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.

Biochemical monitoring of treatment for galactosaemia: biological variability in metabolite concentrations.

Red cell galactose 1-phosphate (Gal-1-P) concentrations and urinary galactitol excretion have been suggested as biochemical indices of dietary compliance in classical transferase-deficient galactosaemia. We report our experience of measuring both in 32 patients over 0-10.9 years (median 3.45). A total of 438 blood specimens for Gal-1-P and 383 urine specimens for galacitol assay were received; 317 pairs of specimens were collected at the same time. Concentrations of both analytes fell rapidly over the first 2-3 months following dietary intervention, to mean (geometric SD) levels of 225 (1.60) mumol/L red cells for Gal-1-P and 388 (1.19) mumol/mmol creatinine for galactitol. Concentrations then fell exponentially over the next 7-8 years, with times to half-disappearance of 6.3 years for Gal-1-P and 6.4 years for galactitol, to levels of 104 (1.58) and 193 (1.36) respectively in patients aged over 10 years. Concentrations of both analytes were independent of the presence of the common Q188R mutation. Mean intra- and inter-individual coefficients of variation (CV) across the range of values studied were 36% and 61% for Gal-1-P, and 37% and 42% for galactitol. Analytical CVs were 3.6% for Gal-1-P and 5.5% for galactitol, indicating that the major source of variability is biological. The correlation coefficient between Gal-1-P and galactitol in paired samples overall was 0.33; the regression equation being [Galactitol] = 0.84[Gal-1-P] + 176. Serial measurements of both Gal-1-P and galactitol may be valuable in monitoring galactosaemia, but high intra-individual biological variability limits their usefulness. Standardization of sample collection times may improve this. Further work is needed to assess the predictive values of both analytes for long-term outcome.

Generalised uridine diphosphate galactose-4-epimerase deficiency.

The generalised form of epimerase deficiency galactosaemia has been described in only two children from unrelated families. Their progress is reported and three other affected children from these families are described. The initial presentation was similar to classic galactosaemia. Despite treatment all have shown poor growth and moderate learning difficulties. Three have sensorineural deafness and four have pronounced dysmorphic features. The two older female patients have normal pubertal development.

Prenatal diagnosis of galactosemia.

The experience from three different European centres with the prenatal diagnosis of galactose-1-phosphate-uridyltransferase (GALT) deficiency is presented and the question whether or not there is a need for prenatal diagnosis of this disorder is discussed. Most prenatal diagnoses (n = 50) have been performed by assay of GALT activity in cultured amniotic fluid cells. The assay used is reliable and clearly distinguishes homozygous affected fetuses (n = 11; 0%-2.3% of mean control enzyme activity) from non-(homozygous)-affected fetuses. The GALT assay for cultured amniocytes was adapted to assay the enzyme directly in chorionic villi. The experience with chorionic villi comprises 23 cases with 5 affected fetuses (0%-4.2% of mean control enzyme activity). In 36 cases galactitol was determined in amniotic fluid supernatant by gas chromatography-mass spectrometry. This method also differentiated affected (n = 11; galactitol 5.9-10.6 mumol/l) and unaffected pregnancies (galactitol 0.23-1.6 mumol/l) clearly and has the advantage of providing a result within a day or two after amniocentesis. Prenatal diagnosis of galactosemia is undertaken rarely and sometimes for the wrong reasons, but it should perhaps be considered more seriously until better methods of treatment are established.

Effects of galactosemia in utero.

There is direct evidence that in galactosemia, due to galactose-1-phosphate uridyltransferase deficiency, galactose, galactose-1-phosphate and galactitol accumulate in the fetus by week 20 of gestation. The metabolic abnormality may develop earlier than this, however, since the key enzymes in galactose metabolism have been shown to be present in normal fetal liver from the 10th week of gestation. There has been a single report of increased galactitol in amniotic fluid obtained at 10 weeks gestation, the outcome being a baby affected with galactosemia. Cataract formation in the fetus is rare and the only direct evidence that galactosemia may have harmful effects in utero. However, it has been concluded that the liver pathology seen in some patients who died in the neonatal period originated prenatally, and some studies have found that galactosemia is associated with reduced birth weight. Reports of two patients with histologically normal ovaries very soon after birth have been cited as evidence against gonadal dysfunction arising during fetal life, but it should be noted that this is not a constant feature in female galactosemics. Other observations, particularly those made from animal models, would suggest that ovarian dysfunction is most likely to have been caused in utero.

UDP-glucose and UDP-galactose concentrations in cultured skin fibroblasts of patients with classical galactosaemia.

A very clear-cut reduction in UDP-galactose (UDPGal) levels in erythrocytes, skin fibroblasts and liver of patients with classical galactosaemia has been reported. As UDPGal is the galactosyl donor in glycoprotein and glycolipid synthesis, it has been suggested that an abnormality in these complex compounds may be the cause of some of the long-term complications of the disease. More recent work on erythrocytes, employing mainly HPLC rather than the enzyme methods used to measure UDPGal originally, casts doubt on the hypothesis because, although some reduction was still found, there was a large overlap between galactosaemic and normal distributions. We have reproduced the experiments on cultured skin fibroblasts at confluency, but measuring UDPGal and UDP-glucose (UDPGlc) by HPLC. There was no reduction in UDPGal levels in galactosaemic compared to control cell lines. The existence of a biologically significant depletion of UDPGal in galactosaemia remains in doubt.

The investigation of UDPGlucose and UDPGalactose concentration in red blood cells of patients with classical galactosaemia.

UDPGlucose (UDPGlc) and UDPGalactose (UDPGal) are nucleotide sugars formed via the galactose metabolic pathway and are essential cofactors for the incorporation of galactose and glucose into complex glycoproteins and glycolipids. It has been proposed that in classical galactosaemia, where the enzyme galactose-1-phosphate uridyl transferase is deficient, the reaction product UDPGal is reduced leading to the long-term complications associated with the disease. We have measured the concentration of UDPGal and UDPGlc in red blood cells by high performance liquid chromatography (HPLC) in 16 children and 15 adult galactosaemics and compared the results with 30 and 27 control children and adults, respectively. The results indicate that UDPGal levels were found to be significantly reduced in galactosaemic patients and UDPGlc/UDPGal ratios significantly increased.

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.

Galactosaemia: results of the British Paediatric Surveillance Unit Study, 1988-90.

Ascertainment of cases of classical galactosaemia over a three year period in the UK and the Republic of Ireland, through the British Paediatric Surveillance Unit, identified 58 proved cases and two in whom the diagnosis was strongly suspected. One patient died at 4 days, and severe morbidity was no more frequent in babies diagnosed clinically because of their symptoms compared with those who were screened for galactosaemia. Treatment of four babies in the non-screened group was delayed until after 5 weeks of age, but it is concluded that all cases of galactosaemia could be diagnosed in an acceptable time without screening, providing clinical vigilance is maintained.

Inherited metabolic diseases in the sudden infant death syndrome.

All sudden, unexpected infant deaths presenting during a two year period within a defined geographical area in Avon and north Somerset were investigated for inherited metabolic disease. Of 95 deaths, 88 were classified as cases of sudden infant death syndrome (SIDS). In addition to the normal postmortem investigations, samples of cerebrospinal fluid, urine, vitreous humour, and skin were collected for metabolic studies. No abnormal organic acid metabolites were found in the fluids from the 88 cases of SIDS. Fatty acid oxidation was assessed in skin fibroblasts from 70 cases of SIDS, but no examples of medium chain acyl CoA dehydrogenase (MCAD) deficiency were found. One case with abundant glycogen in the liver was subsequently diagnosed as having glycogen storage disease type 1c. These findings suggest that the incidence of MCAD deficiency and other metabolic diseases in SIDS is much lower than previously claimed.

Molecular heterogeneity at the phenylalanine hydroxylase locus in the population of the south-west of England.

The phenylalanine hydroxylase gene locus has been studied in 35 independent phenylketonuric families in the south-west of England using RFLP haplotype patterns and allele specific oligonucleotide probes. Haplotype 3 was the most common pattern on mutant chromosomes and there was strict linkage disequilibrium between this haplotype and the splice mutation in exon 12. The R408W mutation in exon 12 occurred on both haplotypes 1 and 2. The R126Q mutation in exon 7 was found only on a rare haplotype 28 pattern. No gene carried the R158Q mutation. More than 60% of mutant genes did not carry these four mutations which were originally described in other European populations. We suggest that the splice mutation arose as a single event and spread throughout northern Europe by population migration and admixture. In addition, we believe the haplotype/mutation associations seen in our population are a reflection of the mixed ancestry of the inhabitants of the British Isles.

Genetic analysis of treated and untreated phenylketonuria in one family.

We describe a family in which four subjects in two generations have a disorder of phenylalanine metabolism. Two first cousins had different biochemical presentations in the neonatal period. The older child was thought to have a more severe form of phenylketonuria (PKU), and the younger child a milder form. While carrying out family studies we discovered that their mutual grandfather and his older unmarried brother, both of normal intelligence, had a marked and previously undiagnosed hyperphenylalaninaemia. DNA analysis using RFLP haplotypes has shown that there are four independent mutant PKU alleles in this family which are found on three haplotype patterns. None of the affected family members carries a previously defined mutation at the phenylalanine hydroxylase (PAH) locus and so DNA analysis was not able to explain the apparently different biochemical phenotypes in the affected members of this family.