of galactose-1-phosphate uridylyltransferase. Human molecular genetics. 2009;18(9):1624-32. • Fernhoff PM. Duarte galactosemia: how sweet is it? . 2010;56(7):1045-6. • Ficicioglu C, Thomas N, Yager C, Gallagher PR, Hussa C, Mattie A, Day-Salvatore DL, Forbes BJ. Duarte (DG) galactosemia: a pilot study of biochemical and neurodevelopmental assessment in children detected by newborn screening. Mol Genet Metab. 2008;95(4):206-12. • Ficicioglu C, Yager C, Segal S. Galactitol and galactonate in red blood cells of children with the Duarte/galactosemia genotype. Mol Genet Metab. 2005;84(2):152-9. • Fridovich-Keil J, Gambello M, Singh R, Sharer J. Duarte Variant Galactosemia. In: Pagon R, Adam M, Ardinger H, Bird T, Dolan C, Fong C, Smith R, Stephens K, editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 2014. • Lynch M, Potter N, Coles C, Fridovich-Keil J. Developmental outcomes of school-age children with Duarte galactosemia: A pilot study. JIMD Reports. 2015; 19:75-84. • Powell KK, Van Naarden Braun K, Singh RH, Shapira SK, Olney RS, Yeargin-Allsopp M. Long- term speech and language developmental issues among children with Duarte galactosemia. Genetics in : official journal of the American College of . 2009;11(12):874-9. • Pyhtila BM, Shaw KA, Neumann SE, Fridovich-Keil JL. Newborn screening for galactosemia in the United States: looking back, looking around, and looking ahead. JIMD Rep. 2015;15:79-93. • Duartegalactosemia.org • https://clinicaltrials.gov/ct2/show/NCT02519504?term=duarte+galactosemia&rank=1 Inclusion and Exclusion Criteria

Inclusion Criteria • Child is 6-12 years old at the time of direct assessment • Child has DG (case) or child does not have any form of galactosemia but is the sibling of a child with DG (control) • Both child and at least one parent/guardian are conversant in English • Both child and at least one parent/guardian are available to participate in one of the Direct Testing Blocks offered

Exclusion Criteria • Any severe developmental disorder, severe intellectual disability, or severe medical condition that could affect the same developmental outcomes being tested for possible association with DG (10 children, including 6 cases and 4 controls, were excluded for this reason) • Unable to complete Part 2 testing due to a severe hearing, visual, or motor disability (0 children were excluded for this reason) • Child participated in our previous pilot study (7 children were excluded for this reason) • Child DNA sample unavailable or GALT genotype inconclusive (6 children were excluded for this reason) Milk vs. low-galactose formula consumption in infancy by 206 cases and 140144 controls

120

100

80

60 milk galformula

40 gal formula milk

20 - Low low -

0 Cases Controls Children with DG Controls Demographics (n=350)