The role of human demographic history in determining the distribution and frequency of transferase-deficient galactosaemia mutations.

Classical or transferase-deficient galactosaemia is an inherited metabolic disorder caused by mutation in the human Galactose-1-phosphate uridyl transferase (GALT) gene. Of some 170 causative mutations reported, fewer than 10% are observed in more than one geographic region or ethnic group. To better understand the population history of the common GALT mutations, we have established a haplotyping system for the GALT locus incorporating eight single nucleotide polymorphisms and three short tandem repeat markers. We analysed haplotypes associated with the three most frequent GALT gene mutations, Q188R, K285N and Duarte-2 (D2), and estimated their age. Haplotype diversity, in conjunction with measures of genetic diversity and of linkage disequilibrium, indicated that Q188R and K285N are European mutations. The Q188R mutation arose in central Europe within the last 20 000 years, with its observed east-west cline of increasing relative allele frequency possibly being due to population expansion during the re-colonization of Europe by Homo sapiens in the Mesolithic age. K285N was found to be a younger mutation that originated in Eastern Europe and is probably more geographically restricted as it arose after all major European population expansions. The D2 variant was found to be an ancient mutation that originated before the expansion of Homo sapiens out of Africa.

Serum markers of bone turnover in children and adolescents with classic galactosemia.

PURPOSE: Classic galactosemia is an inherited metabolic disease resulting from galactose-1-phosphate uridyltransferase (GALT) deficiency. Dietary lactose exclusion reverses many clinical manifestations of acute phase of the disease. Unfortunately most of the patients, despite dietary treatment, develop long-term complications among them disturbances of bone mineralization resulting in decrease of bone mineral density (BMD). The aim of our study was to assess bone formation and resorption processes with bone turnover markers in children and adolescents with galactosemia. MATERIALS AND METHODS: We studied 62 galactosemic children (mean age+/-SD 5.9+/-2.7 years) and adolescents (mean age+/-SD 15.6+/-2,4 years). The clinical diagnosis had been confirmed by the absence of GALT activity in erythrocytes. All patients were diagnosed in the neonatal period and had good dietary control. Healthy children (n=70) were the reference group. Serum osteocalcin (OC), bone alkaline phosphatase (BALP), collagen type I crosslinked C-telopeptide (CTX-I), 25(OH)D metabolite of vitamin D were determined by ELISA assays. RESULTS: We observed similar mean values of bone formation markers in children with galactosemia as compared to the age-matched controls. The level of bone resorption marker CTX-I in these patients was lower by about 20% (p<0.001) than in healthy children. On the contrary we obtained slightly higher values of CTX-I in adolescents with galactosemia in comparison to the age-matched controls. In these patients the values of OC and BALP were significantly higher than in healthy adolescents (111.8+/-52.1 microg/L versus 82.3+/-43.0 microg/L, p<0.02; and 95.4+/-45.7 U/L versus 72.6+/-40.6 U/L, p<0.05 respectively). CONCLUSION: Our results suggest that bone turnover in galactosemic patients elevates from childhood to adolescence, whereas in healthy individuals there is a decline during aging. Further studies on adults with galactosemia are necessary to assess bone status in these patients.

Should newborn mutation scanning for hyperphenylalaninaemia and galactosaemia be implemented? A Polish experience.

OBJECTIVE: To elucidate whether screening for mutations causing hyperphenylalaninaemia (HPA) and classic galactosaemia could provide important, additional information on a clinical phenotype. METHOD: Genotypes that cause disease at the phenylalanine hydroxylase (PAH) gene and galactose-1-phosphate uridyltransferase (GALT) gene in a group of 101 hyperphenylalaninaemic and 77 patients with classic galactosaemia were established. The PAH and GALT mutations were identified in genomic DNA extracted from whole blood leucocytes using single stranded conformational analysis and direct fluorescent sequencing of polymerase chain reaction (PCR) products. RESULTS: Mild HPA and mild phenylketonurea (PKU) were caused by divergent genotypes. In the studied group a total of 26 different mild and intermediate PAH mutations were identified, most of them being rare ones. Classic galactosaemia was caused by two frequent mutations, accounting for 82% of all mutated alleles. CONCLUSIONS: Identification of mild or intermediate mutations causing HPA could provide fast and reliable information about future clinical outcome of a newborn infant. Molecular diagnosis of HPA should be preceded by biochemical analysis and implemented to differentiate mild forms of HPA and cases of ambiguous classification. Because of multiple rare mutations scattered on all exons, scanning of the entire PAH coding sequence could be useful and cost beneficial. Routine genotyping is not proposed in classic phenylketonuria and classic galactosaemia, as it provides limited additional, prospective information on the clinical phenotype.

[Homocysteine and some lipid parameters in hypercholesterolemic children]. / Homocysteina i wybrane parametry przemian lipidowych u dzieci z hipercholesterolemia.

High serum homocysteine (tHcy) concentration is increasingly recognised as independent risk factor for atherosclerosis, early coronary heart disease (CHD) and other vascular diseases. It has been proved that adult cardiovascular disease begins in childhood. In the presented studies we determined concentrations of homocysteine, lipids and lipoproteins in plasma of hypercholesterolemic and normocholesterolemic children. In hypercholesterolemic children total cholesterol, LDL cholesterol, apolipoprotein B and triglycerides were significantly higher, whereas HDL cholesterol and apolipoprotein A-I were lower in comparison to the control group. Total serum homocysteine in children with positive family history for cardiovascular disease CHD(+) was significantly higher than in the control groups, and in CHD(-) group. It was respectively 7.3 micromol/1 versus 5.45 micromol/l versus 5.21 micromol/l. The results obtained in our study indicate that in hypercholesterolemic children with positive family history for CHD, the concentration of tHcy can be considered as a separate predictive risk factor for premature cardiovascular disease.

Serum homocysteine and lipoprotein (a) concentrations in hypercholesterolemic and normocholesterolemic children.

The relationship between lipids, lipoproteins, total homocysteine, and lipoprotein (a) was studied in hypercholesterolemic and normocholesterolemic children. In hypercholesterolemic children, concentrations of total cholesterol, low-density lipoprotein (LDL) cholesterol, apolipoprotein B, and triglycerides were significantly higher compared to levels in controls, whereas concentrations of high-density lipoprotein (HDL) cholesterol and apolipoprotein A-I were lower compared to those in the control group. Total serum homocysteine concentrations in children with a positive family history for cardiovascular disease CHD(+) (7.28 micromol/L) were significantly higher than those in the control group (5.45 micromol/L), and in the group of CHD(-) children (5.25 micromol/L). The median value of lipoprotein (a) in patients was 31.5 mg/dL (range, 11-209 mg/dL) and in the control group, 19 mg/dL (range, 11-95 mg/dL). Concentrations of Lp (a), exceeding 30 mg/dL, were present in 45% of CHD(+) children, in 29% of CHD(-) children, and in only 11% of the control group.

[Screening programme for hyperlipidemia in children and adolescents. Prophylactic aspects of atherosclerosis].

Atherosclerotic cardiovascular diseases (CVD), mainly coronary heart disease (CHD) remain the leading cause of death in adult populations of many countries. The following risk factors for atherosclerosis were identified: hypercholesterolemia, hypertension, cigarette smoking and obesity. Scientific reports and epidemiological studies have shown that atherosclerosis begins in childhood. Therefore consensus was obtained that the earlier the prevention begins the better results are achieved. But there are many controversies around early identification of hypercholesterolemia in children. Three options were considered: cholesterol mass screening, selective cholesterol screening and no screening at all. The most acceptable is selective screening performed in children of high risk families (CVD or hypercholesterolemia in the family). It is recommended by the US Expert Panel for the National Cholesterol Education Program for Children and Adolescents (NCEP-Peds). According to the NCEP-Peds, screening should include the following groups: I) children whose parents or grandparents have a history of CVD (under the age of 55 years), 2) children whose parents have a raised blood cholesterol concentration (above 240 mg/dl), 3) children with negative or unknown family history, but having other risk factors (hypertension, obesity, cigarette smoking, high-fat diet). The experts recommend that the examination should be performed in children after the age of 2 years. The NCEP-Peds guidelines set total cholesterol levels in serum for children and adolescents from families at risk, below 170 mg/dl, as acceptable. Total cholesterol level between 170 and 199 mg/dl is classified as borderline and 200mg/dl and above--as high.

[Effectiveness of the screening programme for galactosemia. New strategy in Poland].

Galactosemia is an autosomal recessive disease related to deficiency of one of three different enzymes involved in the metabolism of galactose: galactokinase (GALK), galactoso-J-phosphate uridyltransferase (GALT) or UDP-galactose-4-epimerase (GALE). Classic galactosemia is due to GALT deficiency and is the most common. Longitudinal studies have shown that in spite of early diagnosis and early treatment of children with galactosemia detected in the mass screening programme, the results are poor and mental retardation as well as other complications are of similar severity as in children diagnosed clinically without screening. In many investigations it was also proved that some impairments developed already in the prenatal period. Therefore, many countries among them also Poland, stopped mass screening for galactosemia. At present, in Poland the procedure strategy in galactosemic children and their families include: diagnosis of new cases on the basis of clinical symptoms, selective screening in high-risk families, prophylactic lactose-free diet for mothers during pregnancy. Such management can help to prevent clinical manifestations in newborns and prevent death in the early period of life.

[Early diagnosis of inborn errors of metabolism. New technologies].

Inborn errors of metabolism, in spite of their not very high incidence play more and more evident role in the causes of infant's mortality and morbidity. Therefore early diagnosis and early treatment becomes an important issue in contemporary medicine. The criteria for newborn mass screening, selective screening and supplementary screening as well as the new technologies, among others mass spectrometry, Tandem MS, were discussed.

[Evaluation of the usefulness for neonatal mass screening in light of 35 years personal experience]. / Ocena przydatnosci badan przesiewowych u noworodków w swietle 35 lat doswiadczen wlasnych.

The results and the significance of neonatal mass-screening programmes for inborn errors of metabolism, conducted by the National Research Institute of Mother and Child (NRIMC), are discussed. As the first in Poland, in 1964, mass-screening for phenylketonuria (PKU) was introduced. The BIA-Guthrie test was used. Other Guthrie tests (GBIA) were applied in homocystinuria, tyrosinemia, histidinemia and leucinosis (Maple Syrup Urine Disease-MSUD). In the middle of the 60. the Beutler and Baluda test was introduced for galactosaemia, as well as the Efron urine test in infant screening for different inborn errors of metabolism. In the middle of the 70., neonatal mass-screening for cystic fibrosis (CF, mucoviscidosis) was started. Meconium tests and the sweat test with ion selective chloride electrode were used. Apart from inborn errors of metabolism, we also introduced a screening programme for neuroblastoma in which vaniline mandelic acid (VMA) in urine was estimated and for congenital hypothyroidism were TSH level was assessed. The results of screening are shown in the tables and in the figures. In our opinion the best clinical results are obtained with screening for congenital hypothyroidism and for PKU, since very early detection and treatment in these diseases prevents severe mental retardation. We therefore consider that both these screening programmes should be treated as obligatory examinations in all neonates. Taking into consideration the fact that there are different types of hyperhenylalaninemias, the principles of differential diagnosis are discussed. Molecular genetic investigations, carried out in the NRIMC Department of Genetics proved to be a very important procedure in the verification of diagnosis of different mutations. The authors also discuss the problem of dietary treatment duration in PKU. In our opinion the hypophenyloalanine diet regimen in girls, should not be discontinued during adolescence, since there is the problem of maternal PKU and the possibility of foetal damage. The results of our own investigations of maternal PKU are discussed. The significance of mass-screening for galactosemia is still under discussion. In our opinion, mass-screening for galactosemia is not useful and we have discontinued it. Selective screening has been started combined with molecular genetic studies in high risk families. In the future, we plan to prepare guidelines on the principles of diagnosis and treatment of galactosemia in children and women in the reproductive age. Mass-screening for cystic fibrosis is also still under discussion. The results of the early screening programmes were not satisfactory and the tests were discontinued. In 1998, after reorganisation of the whole system, CF screening, using tripsin-radioimmune assays, was started again. The new screening programme is combined with molecular genetic investigation of different mutations. It is still too early to assess the importance and success of this CF mass-screening programme. We decided to discontinue the screening for homocystinuria, histidinemia, tyrosinemia, leucinosis and for neuroblastoma, since these programmes did not comply with criteria of mass-screening. In 1997, major reorganisation of screening programmes for inborn errors of metabolism, at NRIMC, was undertaken. The Guthrie test for PKU was changed to a quantitative colorimetric method. The immuno-luminometric method is used for TSH estimation. The whole system is based on complete computer control of all the steps of screening, from blood sampling on filter paper until the final diagnosis. The advantages of this modern system of organisation of the screening programme are discussed.

Preliminary results of UDP galactose pyrophosphorylase in the erythrocytes of healthy persons and in patients with galactosaemia.

Direct chromatographic isolation of UDP galactose and galactose-1-phosphate was used for determination of the activity of UDP galactose pyrophosphorylase in erythrocytes. The activity was determined by measuring the amount of UDP galactose produced from galactose-1-phosphate and uridine triphosphate. In homozygotes with galactosaemia the activity of the enzyme was nearly ten times lower than in controls, so this difference was highly significant statistically (p less than or equal to 0.001) and the respective values were 0.0051 +/- 0.0003 and 0.0418 +/- 0.0038 mumol of UDP galactose formed during 1 hour by 1 ml of erythrocytes (300 mg of haemoglobin). In heterozygotes with galactosaemia the activity of the enzyme had intermediate values between those in homozygotes and healthy controls.