1. When do we speak about galactosaemia? Galactose Glucose (D) Lactose at the root of the intestinal villi ( lactase (β galactosidase ) enzyme Glucose ( disaccharide ) monosaccharide + H 2 O hydrolysis inhibitid

2. galactose aldosereductase galactitol galactokinase galactose-1-P gal.-1-P uridyl transferase. UDP galactose UDP gal.-4-epimerase UDP glucose UDPG pyrophosporilase glucose-1-P Total galactose The process of conversion

3. cytosol GLUT GALT glucose galactose cytosol The glucose-6-phosphate and the galactose-1-phosphate fall into an ambush in the cell. THE PHOSPHORYLATION impermeable

4. Determination of galactosaemia : based on Guthrie microbiological metabolic inhibition method Principle: the blood-soaked filter paper spots are placed on agarose medium witch contains galactose sensitive mutant E.coli (W5) strain „Microbiological newborn screening”

7. Weidemann test: galactose over 20 mg % Principle: The galactose – dehydrogenase ( Gal-DH ) enzyme transfers H + from galactose to NAD, to form fluorescent NADH Control (mg % ) Sample 20 25 30 35 40

8. Interfering factors: False-positive result: 1. if antibiotic was given to the newborn which inhibits the growth of E-coli 2. any contamination which gives a clear zone around the disk False-negative result: 1. if the blood sample was taken before breast feeding was started 2. if the filter paper was not blood soaked properly 3. if the baby was transfused

9. Total Galactose Screening Assay / galactose and galactose-1- phosphate / R &D Diagnostics Ltd Greece alkaline phosphatase galactose-1- phosphate Galactose galactose dehydrogenase Galactose + NAD+H 2 O Galaktono (1,4) lakton + + NADH + + H + diaphorase NADH + NBT ( yellow )NBTH ( violet ) + NAD / nitroblu-terazolium // formazan /

10. screening plate of galactosaemia

11. Beutler probe : determination the galactose-1-phosphate – deficit of galactose-1-phosphate – uridil- transferase uridil- transferase ( G-1-PUT ) enzyme to confirm the diagnosis of classical galactosaemia Samples: heparinised total blood collected into gas capillary and a healthy control blood which is collected, transported, stored, and measured under the same conditions

12. Principle: In the presence of G-1-PUT the NADP is reduced forming fluorescent NADPH blood + galactose-1-phosphate + NADP 30‘ 60‘ 90‘ 120‘ Control: Sample:

13. Time ( minute ) 0 30 60 90 120 Control Galaktosaemia. heterozygote Galaktosaemia classic Duarte variant Duarte variant heterozygote

14. Detection of Galactose-1-phosphate uridil- transferase /GALT/ enzyme GALT GAL-1-P + UDPG Galactose-1-phosphate uridil-transferase G-1-P + UDP-GAL PGM G-1-P phosphoglucomutase G-6-P G-6-PD G-6-P + NADP glucose-6-phosphate dehydrogenase 6-PGA + NADPH + 6-PGD 6-PGA + NADP 6-fphosphogluconate dehydrogenase R-5-P + NADPH +

15. Results GALT enzyme aktivity : Normal : > 3,8 U/gHb Transitional: 2,9 -3,8 U/gHb Abnormal: < 2, 9U/gHb Beteg B.I.T.K.A.G.M.I.M.AF.G.S.A.S.I.T.A. GALT3.403.111.052.933.511.773.341.751.25 Galactose tolerance test ( 1,5 g/kg laktóz; max 50g ) 30 perc 3,673,103,123,473,83 60 perc 4,013,333,223,914,11 120 perc 3,813,523,283,883,97

16. Screening year Screened saces Screening year Screened saces Screening year Screened saces 19861 G1PUT19962 G1PUT 5 G-kinase 20064 G-kinase 19871 G1PUT19972 G1PUT 3 G-kinase 20074 G-kinase 19881 G1PUT19983 G1PUT 2 G-kinase 2 Duarte? 20084 G-kinase 19891 G1PUT19992 Duarte? 3 G-kinase 20094 G-kinase 1990020003 G1PUT 1 G-kinase 20104 G-kinase 19911 G1PUT 2 G-kinase 20010 2 G-kinase 20111 G1PUT 19921 G-kinase20022 G1PUT 2 G-kinase 19931 G1PUT 1 G-kinase 20031 G1PUT 2 G-kinase 1 Duarte? 19941 G1PUT 1 G-kinase 20042 G1PUT 0 19951 G1PUT 1 G-kinase 20051 G1PUT 10 G-kinase Screened galactosaemic cases

17. Most common point mutations G-1-PUTransferase Galactokinase GALT GalK1 GalK2 9p13 17q24 15q21.1 Classical Duarte Q188R G1391A S142I G171V K285N N314D G148C P28T

18. countryScreening starting Austria1966 Ireland1972 United Kingdom1972 Only in Scotland Switzerland1975 Hungary1975 Belgium1978 Only the Flemish provinces Germany1978 Not compulsory in some provinces Denmark2002 Franceno Netherlandsno Norwayno Polandno Spainno Newborn screening of galactosaemia in Europe