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

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

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

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”

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

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

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 /

screening plate of galactosaemia

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

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:

Time ( minute ) Control Galaktosaemia. heterozygote Galaktosaemia classic Duarte variant Duarte variant heterozygote

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 +

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. GALT 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, perc 3,813,523,283,883,97

Screening year Screened saces Screening year Screened saces Screening year Screened saces G1PUT19962 G1PUT 5 G-kinase G-kinase G1PUT19972 G1PUT 3 G-kinase G-kinase G1PUT19983 G1PUT 2 G-kinase 2 Duarte? G-kinase G1PUT19992 Duarte? 3 G-kinase G-kinase G1PUT 1 G-kinase G-kinase G1PUT 2 G-kinase G-kinase G1PUT G-kinase20022 G1PUT 2 G-kinase G1PUT 1 G-kinase G1PUT 2 G-kinase 1 Duarte? G1PUT 1 G-kinase G1PUT G1PUT 1 G-kinase G1PUT 10 G-kinase Screened galactosaemic cases

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

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