Contingent first-trimester screening for aneuploidies with cell-free DNA in a Danish clinical setting.

OBJECTIVES: The primary aim of this study was to compare the screening performance for trisomy 21 (T21) between combined first-trimester screening (cFTS) with referral for invasive testing at a T21 risk ≥ 1 in 300, and contingent screening consisting of referral for invasive testing at a cFTS-T21 risk ≥ 1 in 100 and referral for cell-free DNA (cfDNA) testing at a cFTS-T21 risk between 1 in 100 and 1 in 1000. Secondary aims were to compare the incidence of fetuses diagnosed with trisomy 18 (T18), trisomy 13 (T13) or sex chromosome aneuploidy, and examine the association between fetal fraction of cfDNA in maternal blood and maternal/fetal characteristics. METHODS: Women with a singleton pregnancy and a cFTS-T21 risk of ≥ 1 in 1000 were recruited consecutively from two Danish hospitals between August 2014 and May 2015. First-trimester combined screening was based on maternal age, nuchal translucency thickness and levels of pregnancy-associated plasma protein A (PAPP-A) and ß-human chorionic gonadotropin (ß-hCG). Blood samples for cfDNA testing were analyzed for risks of T21, T18, T13 and sex chromosomal aneuploidies. cfDNA analysis was conducted blinded to the cFTS assessment and karyotype results. Pregnancy outcomes and pre- and postnatal karyotypes were obtained from the Danish Fetal Medicine Database. RESULTS: Among 6449 women who underwent cFTS risk assessment, 869 (13.5%) had a T21 risk of ≥ 1 in 1000 and 597 were included for cfDNA testing. Among these, there were 15 cases of T21, one case of T18 and two cases of T13. The sensitivity for detection of T21 was 100% using both screening strategies, while specificity increased significantly (P < 0.0001) from 97.0% using the cFTS strategy to 98.8% using the contingent approach. The sensitivity for detection of T21, T18 and T13 increased from 94.4% using the cFTS strategy to 100% using the contingent approach, with overlapping CIs, while specificity increased significantly (P < 0.0001) from 97.1% for cFTS to 98.9% for the contingent strategy. Seven pregnancies were categorized as being at increased risk of a sex chromosomal aneuploidy by cfDNA testing but chromosome analysis was discordant, corresponding to a false-positive rate of 1.2%. The fetal fraction decreased significantly with increasing maternal weight and increased significantly with the level of ß-hCG and PAPP-A and among female fetuses, in both univariate and multivariate analyses. CONCLUSIONS: In a clinical setting with efficient cFTS, contingent screening offering women with a cFTS risk of ≥ 1 in 100 an invasive test and women with a risk from 1 in 100 to 1 in 1000 a cfDNA test had the same sensitivity for T21, T18 and T13, but significantly increased specificity, when compared with offering an invasive test to all women with a risk of ≥ 1 in 300. Implementing contingent screening would therefore reduce significantly the number of invasive tests performed at no loss of sensitivity. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.

Increased nuchal translucency thickness and risk of neurodevelopmental disorders.

OBJECTIVE: To investigate the association between fetal nuchal translucency (NT) thickness and neurodevelopmental disorders in euploid children. METHODS: This study included 222 505 euploid children who had undergone routine first-trimester screening during fetal life. Children were divided according to prenatal NT into three groups: NT < 95th percentile (n = 217 103 (97.6%)); NT 95th -99th percentile (n = 4760 (2.1%)); and NT > 99th percentile (n = 642 (0.3%)). All children were followed-up to a mean age of 4.4 years. Information on diagnoses of intellectual disability, autism spectrum disorders (ASD), cerebral palsy, epilepsy and febrile seizures was obtained from national patient registries. RESULTS: There was no excess risk of neurodevelopmental disorders among euploid children with first-trimester NT 95th -99th percentile. For children with NT > 99th percentile, there were increased risks of intellectual disability (odds ratio (OR), 6.16 (95% CI, 1.51-25.0), 0.31%) and ASD (OR, 2.48 (95% CI, 1.02-5.99), 0.78%) compared with children with NT < 95th percentile (incidence of 0.05% for intellectual disability and 0.32% for ASD), however, there was no detected increase in the risk of cerebral palsy (OR, 1.91 (95% CI, 0.61-5.95), 0.47%), epilepsy (OR, 1.51 (95% CI, 0.63-3.66), 0.78%) or febrile seizures (OR, 0.72 (95% CI, 0.44-1.16), 2.65%). CONCLUSIONS: In a large unselected cohort of euploid children, there was no increased risk of neurodevelopmental disorders among those with a first-trimester NT 95th -99th percentile. Among euploid children with first-trimester NT > 99th percentile, there were increased risks of intellectual disability and ASD, but the absolute risk was reassuringly low (< 1%). Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.

X-linked congenital ptosis and associated intellectual disability, short stature, microcephaly, cleft palate, digital and genital abnormalities define novel Xq25q26 duplication syndrome.

Submicroscopic duplications along the long arm of the X-chromosome with known phenotypic consequences are relatively rare events. The clinical features resulting from such duplications are various, though they often include intellectual disability, microcephaly, short stature, hypotonia, hypogonadism and feeding difficulties. Female carriers are often phenotypically normal or show a similar but milder phenotype, as in most cases the X-chromosome harbouring the duplication is subject to inactivation. Xq28, which includes MECP2 is the major locus for submicroscopic X-chromosome duplications, whereas duplications in Xq25 and Xq26 have been reported in only a few cases. Using genome-wide array platforms we identified overlapping interstitial Xq25q26 duplications ranging from 0.2 to 4.76 Mb in eight unrelated families with in total five affected males and seven affected females. All affected males shared a common phenotype with intrauterine- and postnatal growth retardation and feeding difficulties in childhood. Three had microcephaly and two out of five suffered from epilepsy. In addition, three males had a distinct facial appearance with congenital bilateral ptosis and large protruding ears and two of them showed a cleft palate. The affected females had various clinical symptoms similar to that of the males with congenital bilateral ptosis in three families as most remarkable feature. Comparison of the gene content of the individual duplications with the respective phenotypes suggested three critical regions with candidate genes (AIFM1, RAB33A, GPC3 and IGSF1) for the common phenotypes, including candidate loci for congenital bilateral ptosis, small head circumference, short stature, genital and digital defects.

A novel microdeletion syndrome at 3q13.31 characterised by developmental delay, postnatal overgrowth, hypoplastic male genitals, and characteristic facial features.

BACKGROUND: Congenital deletions affecting 3q11q23 have rarely been reported and only five cases have been molecularly characterised. Genotype-phenotype correlation has been hampered by the variable sizes and breakpoints of the deletions. In this study, 14 novel patients with deletions in 3q11q23 were investigated and compared with 13 previously reported patients. METHODS: Clinical data were collected from 14 novel patients that had been investigated by high resolution microarray techniques. Molecular investigation and updated clinical information of one cytogenetically previously reported patient were also included. RESULTS: The molecular investigation identified deletions in the region 3q12.3q21.3 with different boundaries and variable sizes. The smallest studied deletion was 580 kb, located in 3q13.31. Genotype-phenotype comparison in 24 patients sharing this shortest region of overlapping deletion revealed several common major characteristics including significant developmental delay, muscular hypotonia, a high arched palate, and recognisable facial features including a short philtrum and protruding lips. Abnormal genitalia were found in the majority of males, several having micropenis. Finally, a postnatal growth pattern above the mean was apparent. The 580 kb deleted region includes five RefSeq genes and two of them are strong candidate genes for the developmental delay: DRD3 and ZBTB20. CONCLUSION: A newly recognised 3q13.31 microdeletion syndrome is delineated which is of diagnostic and prognostic value. Furthermore, two genes are suggested to be responsible for the main phenotype.

Corpus callosum abnormalities, intellectual disability, speech impairment, and autism in patients with haploinsufficiency of ARID1B.

Corpus callosum abnormalities, intellectual disability, speech impairment, and autism in patients with haploinsufficiency of ARID1B. Corpus callosum abnormalities are common brain malformations with a wide clinical spectrum ranging from severe intellectual disability to normal cognitive function. The etiology is expected to be genetic in as much as 30-50% of the cases, but the underlying genetic cause remains unknown in the majority of cases. By next-generation mate-pair sequencing we mapped the chromosomal breakpoints of a patient with a de novo balanced translocation, t(1;6)(p31;q25), agenesis of corpus callosum (CC), intellectual disability, severe speech impairment, and autism. The chromosome 6 breakpoint truncated ARID1B which was also truncated in a recently published translocation patient with a similar phenotype. Quantitative polymerase chain reaction (Q-PCR) data showed that a primer set proximal to the translocation showed increased expression of ARID1B, whereas primer sets spanning or distal to the translocation showed decreased expression in the patient relative to a non-related control set. Phenotype-genotype comparison of the translocation patient to seven unpublished patients with various sized deletions encompassing ARID1B confirms that haploinsufficiency of ARID1B is associated with CC abnormalities, intellectual disability, severe speech impairment, and autism. Our findings emphasize that ARID1B is important in human brain development and function in general, and in the development of CC and in speech development in particular.

First-trimester screening for trisomy 21 in Denmark: implications for detection and birth rates of trisomy 18 and trisomy 13.

OBJECTIVES: In Denmark a new national guideline for prenatal screening and diagnosis was issued in 2004 according to which all pregnant women should be offered a first-trimester combined risk assessment for trisomy 21 (T21). The aim of this study was to investigate whether the new screening strategy for T21 has changed the gestational age at which trisomy 18 (T18) and trisomy 13 (T13) are diagnosed prenatally, and the number of infants born with T18 or T13. METHODS: We collected from the Danish Cytogenetic Central Register information on all prenatal and postnatal chromosome analyses for T18 or T13, registered from 1997 to 2007. Information on first-trimester screening results was collected from each department of obstetrics and gynecology performing the nuchal translucency scans. The cut-off used for referral to invasive diagnostic testing for T21 and for T18/T13 was 1 : 300 and 1 : 150 at screening, respectively. RESULTS: In total, there were 435 cases with T18 and 168 cases with T13 between 1997 and 2007 in Denmark. The estimated incidence of T18 and T13 at the time of delivery was calculated as 2.5 and 1.6 per 10 000 deliveries, respectively. The number (proportion) of cases diagnosed before week 18 increased significantly, from 63 (59.4%) in 1997 and 1998 to 90 (80.4%) in 2006 and 2007 (P < 0.001). In addition, the number of T18 and T13 cases diagnosed prenatally after week 22 or postnatally decreased significantly, from 34 (32.1%) in 1997 and 1998 to seven (6.3%) in 2006 and 2007 (P < 0.0001). For women participating in first-trimester risk assessment in 2006 and 2007, the detection rate of T18 and T13 was 78.8% (95% CI, 71.0-86.7%). CONCLUSION: The number of T18 and T13 fetuses diagnosed before week 18 increased significantly after the introduction of a combined first-trimester screening strategy for T21 in Denmark. In addition, the total number of fetuses diagnosed late in pregnancy and infants born with T18 or T13 decreased significantly. The national detection rate for T18 and T13 in the first trimester is comparable with detection rates found in modeled datasets and other prospective studies.

Wood smoke in a controlled exposure experiment with human volunteers.

Exposure to wood smoke in the general population is increasing and concurrently, also our awareness. This article describes a wood-smoke generating system for studying human exposure to wood smoke and symptoms related to this exposure. Twenty nonsmoking atopic human participants with normal lung function and normal bronchial reactivity were randomly exposed for 3 h at three different exposure conditions; clean filtered air (control exposure) and wood smoke with a characteristic particulate matter (PM) concentration of 200 µg/m³ (low) and 400 µg/m³ (high) under controlled environmental conditions. The range for PM2.5 load observed for single experiments was 165-303 µg/m³ for the low exposure and 205-662 µg/m³ for the high exposure, whereas particle loads during clean air exposure most often were below the detection limit (< 20 µg/m³). Health effects were evaluated in relation to rated changes in symptoms and environmental perception using a computerized questionnaire and a potentiometer. Subjective symptoms were generally weak, but when combining the effect of each of the symptoms into categorical symptom indices, significant effects were found for "environmental perception" (p = 0.0007), "irritative body perceptions" (p = 0.0127), "psychological/neurological effects" (p = 0.0075) and "weak inflammatory responses" (p = 0.0003). Furthermore, significant effects (p = 0.0192) on self-reported general mucosa irritation were found. In conclusion, exposure to wood smoke affected symptom rating and caused irritated mucosas in humans. The knowledge gained in this study on subjective-rated symptoms may be important for understanding human response to wood-smoke exposure.

Diagnostic yield by supplementing prenatal metaphase karyotyping with MLPA for microdeletion syndromes and subtelomere imbalances.

OBJECTIVE: The aim of the study was to retrospectively assess the relevance of using multiplex ligation-dependent probe amplification (MLPA) for detection of selected microdeletion syndromes (22q11, Prader-Willi/Angelman, Miller-Dieker, Smith-Magenis, 1p-, Williams), the reciprocal microduplication syndromes and imbalance at the subtelomere regions of chromosomes in a routine prenatal setting. METHOD: A total of 530 prenatal samples were analysed by commercial MLPA kits (SALSA P064, P036 and P069) in addition to rapid aneuploidy testing and G-band karyotyping. RESULTS: Among the prenatal samples with a normal metaphase karyotype, nine submicroscopic imbalances were detected: seven 22q11 deletions (Velocardiofacial/DiGeorge syndrome), one 15q11 deletion (Prader-Willi syndrome) and one terminal deletion of the short arm of chromosome 4 (Wolf-Hirschhorn syndrome). All imbalances were found in amniocentesis (AC) taken due to fetal structural malformation and/or other ultrasound scan (US) detected abnormality. The diagnostic yield was 4.1% in the subgroup with structural malformation and 1.6% in the subgroup with other US abnormality. CONCLUSION: The data set substantiates that additional MLPA analyses for selected microdeletions and subtelomere imbalances are valuable in routine prenatal diagnostics, when a malformation(s) and/or other abnormalities are detected by US. In contrast, the additional MLPA analyses gave no diagnostic yield in case of increased nuchal translucency (NT).

A new microduplication syndrome encompassing the region of the Miller-Dieker (17p13 deletion) syndrome.

BACKGROUND: The use of array comparative genome hybridisation (CGH) analyses for investigation of children with mental retardation has led to the identification of a growing number of new microdeletion and microduplication syndromes, some of which have become clinically well characterised and some that await further delineation. This report describes three children with de novo 17p13.1 duplications encompassing the PAFAH1B1 gene, who had similar phenotypic features, including mild to moderate developmental delay, hypotonia and facial dysmorphism, and compares them to the few previously reported cases with this duplication. METHODS: Multiplex ligation-dependent probe amplification (MLPA) or array-CGH was used to diagnose three developmentally delayed children with duplications of 17p13. The duplications were characterised further using Agilent array technology, revealing duplication sizes from 1.8 to 4.0 Mb, with a region of overlap corresponding to 1.8 Mb. Detailed clinical information was obtained from patient files and personal examinations. RESULTS: The developmental delay and similar clinical features in the three patients were most likely due to a common microduplication of 17p13. CONCLUSIONS: In contrast to patients with deletion of the region (Miller-Dieker syndrome) the patients reported here had mild to moderate retardation and displayed no lissencephaly or gross brain malformations. Further cases with similar duplications are expected to be diagnosed, and will contribute to the delineation of a potential new microduplication syndrome of 17p13.

Nasal hyperresponders and atopic subjects report different symptom intensity to air quality: a climate chamber study.

UNLABELLED: Short-term exposure to dust and dust added with beta-(1,3)-d-glucan or aldehydes may cause sensory reactions. In random order, we exposed 36 volunteers in a climate chamber to clean air, office dust, dust with glucan, and dust with aldehydes. Three groups of subjects were exposed, eleven were non-atopic with nasal histamine hyperreactivity, 13 were non-atopic, and 12 were atopic. Subjective ratings of symptoms and general health were registered four times during four 6-h exposure sessions. Six symptom intensity indices were constructed. The nasal hyperreactive group had a high and time-dependent increase of mucous membrane irritations, whereas the atopic group had a low and stable rate of irritations with exposure time, close to the reference group (P = 0.02 for differences between the groups with respect to time under exposure for Weak Inflammatory Responses and P = 0.05 for Irritative Body Perception, significance mainly because of the nasal hyperreactive group). Exposure to dust, with or without glucan or aldehydes, showed increased discomfort measured by the index for Constant Indoor Climate, and dust with glucan had a similar effect for the index for Lower Respiratory Effects. For Psychological and Neurological Effects these were dependent on group affiliation, thus preventing a uniform statement of exposure effects for all three investigated groups. PRACTICAL IMPLICATIONS: Opportunities for identifying persons with high or low sensitivity to low-level exposures are important in preventive medicine and will reduce intra-group variability and thus increase the power of experimental and epidemiological studies searching for correlations between exposures and health effects. The contrast between nasal hyperreactive on one side and atopic and reference subjects on the other side is particularly important. The atopic group indicated a non-homogenous reaction depending on their hyperreactive status, a finding that could be important but needs further confirmation.

Effects on human eyes caused by experimental exposures to office dust with and without addition of aldehydes or glucan.

UNLABELLED: Thirty-six volunteers (in three susceptibility groups: 11 subjects were non-allergic with nasal histamine hypersensitivity, 13 were non-allergic with normal sensitivity, and 12 were pollen allergic with or without nasal hypersensitivity) were exposed for three and a half hours in a climate chamber. Each subject was exposed to clean air (dust 45 +/- 38 microg/m(3) total suspended particle, TSP), house dust at 357 +/- 180 microg/m(3) TSP, house dust 382 +/- 175 microg/m(3) TSP with added glucan (50 ng/m(3)) and house dust 394 +/- 168 microg/m(3) TSP with added aldehydes corresponding to a gaseous phase of 300 microg/m(3) in the air. The study was explorative by nature. No significant effects of exposures as such were seen on break-up time, conjunctival epithelial damage score and Trolox Equivalent Antioxidant Capacity (TEAC) in tear film and subjective ratings. However, in TEAC a significant different time course was seen during exposures to aldehyde-containing dust indicating a subacute and late response to the exposures. Perceived eye irritation increased significantly during exposures to normal dust. The perception ratings were highly correlated, whereas no correlation was found between the subjective responses and the objective measurements. PRACTICAL IMPLICATIONS: The findings indicate that measurement effects on the eyes are rather insensitive measures of short time effects of office dust exposures.

An automated method for measuring static pressure-volume curves of the respiratory system and its application in healthy lungs and after lung damage by oleic acid infusion.

Elastic pressure/volume (PV) curves of the respiratory system have attracted increasing interest, because they may be helpful to optimize ventilator settings in patients undergoing mechanical ventilation. Clinically applicable methods need to be fast, use routinely available equipment, draw the inspiratory and expiratory PV curve limbs, separate the resistive and viscoelastic properties of the respiratory system from the elastic properties, and provide reproducible measurements. This paper presents a computer-controlled method for rapid measurements of static PV curves using a long inflation-deflation with pauses, and its evaluation in six pigs before and after lung damage caused by oleic acid. The method is fast, i.e. 20.5 +/- 1.9 s (mean +/- SD) in healthy lungs and 17.7 +/- 4.1 s in diseased lungs, this including inspiratory and expiratory pauses of 1.1 s duration. In addition the only equipment used was a clinical ventilator and a PC. For healthy and damaged lungs expiratory PV curve limbs were very reproducible and were at higher volume than the inspiratory limbs, indicating hysteresis. For damaged lungs inspiratory PV limbs were reproducible. For healthy lungs the inspiratory limbs were reproducible but only after the first inflation-deflation. It is possible that during the first inflation alveoli are recruited which are not derecruited on deflation, shifting the inspiratory limb of the PV curve. The paused long inflation-deflation technique provides a quick, automated measurement of static PV curves on both inspiratory and expiratory limbs using routinely available equipment in the intensive care unit.

Reproduction of MIGET retention and excretion data using a simple mathematical model of gas exchange in lung damage caused by oleic acid infusion.

The multiple inert-gas elimination technique (MIGET) is a complex mathematical model and experimental technique for understanding pulmonary gas exchange. Simpler mathematical models have been proposed that have a limited view compared with MIGET but may be applicable for use in clinical practice. This study examined the use of a simple model of gas exchange to describe MIGET retention and excretion data in seven pigs before and following lung damage caused by oleic acid infusion and subsequently at different levels of positive end-expiratory pressure. The simple model was found to give, on average, a good description of MIGET data, as evaluated by a chi(2) test on the weighted residual sum of squares resulting from the model fit (P > 0.2). Values of the simple model's parameters (dead-space volume, shunt, and the fraction of alveolar ventilation going to compartment 2) compared well with the similar MIGET parameters (dead-space volume, shunt, log of the standard deviation of the perfusion, log of the standard deveation of the ventilation), giving values of bias and standard deviation on the differences between dead-space volume and shunt of 0.002 +/- 0.002 liter and 7.3 +/- 2.1% (% of shunt value), respectively. Values of the fraction of alveolar ventilation going to compartment 2 correlated well with log of the standard deviation of the perfusion (r(2) = 0.86) and log of the standard deviation of the ventilation (r(2) = 0.92). These results indicate that this simple model provides a good description of lung pathology following oleic acid infusion. It remains to be seen whether physiologically valid values of the simple model parameters can be obtained from clinical experiments varying inspired oxygen fraction. If so, this may indicate a role for simple models in the clinical interpretation of gas exchange.

Thrombolytic therapy in diabetic patients with acute myocardial infarction.

OBJECTIVE: To compare the frequency of thrombolytic therapy in diabetic and nondiabetic patients with acute myocardial infarction (MI) and to examine why some diabetic patients do not receive thrombolytic therapy. RESEARCH DESIGN AND METHODS: Retrospective study of all diabetic patients with acute MI admitted to the coronary care unit of Aalborg Hospital within a 3-year period. RESULTS: Only 35% (43 of 123) of patients with diabetes compared with 47% (404 of 856) of patients without diabetes received thrombolytic therapy (P < 0.002). There was no difference in the percentage of patients thrombolyzed among patients admitted to the hospital within 12 h after onset of symptoms. Of diabetic patients who were not thrombolyzed, 60% (48 of 80) arrived at the hospital later than 12 h after onset of symptoms. Among patients who arrived late, 63% (35 of 56) had Q wave infarction and 84% (47 of 56) had symptoms typical of acute MI. Mortality was 29% (16 of 56) in this group. Only one patient did not receive thrombolytic therapy due to diabetic retinopathy. CONCLUSIONS: Significantly fewer diabetic patients received thrombolytic therapy compared with patients without diabetes. The main reason diabetic patients did not receive thrombolytic therapy was late arrival to the hospital.

Mutations in PMM2 that cause congenital disorders of glycosylation, type Ia (CDG-Ia).

The PMM2 gene, which is defective in CDG-Ia, was cloned three years ago [Matthijs et al., 1997b]. Several publications list PMM2 mutations [Matthijs et al., 1997b, 1998; Kjaergaard et al., 1998, 1999; Bjursell et al., 1998, 2000; Imtiaz et al., 2000] and a few mutations have appeared in case reports or abstracts [Crosby et al., 1999; Kondo et al., 1999; Krasnewich et al., 1999; Mizugishi et al., 1999; Vuillaumier-Barrot et al., 1999, 2000b]. However, the number of molecularly characterized cases is steadily increasing and many new mutations may never make it to the literature. Therefore, we decided to collate data from six research and diagnostic laboratories that have committed themselves to a systematic search for PMM2 mutations. In total we list 58 different mutations found in 249 patients from 23 countries. We have also collected demographic data and registered the number of deceased patients. The documentation of the genotype-phenotype correlation is certainly valuable, but is out of the scope of this molecular update. The list of mutations will also be available online (URL: http://www.kuleuven. ac.be/med/cdg) and investigators are invited to submit new data to this PMM2 mutation database.

Absence of homozygosity for predominant mutations in PMM2 in Danish patients with carbohydrate-deficient glycoprotein syndrome type 1.

Carbohydrate-deficient glycoprotein syndrome type 1 (CDG1; McKusick No. 212065) is an autosomal recessively inherited disease characterised clinically by central nervous system dysfunction and biochemically by hypoglycosylation of many serum proteins. Most patients with CDG1 have deficient activity of phosphomannomutase. The locus for this enzyme has been mapped to 16p13, and a gene, PMM2, encoding phosphomannomutase has been isolated. We identified 34 mutations on 36 disease chromosomes in 18 unrelated Danish patients with CDG1. All patients have less than 15% residual activity of phosphomannomutase. Two mutations account for 88% of all mutations: F119L and R141H were each found in 16 out of 36 CDG1 alleles. These two mutations were found to be in linkage disequilibrium with two different alleles of the marker D16S3020, suggesting that there is one ancestral origin for each mutation. Two new mutations, G117R and D223E, were identified also. Surprisingly, no patient was homozygous for either of the two common mutations, suggesting that homozygosity for these mutations is either lethal or so benign that such patients are not detected.

Carbohydrate-deficient glycoprotein syndrome type 1A: expression and characterisation of wild type and mutant PMM2 in E. coli.

We have identified the PMM2 genotypes of 22 unrelated Danish patients with carbohydrate-deficient glycoprotein syndrome type 1A: R141H/F119L (18), R141H/C192G (1), F119L/F119L (1), F119L/G117R (1) and D223E/T237R (1). The lack of patients homozygous for R141H is statistically highly significant, but unexplained. In order to investigate the effect of PMM2 mutations on phosphomannomutase (PMM2) activity, PMM2-cDNA was cloned into a pET3a vector. Following introduction of mutations into PMM2-cDNA by site-specific mutagenesis, wild type and mutant PMM2-cDNA were expressed in E. coli Bl21(DE3) cells, and the activity of PMM2 was determined by an enzymatic assay using mannose 1-phosphate as substrate. Recombinant R141H, G117R, and T237R PMM2 had no detectable catalytic activity, and the F119L PMM2 had 25% of the activity of the wild type. The activity of the C192G and D223E PMM2 was in the normal range, but the affinity for their substrate was lower, and the proteins were more sensitive to increased temperatures. Each patient has at least one mutation which retains residual PMM2 activity. Our results support the hypotheses that a genotype conveying residual PMM2 catalytic activity is required for survival, and that homozygosity for R141H impairs PMM2 to a degree incompatible with life.

Lack of Hardy-Weinberg equilibrium for the most prevalent PMM2 mutation in CDG-Ia (congenital disorders of glycosylation type Ia).

The R141H mutation in the PMM2 gene is the most frequent mutation in type Ia of the congenital disorders of glycosylation (formerly carbohydrate-deficient glycoprotein syndromes)(CDG-Ia). However, it has never been observed in the homozygous state. Homozygosity for this mutation is probably incompatible with life. In this study, we determined the frequency of R141H in two normal populations: in neonates of Dutch origin 1/79 were carriers, whilst in the Danish population, a carrier frequency of 1/60 was found. These figures are clearly in disequilibrium with the frequency of CDG-Ia that has been estimated at 1/80,000 to 1/40,000 in these populations. Haplotype analysis of 43 patients with the R141H mutation of different geographic origins indicated that the R141H is an old mutation in the Caucasian population. Based on the new data, the disease frequency has been calculated at 1/20,000 in these populations. It is concluded that the disease is probably underdiagnosed.

Molecular genetic basis and prevalence of glycogen storage disease type IIIA in the Faroe Islands.

Glycogen storage disease type IIIA (GSD IIIA) is caused by mutations of the amyloglucosidase gene (AGL). For most populations, none of the AGL mutations described to date is particularly frequent. In this paper, we report that six children with GSD IIIA from the Faroe Islands were found to be homozygous for the novel nonsense mutation c.1222C>T (R408X) of the AGL gene. This mutation is easily detected by restriction enzyme digest with NsiI after mismatch PCR. Investigating five intragenic polymorphisms, we could show that this mutation was always associated with the same haplotype. The c.1222C>T mutation could be detected on two chromosomes of another 50 unselected GSD IIIA patients of other European or North American origin which means that this mutation plays a minor role worldwide. From the fact that we are currently aware of a total of 14 GSD IIIA cases in the Faroese population of 45 000, the observed prevalence is 1 : 3100. While the novel AGL mutation c.1222C>T was not detectable among 198 German newborns, nine out of 272 children from the Faroese neonatal screening program were found to be heterozygous for this mutation. Thus, the calculated prevalence is 1 : 3600 (95% CI 1:700-1:6400). We conclude that due to a founder effect, the Faroe Islands have the highest prevalence of GSD IIIA world-wide. The detection of the molecular defect has facilitated the diagnosis and has offered the opportunity for prenatal diagnosis in this patient group.

Recurrent fetal polycystic kidneys associated with glutaric aciduria type II.

A woman had two pregnancies terminated in the 20th and 21st weeks of gestation after ultrasonographic detection of enlarged hyperechoic kidneys in both fetuses. The combination of polycystic kidneys and steatotic liver found at autopsy suggested glutaric aciduria type II (GA II), which was confirmed by biochemical investigation. GA II or multiple acyl-CoA dehydrogenase deficiency is an autosomal recessively inherited defect of mitochondrial energy metabolism, which usually results in neonatal death. When pregnancy is terminated because of enlarged hyperechoic kidneys in the fetus, autopsy is crucial for establishing the correct diagnosis. The combination of polycystic kidneys and steatotic liver should bring GA II to mind, and prompt appropriate biochemical investigations so that genetic counselling and first trimester diagnosis can be offered in future pregnancies.