Complete genomic structure and mutational spectrum of PHKA2 in patients with x-linked liver glycogenosis type I and II.

X-linked liver glycogenosis (XLG) is probably the most frequent glycogen-storage disease. XLG can be divided into two subtypes: XLG I, with a deficiency in phosphorylase kinase (PHK) activity in peripheral blood cells and liver; and XLG II, with normal in vitro PHK activity in peripheral blood cells and with variable activity in liver. Both types of XLG are caused by mutations in the same gene, PHKA2, that encodes the regulatory alpha subunit of PHK. To facilitate mutation analysis in PHKA2, we determined its genomic structure. The gene consists of 33 exons, spanning >/=65 kb. By SSCP analysis of the different PHKA2 exons, we identified five new XLG I mutations, one new XLG II mutation, and one mutation present in both a patient with XLG I and a patient with XLG II, bringing the total to 19 XLG I and 12 XLG II mutations. Most XLG I mutations probably lead to truncation or disruption of the PHKA2 protein. In contrast, all XLG II mutations are missense mutations or small in-frame deletions and insertions. These results suggest that the biochemical differences between XLG I and XLG II might be due to the different nature of the disease-causing mutations in PHKA2. XLG I mutations may lead to absence of the alpha subunit, which causes an unstable PHK holoenzyme and deficient enzyme activity, whereas XLG II mutations may lead to in vivo deregulation of PHK, which might be difficult to demonstrate in vitro.

Maternal mild hyperphenylalaninaemia: an international survey of offspring outcome.

Maternal phenylketonuria (PKU) has adverse effects on the offspring including microcephaly, mental retardation, congenital heart disease, and intrauterine growth retardation. Maternal non-PKU mild hyperphenylalaninaemia (MHP) is believed to be benign, but whether there may be long-term consequences to offspring is unclear. In an international survey we have obtained information about 86 mothers with MHP (blood phenylalanine 167-715 mumol/L), their 219 untreated pregnancies, and 173 offspring. Spontaneous fetal loss (13% of pregnancies), congenital heart disease (2.3% of offspring), and severe non-cardiac anomalies (2.9% of offspring) occurred at frequencies within expected limits for the general population. For weight and length at birth the median percentile was the 50th but that for birth head circumference was the 25th. Median z-scores for birth length and head circumference were significantly lower for offspring of mothers with phenylalanine concentrations above 400 mumol/L than for those whose mothers had lower values (p = 0.05 and p = 0.005, respectively). The median intelligence quotient (IQ) of the offspring (3-27 years) was 100 for those whose mothers had higher phenylalanine concentrations and 108 for those of the lower phenylalaninaemia group. However, offspring IQ correlated slightly more closely with maternal IQ (r = 0.53, p < 0.001) than with maternal phenylalanine concentration (r = 0.45, p = 0.02). Maternal MHP does not seem to have serious consequences for the fetus. A maternal phenylalanine concentration of less than 400 mumol/L does not warrant intervention. Nevertheless, maternal blood phenylalanine above this value is associated with slightly lower birth measurements and offspring IQ than lower maternal blood phenylalanine concentrations.

X-linked liver glycogenosis: localization and isolation of a candidate gene.

X-linked phosphorylase kinase (PHK) deficiency causes X-linked liver glycogenosis (XLG) which is the most frequent liver glycogen storage disorder in man. Recently we assigned XLG to the Xp22 chromosomal region by linkage analysis in two families segregating XLG. In this study a further localization of XLG in Xp22 was performed by extending the number of Xp22 markers, by extension of the number of family members from the two families of our previous study and by linkage analysis in four additional XLG families. Two-point linkage analysis revealed lod scores of 4.60, 5.73, 5.28, 8.62 and 5.14 for linkage between XLG and the DNA markers pXUT23 and pSE3.2-L(DXS16), pD2(DXS43), pTS247-(DXS197) and pPA4B(DXS207), respectively, all at 0% recombination. Linkage heterogeneity was not observed in this set of families. Multipoint linkage analysis increased the lod score for linkage between XLG and Xp22 to 16.79 relative to DXS197/DXS207. The position of the XLG gene was confirmed by analysis of recombinational events locating the XLG gene between DXS85 and DXS41. The XLG gene could not be mapped more precisely in this chromosomal region of approximately 20cM because of the absence of recombinational events between the XLG gene and the Xp22 markers. As we have previously shown that the rabbit liver alpha subunit of PHK (PHKA2) hybridizes to human Xp22, we isolated a human PHKA2 cDNA from a human hepatoma lambda gt11 cDNA library. Fluorescent in situ hybridization mapped human PHKA2 to Xp22. As this physical mapping coincides with the genetic mapping of XLG by linkage analysis, PHKA2 most probably harbours the mutation(s) responsible for XLG.

Geleophysic dysplasia.

We describe 2 children with geleophasic dysplasia. Prominent cardiac disease in one of the patients caused death at an early age. The history of consanguinity in one of the families supports autosomal recessive mode of inheritance. Histological and ultrastructural changes suggest that a disturbance in the relations between cell membrane and extracellular matrix may be involved in the pathogenesis.

Cutaneous presentation of sarcoidosis in an infant.

A 4-month-old infant developed a symptomless erythematous maculopapular rash on her abdomen spreading to involve other areas of her trunk and limbs. Skin biopsy showed the features of sarcoidosis and she later developed sarcoid uveitis and arthritis requiring systemic steroid therapy.

Alpha- and beta-mannosidoses.

Clinical, pathological and biochemical findings in the mannosidoses are described. Family studies showed granulocyte-rich white cell fractions to be the tissue of choice for carrier detection in beta-mannosidosis. Metabolic labelling studies using [3H] mannose demonstrated accumulation of Man beta 1-4GlcNAc in cultured skin fibroblasts from a patient with this condition. Alternative methods of egress from lysosomes were suggested for this compound by its secretion into culture medium and apparent reduction of storage with time in cultures. beta-mannosidase deficient goats are not thought to be a true animal model of the human condition, as although they showed a similar enzyme deficiency, the clinical presentation is much more severe and the major storage material (Man beta 1-4GlcNAc beta 1-4GlcNAc) is different.

Chorionic villus sampling: diagnostic uses and limitations of enzyme assays.

Control ranges for enzymes in uncultured chorionic villi were established, based on: (1) 21 of 22 enzymes (mainly lysosomal) in villi had similar properties to the enzyme in cultured fibroblasts; (2) isoenzyme patterns in villi were similar to those in fibroblasts for five lysosomal enzymes but different for aryl sulphatases; (3) control ranges were determined for 12 enzymes in abortion villi and for 21 enzymes in biopsy villi, values tending to be higher in the latter for those enzymes studied in both types of sample; (4) storage of samples under various conditions revealed no major changes in activity of seven lysosomal enzymes. A number of potential pitfalls in the use of chorionic villus samples for diagnosis of metabolic disorders by enzyme assay are described: (1) the presence of aryl sulphatase C in chorionic villi, an isoenzyme which may interfere in assays of aryl sulphatase A; (2) the presence of maternal enzyme in chorionic villus material illustrated by the detection of the A isoenzyme of B-hexosaminidase in chorionic villus from a pregnancy affected with Sandhoff's disease; (3) the finding of falsely normal levels of alpha-iduronidase in chorionic villus samples from a pregnancy affected with Hurler's disease, probably due to contamination with maternal tissue which has relatively high levels of this enzyme compared with fetal chorionic material: (4) the inadequacy of indirect assays of incorporation of radiolabel into macromolecules using chorionic villi, for example [14C]propionate incorporation for prenatal diagnosis of methylmalonic aciduria. Provided that such pitfalls are recognized and great care is taken in selection of villus samples and interpretation of results, chorionic villus sampling allows reliable prenatal diagnosis of a large number of disorders using enzyme assays.

A clinician's view of the mass screening of the newborn for inherited diseases: current practice and future considerations.

The case for or against mass screening for inherited diseases is discussed. There is universal acceptance for mass screening for phenylketonuria and congenital hypothyroidism. The case for mass screening for galactosaemia and for maple syrup urine disease is not very strong; they could be considered under the heading of 'urgent screening of the sick newborn'. It is difficult to find good arguments for mass screening for congenital adrenal hyperplasia. For screening for glucose-6-phosphate dehydrogenase deficiency and sickle cell disease, the established criteria for mass screening do not apply. A simple tool for early detection is now available and the population afflicted with a mutant gene which causes major health problems should receive special attention from its government. It is too early to offer any comment about cystic fibrosis screening; further developments must be awaited.

First trimester diagnosis of methylmalonic aciduria.

We have studied methylmalonyl CoA mutase activity in control chorionic villi to establish the potential use of assays performed directly on this tissue for prenatal diagnosis of methylmalonic aciduria. We report the detection of a fetus affected with the apo-mutase deficient form of this condition at 9 weeks' gestation. Methylmalonyl CoA mutase was markedly deficient in chorionic villi, approximately 2.5 per cent of the mean control value. However, incorporation of label from [14C]-propionate into protein was 10 and 40 per cent of the mean control value, respectively, in two portions of the same biopsy, highlighting potential problems in the use of this indirect assay. Normal results were obtained in chorionic villus samples from four other pregnancies 'at risk' for methylmalonic aciduria which were subsequently shown to be unaffected with this condition. The diagnosis in the affected pregnancy was confirmed by demonstration of a marked deficiency of methylmalonyl CoA mutase activity in villi obtained at termination and in cultured fetal fibroblasts. Reduced incorporation of [14C]-propionate label into protein was also found in these tissues.

First trimester prenatal diagnosis of Sandhoff's disease.

Chorionic villus sampling was performed on two patients with a previous family history of Sandhoff's disease. Total beta-hexosaminidase (Hex) activity in case 1 was within the normal range (case 1: 6365 mumol/h/g protein; control range: 3227-24 495 mumol/h/g protein). The beta-hexosaminidase isoenzyme pattern was found to be normal. These results were confirmed on cultured amniotic fluid cells. In case 2, the total Hex activity was 672 mumol/h/g protein, i.e., 7 per cent of the control mean (10,085 mumol/h/g protein), and chromatography demonstrated that more than 50 per cent of this activity was due to the abnormal isoenzyme beta-hexosaminidase S (Hex S). The fetus was predicted to be affected by Sandhoff's disease and this was confirmed on fetal tissues after termination of pregnancy. This study demonstrates that a fetus affected by Sandhoff's disease can be reliably diagnosed during the first trimester of pregnancy.

Human beta-mannosidase deficiency: biochemical findings in plasma, fibroblasts, white cells and urine.

Marked deficiencies of beta-mannosidase activity were demonstrated in plasma, leukocytes, fibroblasts and urine of a patient with beta-mannosidosis, similar deficiencies were observed in the proband's sibling. All other lysosomal enzymes measured, including sulphamidase, exhibited normal activity. Both parents showed reduced plasma and leukocyte beta-mannosidase activity. Urinary glycosaminoglycan excretion was normal but TLC of urinary oligosaccharides revealed an abnormal band with the mobility of a disaccharide. This finding was confirmed by Bio-Gel P2 column chromatography. Further purification of this compound revealed two disaccharides, both of which yielded mannose and glucosamine following acid hydrolysis and mannose and N-acetylglucosamine following enzymic digestion. These two compounds are thought to be structural isomers of the disaccharide Man beta-GlcNAc.

Increased excretion of propan-1,3-diol and 3-hydroxypropionic acid apparently caused by abnormal bacterial metabolism in the gut.

Three patients who died in infancy showed an unusual urinary organic acid pattern with excessive excretion of 3-hydroxypropionic acid but none of the other metabolites normally associated with propionyl-CoA carboxylase deficiency. Propan-1,3-diol was present in the urine in all three cases. In the two patients examined propionyl-CoA carboxylase activity was not deficient in cultured skin fibroblasts. A fourth patient, also severely ill, showed similar urinary abnormalities. Feeding a medium-chain triglyceride-rich diet to this patient increased the ratio of 3-hydroxypropionic acid to propan-1,3-diol and resulted also in the appearance of malonic acid in the urine. These abnormal metabolites disappeared on the administration of neomycin and presumably were produced by gut bacteria.

Bone marrow transplantation in the treatment of alpha-mannosidosis.

Bone marrow transplantation was performed in a patient with alpha-mannosidosis. To our knowledge this is the first time such treatment has been attempted. The patient died 18 weeks after successful grafting and specimens of tissues were obtained at necropsy. Alpha-mannosidase activity in spleen and liver was just below normal (spleen 102 mumol/g/hour, control 113-330; liver 29 mumol/g/hour, control 30-131). Splenic alpha-mannosidase activity was indistinguishable from the control enzyme with respect to the Michaelis constant, heat stability, and inhibition by cobalt ions, as was 86% of the liver enzyme. In brain tissue alpha-mannosidase activity was 7% of controls, and less than one third had the properties of the normal enzyme. Oligosaccharides were present only in small amounts in liver and spleen, whereas they were greatly increased in brain tissue. Electron microscopic pictures of liver and spleen tissue showed normal morphology, but brain tissue showed definite vacuolation. These findings suggest that transplantation reversed the somatic changes of alpha-mannosidosis but did not affect lysosomal storage within brain tissue. It is concluded that marrow transplantation may not be a suitable treatment for alpha-mannosidosis.

Krabbe's disease: first trimester diagnosis confirmed on cultured amniotic fluid cells and fetal tissues.

Chorionic villi obtained during the first trimester from a pregnancy at risk for Krabbe's disease were shown to have reduced cerebroside-beta-galactosidase (E.C.3.2.1.46) activity using the artificial substrate trinitrophenylaminolauryl galactocerebroside (TNPAL-galactocerebroside). Assay of this enzyme in cultured amniotic fluid cells following amniocentesis, performed at the patient's request confirmed the diagnosis. Termination of pregnancy was performed and subsequent enzyme studies of the fetal tissues were consistent with the diagnosis of Krabbe's disease, thus confirming that chorionic villi can be used for first trimester diagnosis of this condition.