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.

Mutational spectrum of classical galactosaemia in Spain and Portugal.

Classical galactosaemia is an autosomal recessive inherited metabolic disorder due to deficient galactose-1-phosphate uridyltransferase (GALT). Over 180 different base changes and disease-causing mutations have been reported in the GALT gene. Mutation p.Q188R was found to be the most common molecular defect among caucasian classical galactosaemia patients. We have characterized the spectrum of GALT mutations in a group of 51 Spanish families and 32 Portuguese families with this disease. p.Q188R is also the most prevalent mutation in the Spanish and Portuguese population, accounting for 50% and 57.8% of galactosaemic alleles, respectively. An additional 15 mutations were also identified in Spanish patients, four of which were novel: p.D28H, p.S181A, c.658dupG and c.377+53_1059+87del. In the Portuguese population, 11 different mutations were found, three of which were novel: p.R33H, p.P185S, and p.S192G. The differences observed between the genotypes identified in Portuguese and Spanish galactosaemic populations are notable. Only mutations p.Q188R, p.R148Q and c.820+13g>a were identified in both populations. In spite of the geographical proximity of Spain and Portugal, it seems that they have received genetic influences from different populations. The repeated migrations that occurred in the Iberian Peninsula throughout centuries may explain such variability.

A new human genetic resource: a DNA bank established as part of the Avon longitudinal study of pregnancy and childhood (ALSPAC).

We describe a unique human DNA resource forming part of the Avon Longitudinal Study of Pregnancy and Childhood (ALSPAC), a longitudinal cohort study involving 14 000 children and their families living in a geographically defined area of England. The DNA bank will underpin the search for associations between genetic polymorphisms and common health outcomes. The opportunities to collect blood samples suitable for DNA extraction are necessarily limited, and the samples themselves have often been treated in different ways and have varied storage histories. With the need to maximise yields, the choice of DNA extraction method is critical to the success of the bank and we have investigated the suitability of various commercial and in-house methods of DNA extraction. Various steps have been taken to minimise errors in sample address and identification, including the use of a pipetting robot for dilution and transfer of samples between 96-well arrays to provide aliquots suitable for PCR. The robot has been programmed to cope with concentrated viscous DNA solutions.

The relationship of genotype to cognitive outcome in galactosaemia.

AIMS: To evaluate the cognitive outcome of a cohort of children with galactosaemia in relation to genotype. METHODS: The cohort was drawn from children notified to the British Paediatric Surveillance Unit galactosaemia study which ran from 1988 to 1990. Cognitive outcome was assessed using the Wechsler Intelligence Scale for Children or the Wechsler Preschool and Primary Scale of Intelligence. Parents completed a questionnaire detailing educational status, and the attending paediatrician returned a questionnaire regarding age at diagnosis and biochemical outcome over the previous two years. RESULTS: A total of 45 children were genotyped: 30 were homoallelic for the Q188R mutation, the remainder being heteroallelic for Q188R with K285N (n = 4), L195P (n = 4), or other mutations (n = 7). Psychometric evaluation was available in 34 cases: mean full scale IQ was 79, verbal quotient 79, and performance quotient 82. Genotype was not related to galactose-1-phosphate (Gal-1-P) concentrations. However, children homoallelic for the Q188R mutation had significantly lower IQ scores than those who were heteroallelic (73. 6 v 94.8). This difference was independent of social and demographic influences and Gal-1-P concentrations over the previous two years. CONCLUSIONS: In children with galactosaemia, cognitive outcome appears to relate to genotype rather than metabolic control, as reflected by Gal-1-P concentrations. The value of measuring Gal-1-P concentrations routinely once successfully established on a galactosaemia diet is questionable as concentrations do not appear to affect outcome. In the UK population, homozygosity for the Q188R mutation is invariably associated with a poor outcome, and there is evidence that variability in neurocognitive outcome is at least part dependent on allelic heterogeneity.

Galactosaemia and allelic variation at the galactose-1-phosphate uridyltransferase gene: a complex relationship between genotype and phenotype.

UNLABELLED: More than 160 different base changes have been described at the galactose-1-phosphate uridyltransferase gene and most of these are associated with a disease phenotype. Q188R is the most common mutation in north European populations and those predominantly of European descent. K285N is much rarer but in some countries of east/central Europe it is the second most common mutation. In some populations of northern Europe these two mutations can be found on 70%-80% of mutant chromosomes. Both mutations appear to be associated with a complete loss in enzyme activity and thus, a more severe biochemical phenotype. A single amino acid substitution, N314D, is found on both Duarte 1 and Duarte 2 alleles. Additional base changes that are different on each distinguish D1 from D2 alleles. Whether the differences in galactose-1-phosphate uridyltransferase enzyme activities are associated with the additional molecular changes that distinguish D1 and D2 alleles remains unclear. S135L is found almost exclusively in galactosaemic individuals of African origin. Despite early diagnosis and treatment and adherence to a lactose free diet neurological complications, poor growth and reduced fertility are frequently observed in affected individuals. CONCLUSION: Allelic variation at the galactose-1-phosphate uridyltransferase gene undoubtedly plays a role in defining the biochemical and clinical phenotype. However, clinical galactosaemia is a complex trait in which multiple developmental and metabolic pathways are involved. Ultimately the phenotype is beyond the control of the single gene itself.

Classical galactosemia and mutations at the galactose-1-phosphate uridyl transferase (GALT) gene.

Classical galactosemia is caused by a deficiency in activity of the enzyme galactose-1-phosphate uridyl transferase (GALT), which, in turn, is caused by mutations at the GALT gene. The disorder exhibits considerable allelic heterogeneity and, at the end of 1998, more than 150 different base changes were recorded in 24 different populations and ethnic groups in 15 countries worldwide. The mutations most frequently cited are Q188R, K285N, S135L, and N314D. Q188R is the most common mutation in European populations or in those predominantly of European descent. Overall, it accounts for 60-70% of mutant chromosomes, but there are significant differences in its relative frequency in individual populations. Individuals homoallelic for Q188R tend to have a severe phenotype and this is in keeping with the virtually complete loss of enzyme activity observed in in vitro expression systems. Globally, K285N is rarer, but in many European populations it can be found on 25-40% of mutant chromosomes. It is invariably associated with a severe phenotype. S135L is found almost exclusively in African Americans. In vitro expression results are discrepant, but some individuals carrying S135L appear to exhibit GALT activity in some tissues. Duarte 1 (or Los Angeles) and Duarte 2 (or Duarte) variants carry the same amino acid substitution, N314D, even though D1 is associated with increased erythrocyte GALT activity and D2 with reduced activity. N314D is in linkage disequilibrium with other base changes that differ on the D1 and D2 alleles. N314D does not impair GALT activity in in vitro expression systems. However, there are differences in the abundance of GALT protein in lymphoblastoid cells lines from D2 and D1 individuals. It is unclear whether the specific molecular changes that distinguish the D1 and D2 alleles account for the different activities. The considerable genetic heterogeneity documented to date undoubtedly contributes to the phenotypic heterogeneity that is observed in galactosemia. The additional effects of nonallelic variation and other constitutional factors on phenotypic variability remain to be elucidated.

Sequence variation at the phenylalanine hydroxylase gene in the British Isles.

Using mutation and haplotype analysis, we have examined the phenylalanine hydroxylase gene in the phenylketonuria populations of four geographical areas of the British Isles: the west of Scotland, southern Wales, and southwestern and southeastern England. The enormous genetic diversity of this locus within the British Isles is demonstrated in the large number of different mutations characterized and in the variety of genetic backgrounds on which individual mutations are found. Allele frequencies of the more common mutations exhibited significant nonrandom distribution in a north/south differentiation. Differences between the west of Scotland and southwestern England may be related to different events in the recent and past histories of their respective populations. Similarities between southern Wales and southeastern England are likely to reflect the heterogeneity that is seen in and around two large capital cities. Finally, comparison with more recently colonized areas of the world corroborates the genealogical origin by range expansion of several mutations.

Prediction of multiple hypermutable codons in the human PAH gene: codon 280 contains recurrent mutations in Quebec and other populations.

The predicted mutability profile (MUTPRED) of the phenylalanine hydroxylase (PAH) gene shows that the 48 CpG sites (template and atemplate strands) are either empty of known mutations (7 sites), harbour "PKU" alleles involving CpG doublets (16 sites), or contain mutations that do not involve a C-->T or G-->A substitution in the doublet. These hypermutable sites harbour 32 different mutations in association with at least 66 different haplotypes and hyperphenylalaninemia. The E280K mutation in exon 7 of the PAH gene is a cause of phenylketonuria. It occurs on four different haplotypes in Europeans and on haplotypes 1 and 2 in Quebec. Whereas a single recombination event could explain the two haplotype associations in Quebec, the mutation does involve a CpG dinucleotide. By analyzing multiallelic markers 5' (STR) and 3' (VNTR) to the E280K allele on 12 mutant and 30 normal chromosomes, we conclude that recurrent mutation is the likely origin of E280K in Quebec. The PAH mutation database shows that the allele accounts for 1.5% of PKU chromosomes worldwide.

Experimental design: a useful tool for PCR Optimization.

Because of complex interactions among the components of PCR and the wide and increasing variety of applications in which this technique is used, optimization is necessary for every reaction. Here we describe the use of experimental design techniques (2k fractional factorial design and central composite design) to attain easier, quicker and cheaper PCR optimization of DNA from blood spots. By determining the factors affecting the product yield first (factors screening), the quantity of template DNA needed for PCR and the Mg2+ concentration are easily optimized (factors optimization).

Discordant phenylketonuria phenotypes in one family: the relationship between genotype and clinical outcome is a function of multiple effects.

Four members spanning three generations of one family have phenylketonuria of varying degrees of severity. Two first cousins were screened in the neonatal period and have had dietary phenylalanine restriction since diagnosis, the older patient having been classified as having more severe PKU and the younger one as having mild PKU. Their mutual grandfather and his older brother also have a significant hyperphenylalaninaemia and are of normal intelligence despite never having had restricted phenylalanine intake. Mutation analysis of the phenylalanine hydroxylase (PAH) gene has established that there are four different mutations, two in exon 2 (F39L and L48S) and two in exon 3 (R111X and S67P), which give rise to PKU in this family. In order to establish their relative severity, we screened the PKU populations of western Scotland and the south west of England for these mutations. The exon 3 mutations are rare; however, F39L is relatively common in Scotland and L48S in England. A comparison of diagnostic blood phenylalanine concentrations in subjects carrying L48S/null or F39L/null mutations with those carrying two null mutations suggest that these exon 2 mutations are less deleterious. Thus, in this family, the different biochemical phenotypes can be explained, in part, by different genotypes at the PAH locus but our results show that the relationship between genotype and clinical outcome is more complex and is a function of multiple effects.

Recurrence of the R408W mutation in the phenylalanine hydroxylase locus in Europeans.

The relative frequency of the common phenylalanine hydroxylase (PAH) mutation R408W and its associations with polymorphic RFLP, VNTR, and short-tandem-repeat (STR) sites in the PAH gene were examined in many European populations and one representative North American population of defined European descent. This mutation was found to cluster in two regions: in northwest Europe among Irish and Scottish peoples, and in eastern Europe, including the Commonwealth of Independent States. This allele was significantly less frequent in intervening populations. In eastern European populations, the R408W mutation is strongly associated with RFLP haplotype 2, the three-copy VNTR allele (VNTR 3), and the 240-bp STR allele. In northwestern European populations, it is strongly associated with RFLP haplotype 1, the VNTR allele containing eight repeats (VNTR 8), and the 244-bp STR allele. An examination of the linkage between the R408W mutation and highly polymorphic RFLP, VNTR, and STR haplotypes suggests that recurrence is the most likely mechanism to account for the two different major haplotype associations of R408W in Europe.

Mutation analysis of the phenylalanine hydroxylase gene using heteroduplex analysis with synthetic DNA constructs.

Using heteroduplex analysis generated with synthetic PCR-amplifiable DNA we have screened the PKU populations of southwest England and Wales, western Scotland, and southeast and central England for mutations in exons 3, 7 and 12 of the phenylalanine hydroxylase (PAH) gene. The technique characterized three mutations in exon 12, two in exon 3 and five in exon 7. Altogether over 370 PKU chromosomes were screened. In all geographical regions exon 12 mutations (R408W, IVS12nt1g- > a and Y414C) accounted for about 40% of mutant chromosomes. Exon 3 mutations (principally I65T) were found on between 9 and 12% of mutant alleles and exon 7 mutations accounted for a further 5-7%. Heteroduplex analysis is rapid, simple and safe and three constructs covering three exons can identify between 55 and 60% of mutations in various PKU populations of the UK.

Evidence for origin, by recurrent mutation, of the phenylalanine hydroxylase R408W mutation on two haplotypes in European and Quebec populations.

The R408W mutation in the phenylalanine hydroxylase gene (PAH) of phenylketonuria patients occurs on haplotypes 2.3 and 1.8 in Europeans. The mutation involves a CpG dinucleotide; nonetheless, a single recombination event might also explain the two haplotype associations. By analysis of an STR in the PAH gene 5' to the 408 codon and of the VNTR system in the 3' UTR, we identified unique features of the haplotype 1.8 chromosome harbouring the R408W mutation which are not accounted for by recombination. We conclude that recurrent mutation is the origin of R408W on different PAH haplotypes in Europeans.

Pyridoxine-refractory congenital sideroblastic anaemia with evidence for autosomal inheritance: exclusion of linkage to ALAS2 at Xp11.21 by polymorphism analysis.

A son and daughter of unaffected parents had transfusion dependent, pyridoxine-refractory sideroblastic anaemia from birth. Their haemoglobin levels were 4.3 and 6.4 g/dl, respectively. delta-Aminolaevulinate synthase activity in erythroblasts from fractionated marrow of the sister was 135 pmol delta-aminolaevulinate formed/10(6) erythroblasts/hour (normal range = 110-650 pmol). While mutations of the erythroid-specific delta-aminolaevulinate synthase gene (ALAS2) at Xp11.21 have been reported in patients with X linked sideroblastic anaemia, sequence analysis of the ALAS2 gene in the son did not identify any mutations in the coding region, the intron/exon boundaries, or the 1 kb 5' promoter region. A useful polymorphism was found in the 3' region of the ALAS2 gene, a G to A transition, 220 nt 3' of the AATAAA polyadenylation signal. Mismatch PCR at this site and subsequent discrimination by XmnI restriction analysis of 148 alleles identified the gene frequency of this polymorphism to be 25%. Analysis of the inheritance of this intragenic polymorphism showed that the affected sibs received different maternal alleles at the ALAS2 locus, excluding mutations in this gene as the cause of their sideroblastic anaemia. Furthermore, the absence of a dimorphic erythrocyte population in the mother, coupled with the demonstration of random X inactivation in her peripheral leucocytes, showed that the mother was not the carrier of any X linked sideroblastic anaemia mutation. These results strongly suggest that the sideroblastic anaemia in this family is an autosomal recessive trait.

Biochemical control, genetic analysis and magnetic resonance imaging in patients with phenylketonuria.

Thirteen patients with phenylketonuria, detected by neonatal screening and started on diet within 16 days of age, were investigated between 10 and 18 years of age by magnetic resonance imaging (MRI) of the brain. Biochemical control was assessed from: (1) the life time blood phenylalanine (Phe) control (as determined from (a) the mean yearly exposure to Phe; (b) the accumulated time for each patient that Phe was < 120 mumol/l; (c) > 400 mumol/l; (d) > 800 mumol/l; and (e) > 1200 mumol/l); and (2) the blood Phe control over the 5 years prior to imaging (assessed for each patient by the mean yearly Phe exposure over that period). In all patients the phenylalanine hydroxylase gene locus was studied using restriction fragment length polymorphism haplotypes and mutant genes were screened for a variety of specific mutations which have been reported in other European populations or in populations of north European descent. Two patients had significant abnormalities of cerebral white matter. Although both showed poor biochemical control this did not reach statistical significance when compared to those with normal imaging. DNA haplotype patterns could be assigned to 11 patients and mutant genes were identified in 12. One patient with abnormal imaging and 4 patients without abnormalities had mutations on both chromosomes identified. In these 5 patients there was significant correlation between their genotype and biochemical control. Mutations resulting in residual in vitro enzyme activity were associated with normal imaging.