Galactosemia Screening Using Tandem Mass Spectrometry

Home , Galactosemia

Henrik Simonsen, MD, PhD Denmark Biochemistry

Galactitol UDP-Glucose GALE

Galactose GALK Galactose-1-P GALT UDP-Galactose

Galactonate Glucose-1-P

Glycolysis Glycogen Fragmentation of galactose 1-phosphate

79 100

80 y t i s

n 60 e 97 t n I e v i t

a 40 l e R

20 139 79 259 241 97 0 100 150 200 250 m/z 139 Fragmentation patterns

79 97 100 100

80 80 y y t i t i s s

n 60 Fructose 1-P n 60 e e 97 Galactose 1-P t t n n I I

Fructose 6-P e e v

v Glucose 1-P i i t t

a 40

a 40 l l Glucose 6-P e e R R

20 20 139 259 79 241 139 241 259 0 0 100 150 200 250 m/z 100 150 200 250 m/z

79 79 97 97 Galactose 1-P Fructose 1-P Retrospective screening

2.0

1.5

1.0 ratio (m/z 79 / m/z 97)

0.5 Galactosemia intensity Q188R/N314D Reference Ion

0.0 -1 0 1 2 3 4 5 6 Hexose monophosphates (mmol/L) Analytical procedure

Dried blood spot sample

Extract acetonitrile:water

Pellet debris

Transfer Dilute 1:10

m/z 259>79

Linear regression

In-house retesting if elevated Gal 1-P Participation

Newborns in Denmark 100%

Screened Not screened PKU, CH, Toxoplasmosis 1% 99%

Informed consent No information Supplemental MS/MS Screening 75% 24%

Opt in Opt out 74% 1%

Amino acids Gal 1-P Acylcarnitines Sampling time

20000

15000

10000

5000

0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 Diagnostic Algorithm

MS/MS Gal 1-P

Normal Borderline Elevated <1.2 mmol/L 1.2-2.0 mmol/L >2.0 mmol/L

Resample

Enzymatic GALT genotype Gal 1-P Q188R, N314D

Normal Elevated Sequencing 0-3 micg/mL >3 micg/mL Analytical performance

• Measured samples 9613

• Technical redeterminations 94 = 1%

• Confirmatory redeterminations 29 = 0,3% Screening results

Gal 1-P Gal 1-P Mut1 Mut2 Comment 4,8 1282 Q188R R51L 1,3 7 Q188R N314D 1,9 124 R67C N314D 1,9 0 Q188R N314D 2,3 43 Q188R N314D 1,2 nd N314D N314D Neg. Benedict 1,4 0 N314D N314D 1,5 nd N314D N314D Normal second filterpaper screen 1,7 15 N314D N314D 1,4 1 wt N314D 1,5 1 wt N314D 1,6 0 wt N314D 1,7 nd wt N314D 1,4 0 wt wt 1,5 2 wt wt 1,6 1 wt wt 1,8 1 wt wt 2,0 1 wt wt Gal 1-P levels

2 Gal 1-P (mmol/L)

0 0 1 2 3 4 5 6 C/C C/D D/D D/W W/W Genotypes

Classical Duarte Wild type Sum Classical 1 4 0 5 Duarte 4 4 8 Wild type 5 5 Sum 18 Diagnostic Performance

• Screened 96.000 (est.) • Gal 1-P >2.0 mmol/L 1 patient + 2 • Gal 1-P 1.2-2.0 mmol/L 15

• Clinical incidence: 1 (2 y study period) 1:60.000 (42 y period)

• Sensitivity: 100% (1:1)

• FPR 1:6.400 (0,02%) - 1:32.000 (0,003%)

• PPV: 6% (1:18) - 33% (1:3) Conclusions

• MS/MS is able to detect even weak abnormalities in the GALT gene.

• High sensitivity

• Low false positive rate

• Internal standard is recommended Fragmentation patterns