PMM2 mutation spectrum, including 10 novel mutations, in a large CDG type 1A family material with a focus on Scandinavian families.

Carbohydrate-deficient glycoprotein syndrome type IA (CDG IA) is an autosomal recessive disease characterized clinically by severe involvement of the central and peripheral nervous system, and biochemically by complex defects in carbohydrate residues in a number of serum glycoproteins. CDG IA is caused by mutations in the PMM2 gene located in chromosome region 16p13. In this study, 61 CDG type IA patients (122 chromosomes) were screened for mutations in the PMM2 gene using a combination of SSCP and sequence analysis. More than 95% of the mutations could be detected. All of them were missense mutations. Mutations 422G>A and 357C>A were strikingly more common in the material and comprised 58% of mutations detected. Of the 20 mutations found, 10 were not reported previously. Seven mutations, e.g. 26G>A (five alleles) and 548T>C (seven alleles), were found only in Scandinavian families. The most common genotype was 357C>A/422G>A (36%). Three patients were homozygous, 357C>A/357C>A (two cases), and 548T>C/548T>C (one case). No patients homozygous for the most common mutation 422G>A were detected. The different mutations were clustered e.g., in that most were located in exon 5 (five) and exon 8 (six), while no mutation was detected in exon 2. When the frequencies of each mutation were included, exon 5 comprised 61% (65 chromosomes) of the mutations; in Scandinavian patients the frequency of these mutations was 72%. Thus, analysis of exon five in these patients enables both reliable and time-saving first screening in prenatal diagnostic cases. This could be followed by a second step of additional strategies for the detection of other mutations.

Detailed mapping of the phosphomannomutase 2 (PMM2) gene and mutation detection enable improved analysis for Scandinavian CDG type I families.

The gene for carbohydrate-deficient glycoprotein syndrome type I (CDG1) has previously been localised by us close to marker D16S406 in chromosome region 16p13.2-3. We also presented data indicating a strong founder mutation associated with a specific haplotype in CDG I patients from western Scandinavia. The phosphomannomutase 2 (PMM2) gene was recently put forward as a likely CDG1 candidate gene. We have now shown that the specific haplotype is associated with the PMM2 mutation 357C > A. Using data from radiation hybrid panel we have refined the position of the PMM2 gene to very close to marker D16S3020 in the interval between D16S406 and AFM282ze1 on the distal side and D16S3087 on the proximal side. Due to the severity of the disease many families request prenatal diagnostic services for CDG I. In the meantime, until the mutation spectrum is fully examined, we propose the combined use of mutation analysis and linkage analysis with polymorphic markers as diagnostic tools for Scandinavian CDG I families requesting prenatal diagnosis. Using this strategy we have to date successfully performed 15 prenatal diagnoses for CDG I.

Genome-wide linkage analysis of Scandinavian affected sib-pairs supports presence of susceptibility loci for celiac disease on chromosomes 5 and 11.

Celiac disease (CD) is a common chronic inflammatory disorder of the small intestine with a multifactorial aetiology. HLA is a well-known risk factor, but other genetic factors also influence disease susceptibility. To identify the genes involved in this disorder, we performed a genome-wide scan on 106 well-defined Swedish and Norwegian families with at least two affected siblings. We investigated familial segregation of 398 microsatellite markers, and utilised non-parametric linkage analysis. The strongest linkage with disease was found to the HLA locus (6p) (P<0.000006). There were eight regions besides HLA with a point wise P value below 0.05. Among these eight regions were 11q and 5q, both of which have been suggested in several linkage studies of independent celiac disease families. We also performed a stratification analysis of families according to their HLA genotypes. This resulted in significant differences on chromosome 2q. These results indicate that 11q, 5q and possibly also 2q are true susceptibility regions in CD.

The decrease of IGF-I, IGF-binding protein-3 and bone alkaline phosphatase isoforms during gluten challenge correlates with small intestinal inflammation in children with coeliac disease.

OBJECTIVE: In children with coeliac disease, the ingestion of gluten causes small intestinal inflammation and a clinical picture of malabsorption, weight reduction and short stature. Decreased alkaline phosphatase (ALP) during gluten challenge was found in a previous study. ALP is a marker of bone formation and ALP activities are correlated with growth velocity. The aim of this study was to characterise the previously observed decrease of total ALP by investigating three specific bone ALP isoforms (bone/intestinal, B1 and B2) and three specific liver ALP isoforms (L1, L2 and L3) and, moreover, to correlate these ALP isoforms with other growth factors and growth markers. In addition, we also studied the association with possible weight changes, small intestinal mucosa inflammation, sex, age and gluten dose during gluten challenge. MATERIALS AND METHODS: Bone and liver ALP isoforms, IGF-I, IGF-binding protein (IGFBP)-3 and serum cross-linked carboxy-terminal telopeptide of type I collagen (ICTP) were measured together with change in weight and small intestinal mucosa histopathology in 54 children with earlier enteropathy who participated in a 4-week gluten challenge. RESULTS: We observed a decreased total ALP activity after 4 weeks of gluten challenge, 7.8+/-1.8 to 6.5+/-1.7 microkat/l (means +/- s.d.), which was mainly due to a reduction of the bone ALP isoforms. The sum of all three bone ALP isoforms decreased from 6.3+/-1.7 to 5.1+/-1.6 microkat/l. The decreased activities of the bone ALP isoforms correlated with the observed reductions of IGF-I (r=0.74, P<0.001), IGFBP-3 (r=0.51, P<0.001) and ICTP (r=0.57, P<0.001). The decrease of the growth factors and growth markers correlated with weight reduction, but when influences from the change in weight were adjusted for, the partial correlation of the small intestinal mucosa inflammation was significant for IGF-I (r=-0.56, P<0.001) and IGFBP-3 (r=-0.55, P<0.001). CONCLUSION: The decrease of total ALP was due to a reduction of bone ALP. The decrease of IGF-I and IGFBP-3 was independently correlated with weight change and small intestinal inflammation.

The influence of khat-chewing on birth-weight in full-term infants.

The leaves of the shrub Catha edulis (khat) are widely chewed as part of social life in several countries around the Red Sea and in East Africa. The leaves possess stimulant properties and are also used by pregnant women. The effect of khat on birth-weight has been studied, It was found that healthy full-term, singletons, born after uneventful pregnancies and deliveries, had a significantly lower average birth-weight when the mothers were khat-chewers, either habitually or occasionally (P less than 0.001). Khat-chewing appears to be one of several maternal practices adverse to the fetus.

Stunting and tissue depletion in Yemeni children.

With the aim of assessing whether stunting was associated with depletion of labile tissues such as fat and muscle as an indicator of ongoing malnutrition, we investigated 1176 children 0-7 years of age in PDR Yemen, who participated in a national nutrition survey conducted in 1982-3 and its pilot study from 1978. Arm circumference and triceps fatfold have been measured and upper arm fat area (UFA) and upper arm muscle area (UMA) were calculated to estimate the body stores of fat and muscle. Stunting, defined as a stature shorter than -2 SD of the reference mean, was found in one-third of the children. The average length/height for age deviated progressively from the reference mean up to age group 12-15 months. Exclusively breast-fed infants also deviated in length, although less conspicuously than infants fed in other ways. There was a consistent pattern of smaller UFA and UMA in stunted children compared to equally tall children who were not stunted below 84 cm of height for boys and 102 cm for girls. The difference was statistically significant in a few of the height groups. It is suggested that stunting is accompanied by a slight reduction of fat and muscle tissues during the first years of life.

Complex functional and structural coagulation abnormalities in the carbohydrate-deficient glycoprotein syndrome type I.

Carbohydrate-deficient glycoprotein (CDG) syndrome type I is an autosomal recessive disease with multisystemic manifestations. During childhood the patients may suffer from hemorrhages, which may be lethal, venous thromboses and stroke-like episodes. In this study 15 patients with CDG syndrome type I were examined from the levels and isoform patterns of coagulation factors and inhibitors and fibrinolysis parameters. The screening assays APTT and PTC were unaffected in most cases. In spite of this reduced levels were found particularly for factors II, V, X and XI and for antithrombin and protein C. Low values tended to be associated with elevated liver enzyme levels in serum. The values were at potential clinical risk levels for protein C and/or antithrombin in more than half of the patients, and for factor V and/or factor XI in one third of them. There were no current differences in values between patients who had previously displayed clinical symptoms of coagulation disturbance and those without such symptoms. Partially carbohydrate-deficient isoforms were demonstrated in antithrombin, protein C, protein S and in alpha 2-antiplasmin, but not in factors II, X and fibrinogen. Abnormal isoforms did not appear to reduce the functional activity of the respective glycoproteins. Analysis of individual hemostatic parameters is recommended in these patients in connection with clinical symptoms or elective surgery. The observed variability of the carbohydrate defect in glycoproteins in this disease may be a clue to its pathogenesis.

Denaturing high-performance liquid chromatography is a suitable method for PMM2 mutation screening in carbohydrate-deficient glycoprotein syndrome type IA patients.

The phosphomannomutase 2 gene (PMM2; MIM 601785) has been identified as the carbohydrate-deficient glycoprotein syndrome type 1A gene (CDGS type 1A; MIM 212065). The gene spans 8 exons and 741 bp of coding DNA. Previously, we have identified 20 different mutations in the PMM2 gene using mutation screening with single-stranded conformation polymorphism (SSCP) and sequencing of DNA from 61 CDGS type 1A patients. Because eight of these could not be detected by SSCP, we were not satisfied with the sensitivity of the mutation detection technique used. Thus, we wanted to investigate if denaturing high-performance liquid chromatography (DHPLC) was a more suitable mutation screening method for PMM2. DHPLC was set up for PMM2 by optimizing eight different PCR fragments, one for each exon. The mutation detection was optimized empirically with PCR fragments from controls. First, control samples were run at a universal gradient and after modification and shortening of the gradient, also run at 10 different temperatures, 50-70 degrees C with 2-degree intervals, to enable setting of the temperature with the highest resolution. Then, PCR products with known mutations from the previous study were analyzed, and the results were compared to the control chromatograms for aberrations. We detected 19/20 mutations with DHPLC, and several mutations not detected by earlier screening techniques were readily detected by DHPLC. We conclude that DHPLC is a suitable detection technique for a rapid and reliable first scan of CDGS type 1A patients.

Carbohydrate-deficient glycoprotein syndromes: peculiar group of new disorders.

A new group of metabolic disorders, the carbohydrate-deficient glycoprotein (CDG) syndromes, is reviewed with emphasis on the key condition, the CDG syndrome type I. This disease, an autosomal-recessive multisystem condition, has now been diagnosed in 45 Scandinavian patients. It is characterized by carbohydrate deficiencies of a number of glycoproteins, including uniform changes in transferrin. The transferrin alterations provide a distinct biologic marker and a practical and simple laboratory diagnostic means employing analysis of serum or blood spots from Guthrie-type filter paper. The syndrome presents differently through various life periods. A four-stage grouping system by age has been constructed and is presented. During infancy, internal organ symptoms are dominant; some may be life-threatening. In later childhood and adolescence, static mental deficiency, cerebellar ataxia, slowly progressive lower limb neuropathy, and pigmentary retinal degeneration, as well as secondary skeletal deformities, are the most prominent findings. Two very recently described clinical and biologic variants, CDG syndromes II and III, are summarized and compared to CDG type I.

A method for the determination of ethylenediamine in workroom air.

An ethylenediamine-air mixture was generated in a dynamic gas mixing apparatus, and three different sampling techniques were tested. The analysis was performed using isotachophoresis. Sampling in an impinger flask containing hydrochloric acid (20 mmol/l) gave a quantitative recovery. Desorption losses were noticed when silica gel adsorption tubes were used. Cellulose filter support pads impregnated with oxalic acid were laborious to prepare, and the recovery was high only when freshly prepared filters were used. The use of impingers was found to be the most satisfactory method, and it was used for air monitoring in two factories handling ethylenediamine.

Effect of khat on maternal food intake, maternal weight gain and fetal growth in the late-pregnant guinea pig.

Khat chewing is a widespread male social habit in countries around the southern shore of the Red Sea and in eastern Africa and is also practiced by women, even during pregnancy and lactation. In order to study the potentially adverse effects of khat chewing during pregnancy, guinea pigs were fed 2.2 g/kg of khat leaves daily throughout the third trimester. Control animals were given aspen leaves. Maternal daily food intake was significantly reduced during the first 10 days of feeding and maternal weight gain was slightly lower in the khat group. Khat feeding of the mother significantly reduced the mean birth weight of the offspring by 7% without any effect on litter size or length of gestational period. Since low birth weight is a well-established risk factor for both perinatal and young infant death, khat chewing during pregnancy may be one of the factors contributing to infant mortality.

Effect of khat on uteroplacental blood flow in awake, chronically catheterized, late-pregnant guinea pigs.

In order to investigate effects of khat chewing on uteroplacental blood flow, eight awake, chronically catheterized guinea pigs were fed 2.2 g khat leaves/kg in late pregnancy and regional blood flows were measured with the microsphere technique. Seven animals fed with aspen leaves in the same amounts served as controls. The mean concentration of (+)-norpseudoephedrine in urine 3 h after the end of the feeding was 4.6 micrograms/ml in the khat-fed group with no detectable amounts in the controls. Placental blood flow was reduced by 10% 75 min and by 24% 180 min after khat feeding. Since the khat dose used gave urinary concentrations of (+)-norpseudoephedrine of the same magnitude as those reported in khat chewing women, khat chewing in pregnancy may reduce placental blood flow and impair fetal growth.

Use of khat in lactating women: a pilot study on breast-milk secretion.

Nor-pseudoephedrine, one of the active ingredients of khat (Catha edulis), was found to be excreted in breast-milk in several lactating women who were chewing the leaves of the shrub according to the local customs. The compound could be traced in the urine of one breast-fed infant. It is concluded that the use of khat during lactation should be discouraged until further research has clearly elucidated the potential health hazards.

Growth at the age of 4 years subsequent to early failure to thrive.

Fifty-five children previously investigated for failure to thrive (a rate of weight gain below -2 SD) during at least 6 weeks at 4-18 months of age were followed up and reinvestigated at the age of 4 years. The children were studied in two groups: children with organic causes (OFTT) (n = 21); and children for whom no organic cause was found (nonorganic failure to thrive, NFTT) (n = 34). In children with OFTT, normalization of growth was found for both weight and height attained, as most of the diseases were either amenable to treatment or spontaneously subsided. The only exception was a child with severe encephalopathy. In children with NFTT, much lower values were found, particularly for weight, p less than .01 for both weight and height. Children with a low psychosocial score (less than or equal to 3 adverse factors) showed partial catch-up growth, although significantly lower than that of children with OFFT. Among 13 children with high psychosocial scores (greater than or equal to 4), 6 children had been subjected to strong social and/or psychological intervention. These children showed a more favorable growth pattern compared to children with comparable psychosocial scores where no intervention had been undertaken. The children with NFTT continued to grow slowly, remained meager and seemed to maintain a suboptimal growth pattern, particularly those with higher numbers of risk factors.

Khat-chewing during pregnancy-effect upon the off-spring and some characteristics of the chewers.

In a study of 1,141 consecutive deliveries at delivery centres in the Yemen Arab Republic, the effects of khat (catha edulis) upon the offspring have been studied. The leaves of the shrub khat contain euphorizing compounds and are chewed often, even daily, by many inhabitants. Non-users of khat (n = 427) had significantly fewer low birth-weight babies (less than 2,500 gram) compared to occasional users (n = 223) and regular users (n = 391). The khat-chewing mother was older, of greater parity and had more surviving children than the non-chewers. Significantly more khat-chewers had concomitant diseases. There was no difference in rates of stillbirth or congenital malformations.

[CDGS-1--a recently discovered hereditary metabolic disease. Multiple organ manifestations, incidence 1/80,000, difficult to treat]. / CDGS-1--en nyupptäckt ärftlig metabolisk sjukdom. Manifestationer från många organ, incidens 1/80,000, svårbehandlad.

Carbohydrate-deficient glycoprotein syndrome type 1 (CDGS-1) is an autosomal recessive hereditary metabolic disorder, the gene locus of which is chromosome 16p13. The disorder is characterised by genetic heterogeneity, and by decrease in the gene product, phosphomannomutase 2, though the heterogeneity is far less manifest in affected Swedish families. Its incidence is 1/80,000 live births, and the under-5 mortality rate over 30 per cent. The causes of death are liver failure, cardiac tamponade, haemorrhaging, and severe infection. The characteristic biochemical aberration is the occurrence of deficient carbohydrate chains in many but not all circulating glycoproteins, and the serum and blood concentrations of some glycoproteins may be above or below normal. These changes may improve over time, but never normalise. The clinical picture is generally more problematic during the first years of life when psychomotor retardation is complicated by failure to thrive, liver dysfunction, pericardial effusions, and stroke-like episodes. In addition, strabismus, lipocutaneous anomalies, and gluteal fat pads are always present, and muscular hypotonia and restricted joint mobility are common. Failure to thrive is common, with vomiting and diarrhoea and subsequent slow growth. Inflammation is a constant finding in the liver, and very common in the small bowel. Pancreatic function is also affected. Pericardial effusion has been reported in 50 per cent of the youngest children, requiring pericardectomy in 30 per cent of cases. Haemorrhaging and thromboembolic complications may occur, and the serum concentrations of several factors and inhibitors are low, particularly those of factors V and XI, protein C and antithrombin. Stroke-like episodes occur in about 30 per cent of cases, often following an infection, with coma lasting for hours to several days. Such sequelae as hemiplegia, blindness, and other focal neurological pathology have been observed transiently. Diagnosis is based on the serum carbohydrate-deficient transferrin level, verified by isoelectric focusing. Molecular genetic procedures enable point mutations to be identified and prenatal diagnosis to be performed in many families.