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Background: Apolipoprotein E (APOE) ɛ4 is associated with poor outcome following moderate to severe traumatic brain injury (TBI). There is a lack of studies investigating the influence of APOE ɛ4 on intracranial pathology following mild traumatic brain injury (MTBI). This study explores the association between APOE ɛ4 and MRI measures of brain age prediction, brain morphometry, and diffusion tensor imaging (DTI). Methods: Patients aged 16 to 65 with acute MTBI admitted to the trauma center were included. Multimodal MRI was performed 12 months after injury and associated with APOE ɛ4 status. Corrections for multiple comparisons were done using false discovery rate (FDR). Results: Of included patients, 123 patients had available APOE, volumetric, and DTI data of sufficient quality. There were no differences between APOE ɛ4 carriers (39%) and non-carriers in demographic and clinical data. Age prediction revealed high accuracy both for the DTI-based and the brain morphometry based model. Group comparisons revealed no significant differences in brain-age gap between ɛ4 carriers and non-carriers, and no significant differences in conventional measures of brain morphometry and volumes. Compared to non-carriers, APOE ɛ4 carriers showed lower fractional anisotropy (FA) in the hippocampal part of the cingulum bundle, which did not remain significant after FDR adjustment. Conclusion: APOE ɛ4 carriers might be vulnerable to reduced neuronal integrity in the cingulum. Larger cohort studies are warranted to replicate this finding.

Hereditary hemorrhagic telangiectasia (HHT, Osler-Weber-Rendu disease) is an autosomal dominant inherited disease defined by the presence of epistaxis and mucocutaneous telangiectasias and arteriovenous malformations (AVMs) in internal organs. In most families (~85%), HHT is caused by mutations in the ENG (HHT1) or the ACVRL1 (HHT2) genes. Here, we report the results of genetic testing of 113 Norwegian families with suspected or definite HHT. Variants in ENG and ACVRL1 were found in 105 families (42 ENG, 63 ACVRL1), including six novel variants of uncertain pathogenic significance. Mutation types were similar to previous reports with more missense variants in ACVRL1 and more nonsense, frameshift and splice-site mutations in ENG. Thirty-two variants were novel in this study. The preponderance of ACVRL1 mutations was due to founder mutations, specifically, c.830C>A (p.Thr277Lys), which was found in 24 families from the same geographical area of Norway. We discuss the importance of founder mutations and present a thorough evaluation of missense and splice-site variants.

Objective To characterise a globotriaosylceramide (Gb3) storage cardiomyopathy mimicking Fabry. Methods We investigated five patients from two unrelated families with early adult onset unexplained left ventricular hypertrophy. Endomyocardial biopsy was performed in all patients and diagnostic kidney biopsies in two of them. We measured α-galactosidase A activity in all patients. Three patients were checked for LAMP1 or LAMP2 deficiency and screened for congenital disorders of glycosylation. Gb3 concentration was quantified in plasma, urinary sediment and cardiac muscle. We sequenced the Fabry and Danon genes and looked for other genetic causes by single-nucleotide polymorphism array haplotyping and whole exome sequencing. Results Three patients had a striking fat distribution around the buttocks and upper thighs. All patients developed bradyarrhythmias and needed pacemakers. Cardiac transplantation was performed in three patients due to end-stage heart failure, one patient died before transplantation. The cardiomyocytes contained lysosomal vacuoles with lamellar myelin-like deposits. Interstitial cells had vacuoles containing granular material. Deposits were found in the kidneys without renal dysfunction. The histological pattern was atypical for Fabry disease. Biochemical studies revealed normal activity of α-galactosidase A and other relevant enzymes. There was a selective accumulation of Gb3 in cardiomyocytes, at levels found in patients with Fabry disease, but no mutations in the Fabry gene, and Fabry disease was excluded. Other known lysosomal storage diseases were also excluded. Single-nucleotide polymorphism array haplotyping and whole exome sequencing could not identify the genetic cause. Conclusions We describe a novel familial Gb3-assoociated cardiomyopathy. Autosomal recessive inheritance is likely, but the genetic and metabolic cause remains to be identified.

Objectives Autism spectrum disorders (ASDs) are a heterogeneous group of neurodevelopment disorders with a complex genetic aetiology. The aim of this study was to identify copy number variations (CNVs) with a clinical significance for ASD. Materials and methods Array-based comparative genomic hybridization was applied to detect CNVs in a clinically well-characterized population of 50 children and adolescents with ASD. Results Nine CNVs with predicted clinical significance were identified among eight individuals (detection rate 16%). Three of the CNVs are recurrently associated with ASDs (15q11.2q13.1) or have been identified in ASD populations [3p14.2 and t(8;12)(p23.1;p13.31)]. The remaining regions (15q11.2, 10q21.1, Xp22.2, 16p13.3 and 22q13.1) have not been reported previously as candidate genes for ASD. Conclusion This study identified five novel CNVs among the individuals. The causal relationship between identified CNVs and the ASD phenotype is not fully established. However, the genes involved are associated with ASD and/or other neuropsychiatric disorders, or implicated in synaptic and neuronal activity, thus suggesting clinical significance. Further identification of ASD-associated CNVs is required, together with a broad clinical characterization of affected individuals to identify genotype-phenotype correlations.

Currarino Syndrome is a rare congenital malformation syndrome described as a triad of anorectal, sacral and presacral anomalies. Currarino Syndrome is reported to be both familial and sporadic. Familial CS is today known as an autosomal dominant disorder caused by mutations in the transcription factor MNX1. The aim of this study was to look for genetic causes of Currarino Syndrome in sporadic patients after ruling out other causes, like chromosome aberrations, disease-causing variants in possible MNX1 cooperating transcription factors and aberrant methylation in the promoter of the MNX1 gene. The hypothesis was that MNX1 was affected through interactions with other transcription factors or through other regulatory elements and thereby possibly leading to abnormal function of the gene. We performed whole exome sequencing with an additional 6Mb custom made region on chromosome 7 (GRCh37/hg19, chr7:153.138.664-159.138.663) to detect regulatory elements in non-coding regions around the MNX1 gene. We did not find any variants in genes of interest shared between the patients. However, after analyzing the whole exome sequencing data with Filtus, the in-house SNV filtration program, we did find some interesting variants in possibly relevant genes that could be explaining these patients` phenotypes. The most promising genes were ETV3L, ARID5A and NCAPD3. To our knowledge this is the first report of whole exome sequencing in sporadic CS patients.

Our study aimed to investigate the relationship between exercise-induced pulmonary arterial hypertension and genetic changes related to the transforming growth factor-β (TGF-β) signalling pathway in patients with cardiac septal defects.In a population-based group of 44 patients (age 13-25 years) with either isolated ventricular septal defect (n=27) or isolated atrial septal defect (n=17), right ventricular systolic pressure response to submaximal exercise was studied by echocardiography and classified as normal (≤45 mmHg), borderline (45-50 mmHg) or abnormal (50 mmHg). Three genes related to TGF-β, bone morphogenetic protein receptor type 2 (BMPR2), activin receptor-like kinase 1 (ALK1) and endoglin (ENG), were analyzed by DNA sequencing (only BMPR2) and multiplex ligand-dependent probe amplification (BMPR2, ALK1 and ENG).Pressure response was borderline in five and abnormal in nine patients. Five patients showed mutations in exon 12 of the bone morphogenetic protein receptor type 2 gene. The previously described polymorphism S775N (c. 2324, GA) was found in three patients with normal pressure response. The mutation Y589C (c. 1766, AG), which has not been described previously, was found in two of 14 patients with borderline/abnormal pressure response.Genetic changes in the BMPR2 gene may be overrepresented in patients with cardiac septal defects and exercise-induced pulmonary hypertension.

Patients with the 22q11.2 deletion syndrome display a wide phenotypic variation that is important for clinical follow-up. In this national survey of 60 patients (ages 1 to 54 years) diagnosed by Fluorescence in situ hybridization test, data were collected from medical records, a physical examination, and a semistructured interview. Ultrasound investigation of the kidneys was also performed. In addition, multiplex ligation probe amplification assay was performed to detect deletion size. Phenotypic features leading to the genetic diagnosis were noted. The patients showed a variety of organ malformations including 39 with heart anomalies. Only 20 individuals had been diagnosed with 22q11.2 DS in the first year of life. Four patients had renal and five males had genital malformations. The increased infection susceptibility (excluding otitis media) and most feeding difficulties subsided during early childhood. Speech difficulties started early and were a major problem for many patients at least until 10 years of age. Ten patients developed kyphoscoliosis in late childhood. In teenagers and adults, abnormal social behavior, learning disabilities, and psychiatric symptoms dominated. Our study which also includes adult patients emphasizes a marked change in challenges in individuals with the 22q11.2 deletion syndrome with increasing age.

For lite eller for mye kromosommateriale er den vanligste arsaken til medfodte utviklingsavvik. Kromosomfeil er siden 1960-arene blitt pavist ved lysmikroskopi, som er en noksa grov undersokelse. I 2005 ble DNA-matrisebaserte metoder tatt i klinisk bruk, og man kan na pavise kromosomavvik ned til enkeltgenniva. Slik molekylaer karyotyping gir presis informasjon om hvilke gener som foreligger i endret kopitall, men tolkingen kan vaere vanskelig og forutsetter gode kliniske opplysninger.

Background and Aims: Mutations in the gene encoding the ABCB4 [adenosine triphosphate (ATP)-binding cassette, sub-family B (MDR/TAP), member 4] transporter lower phosphatidylcholine output into bile and contribute to cholesterol gallstone formation by decreasing the solubility of cholesterol in bile. Mutations in ABCB4 have been identified in patients with low phospholipid-associated cholelithiasis. The aim of the present study was to determine the types and frequencies of ABCB4 mutations in cholecystectomized patients aged T/p.R1046X). These two mutations are considered detrimental to ABCB4 protein function. In addition, six missense mutations were found in the ABCB4 gene, and three of these were only present in patients. Conclusion: In our study

Linkage analysis and DNA sequencing in a family exhibiting an X-linked mental retardation (XLMR) syndrome, characterized by microcephaly, epilepsy, ataxia, and absent speech and resembling Angelman syndrome, identified a deletion in the SLC9A6 gene encoding the Na(+)/H(+) exchanger NHE6. Subsequently, other mutations were found in a male with mental retardation (MR) who had been investigated for Angelman syndrome and in two XLMR families with epilepsy and ataxia, including the family designated as having Christianson syndrome. Therefore, mutations in SLC9A6 cause X-linked mental retardation. Additionally, males with findings suggestive of unexplained Angelman syndrome should be considered as potential candidates for SLC9A6 mutations.

Summary Background Several candidate genes have been implicated in the etiology of asthma, including the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). Mutations in the CFTR gene result in derangements of mucociliary clearance. Homozygotes for CFTR mutations develop cystic fibrosis (CF), a disorder characterized mainly by lung and pancreas disease. Objective To investigate whether there was an increased frequency of CFTR mutations in asthma patients. Methods Seven hundred and three subjects aged 10–11 years from the environment and childhood asthma (ECA) study were included in the present study. Possible associations between asthma, reduced lung function, bronchial hyperresponsiveness (BHR), and increased or decreased nitrogen oxide (NO) levels (based on structural parental interview, spirometry, PD 20 methacholine challenge test and exhaled NO measurements), and the five most common CFTR mutations in Norway (ΔF508, R117H, R117C, 4005+2T→C, 394delTT), the modulating polymorphisms IVS8(TG) m T n and the IVS8-5T were investigated. Results No association were found between asthma, reduced lung function, BHR or exhaled NO levels and CF heterozygosity. However, the IVS8(TG) 11 T 7 haplotype was associated with normal lung function. Conclusions Our results do not support the hypothesis that CFTR mutations or polymorphisms play a role in the pathogenesis of asthma in children. However, the distribution of Tn(TG)m haplotypes differed between individuals with reduced lung function and individuals with normal lung function.

We report on a family with three stillborn males, three affected males who were small for gestational age and died within 8 months, and one male who died at age 5 years. This boy had cone-shaped teeth and oligoodontia. He had serious bacterial infections and inflammatory bowel disease. Mutations in the NF-kappaB essential modulator (NEMO) gene have recently been shown to be the cause of a group of ectodermal dysplasia and immunodeficiency disorders (EDA-ID). Analysis of the NEMO gene revealed a nucleotide change in the consensus sequence of the splicing donor site of exon 6 IVS6 + 5G --> A(1027 + 5G --> A), which has not previously been described in EDA-ID. RT-PCR analysis of fibroblast RNA from an aborted affected male fetus demonstrated a skipping of exons 4, 5, and 6 which resulted in a truncated protein of about 35 kDa revealed by NEMO antibody. The skipping of exons 4, 5, and 6 did not affect the ORF of the C-terminal of NEMO encoded by exons 7, 8, 9, and 10, which contains a coiled-coil motif (CC2), a leucin-zipper (LZ), and a zinc finger motif (ZF) sub-domains of NEMO. IkappaBalpha degradation was strongly impaired in the fetal fibroblasts, suggesting an impaired NF-kappaB signaling. One healthy carrier had a completely skewed X-inactivation pattern with the normal X active, whereas the two other carriers had a random X-inactivation pattern. This family may represent a new phenotype within the EDA-ID disorders. From the heterogeneity in X-inactivation phenotype, we conclude that this mutation is not deleterious enough to be lethal for peripheral blood cells.

Lymphedema-cholestasis syndrome (LCS, Aagenaes syndrome) is the only known form of hereditary lymphedema associated with cholestasis. A locus, LCS1, has recently been mapped to chromosome 15q in a Norwegian kindred. In a consanguine Serbian Romani family with a neonate who had a combination of lymphedema and cholestasis with features atypical for Norwegian LCS, haplotype and linkage analysis of markers spanning the LCS1 region argue that a second LCS locus may exist. The infant may represent an instance of a previously undescribed lymphedema-cholestasis syndrome. ( J Pediatr 2003;142:441-7 )

We have searched for the delta F508 mutation in 77 Norwegian cystic fibrosis patients. Of the 154 chromosomes tested, 93 (60%) carried the delta F508 mutation. Haplotypes at the D7S23 locus (KM19 and XV2C markers) were determined. Of 81 chromosomes with the F508 mutation, the B haplotype was found on 77. We found three patients with the G551D and one patient with the R553X mutation in exon 11 of the CFTR locus.

A new X-linked recessive deafness syndrome was recently reported and mapped to Xq22 (Mohr-Tranebjaerg syndrome). In addition to deafness, the patients had visual impairment, dystonia, fractures, and mental deterioration. The female carriers did not have any significant manifestations of the syndrome. We examined X chromosome inactivation in 8 obligate and 12 possible carriers by using a polymerase chain reaction analysis of the methylation-dependent amplification of the polymorphic triplet repeat at the androgen receptor locus. Seven of 8 obligate carriers and 1 of 5 carriers by linkage analysis had an extremely skewed pattern in blood DNA not found in 30 normal females. The X inactivation pattern in fibroblast DNA from 2 of the carriers with the extremely skewed pattern was also skewed but to a lesser degree than in blood DNA. One obligate carrier had a random X inactivation pattern in both blood and fibroblast DNA. A selection mechanism for the skewed pattern is therefore not likely. The extremely skewed X inactivation in 8 females of 3 generations in this family may be caused by a single gene that influences skewing of X chromosome inactivation.

Wiedemann-Beckwith syndrome (WBS) is a syndrome including exomphalos, macroglossia, and generalized overgrowth. The locus has been assigned to 11p15.5, and genomic imprinting may play a part in the expression of one or more genes involved. Most cases are sporadic. An excess of female monozygotic twins discordant for WBS have been reported, and it has been proposed that this excess could be related to the process of X chromosome inactivation. We have therefore studied X chromosome inactivation in 13-year-old monozygotic twin girls who were discordant for WBS. In addition, both twins had Tourette syndrome. The twins were monochorionic and therefore the result of a late twinning process. This has also been the case in previously reported discordant twin pairs with information on placentation. X chromosome inactivation was determined in DNA from peripheral blood cells by PCR analysis at the androgen receptor locus. The affected twin had a completely skewed X inactivation, where the paternal allele was on the active X chromosome in all cells. The unaffected twin had a moderately skewed X inactivation in the same direction, whereas the mother had a random pattern. Further studies are necessary to establish a possible association between the expression of WBS and X chromosome inactivation.

Currarino syndrome (CS) is a clinically variable disorder characterized by anorectal, sacral and presacral anomalies. It is associated with loss-of-function mutations in the motor neuron and pancreas homeobox 1 (MNX1) gene. Inheritance is autosomal dominant, expression variable and penetrance incomplete. We describe a Norwegian family with typical CS in which a heterozygous deletion removes the entire MNX1 gene but no other known genes. We also report MNX1 mutations in three other Norwegian families and confirm that the GCC12 repeat (c.373_375[12]) is a normal allelic variant. This work underscores the importance of dosage analysis of MNX1 when Sanger sequencing is negative.

Bilateral multilocular radiolucencies of the mandible are the main feature of cherubism (OMIM #118400), a rare autosomal dominant disorder primarily affecting the jaw. Typically, symmetrical swelling of the lower face is evident from around three years of age and increases until puberty. The underlying radiolucent lesions consist of vascular fibrotic stroma with scattered multinuclear giant cells. By age 30 years the facial contours are often unremarkable. Missing and displaced teeth as well as premature tooth loss are characteristic. Diagnosis rests upon a combination of clinical, radiographic, histological and molecular findings. SH3BP2 is currently the only gene known to be associated with cherubism. This cross-sectional study describes oral manifestations, quality of life and results of mutation analysis of SH3BP2 in 11 females and 13 males ages five to 84 years with cherubism. One individual with molecularly confirmed Noonan syndrome was excluded from the cohort. Standard statistical tools were used to analyze quality of life data. Mutation analysis was positive in all 22 familial and negative in both sporadic cases. Disease manifestations in mutation carriers varied from none to severe. Although intra-familial variability was marked, we found no evidence of non-penetrance, and females were on average more severely affected than males. Dental sequelae were pronounced; adults lacked a mean of 13 teeth (range 2-28), 13 of 17 individuals aged 16 years and older had removable or fixed dentures and five had dental implants; implant survival rate was 79%. In spite of pronounced disease manifestations and dental sequelae, adult quality of life was good.

Background Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder, which is usually caused by de novo mutations in the MECP2 gene. More than 70% of the disease causing MECP2 mutations are eight recurrent C to T transitions, which almost exclusively arise on the paternally derived X chromosome. About 10% of the RTT cases have a C-terminal frameshift deletion in MECP2. Only few RTT families with a segregating MECP2 mutation, which affects female carriers with a phenotype of mental retardation or RTT, have been reported in the literature. In this study we describe two new RTT families with three and four individuals, respectively, and review the literature comparing the type of mutations and phenotypes observed in RTT families with those observed in sporadic cases. Based on these observations we also investigated origin of mutation segregation to further improve genetic counselling. Methods MECP2 mutations were identified by direct sequencing. XCI studies were performed using the X-linked androgen receptor (AR) locus. The parental origin of de novo MECP2 frameshift mutations was investigated using intronic SNPs. Results In both families a C-terminal frameshift mutation segregates. Clinical features of the mutation carriers vary from classical RTT to mild mental retardation. XCI profiles of the female carriers correlate to their respective geno-/phenotypes. The majority of the de novo frameshift mutations occur on the paternally derived X chromosome (7/9 cases), without a paternal age effect. Conclusions The present study suggests a correlation between the intrafamilial phenotypic differences observed in RTT families and their respective XCI pattern in blood, in contrast to sporadic RTT cases where a similar correlation has not been demonstrated. Furthermore, we found de novo MECP2 frameshift mutations frequently to be of paternal origin, although not with the same high paternal occurrence as in sporadic cases with C to T transitions. This suggests further investigations of more families. This study emphasizes the need for thorough genetic counselling of families with a newly diagnosed RTT patient.

To establish the phenotypic variation and frequency of SPAST mutations or deletions in Norwegian patients with hereditary spastic paraplegia (HSP), we examined 59 unrelated patients with HSP and screened for DNA point mutations and microdeletions in SPG4. Forty-one had a familial history, 35 had a clear dominant inheritance, six had other affected sibs and 18 were sporadic. We found 12 mutations in SPG4, seven of them novel, and four different heterozygous exon deletions, two of them novel. Mutations were found in 16 families showing autosomal dominant (AD) inheritance, and in one sporadic case. In two non-SPG4 families the S44L polymorphism/modifier was found in both affected and unaffected individuals. This is the first study of Norwegian patients with HSP since the 1970s, and the first report on SPG4 in Norway. Our results show that SPG4 mutations and deletions are a significant cause of HSP in our population and warrant SPG4 screening in AD families and selected sporadic cases.

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy) is a neurovascular disease caused by mutations of the notch3 gene, manifesting with strokes or stroke-like episodes, psychiatric symptoms, migraine and dementia. The diagnosis can be confirmed by screening exons of this gene. Involvement of the anterior temporal lobe and external capsule on MRI and presence of granular osmiophilic material on skin biopsy may help in diagnosis. We present two Norwegian families with eight members who have symptoms indicating CADASIL. The mutation R182C was demonstrated in exon 4 in seven; one refused gene testing. Two brothers without symptoms also tested positively for this gene mutation.

In a systematic screening for mutations in the gene encoding the cystic fibrosis transmembrane regulator among Danish cystic fibrosis (CF) patients, we identified a mutation in exon 3 (394delTT); this mutation was found to be relatively common in Denmark. We therefore screened for 394delTT in Sweden and Norway, where it turned out to be the second most frequent mutation, accounting for 4% of all CF mutations. It also occurs with a high frequency in Finland, but has not been found in larger surveys of mutations in the CFTR gene. Thus, 394delTT seems to be a specific Nordic CF mutation.

The toxic lectin modeccin, which inhibits protein synthesis in eukaryotic cells, is cleaved upon treatment with 2-mercaptoethanol into two peptide chains which move in polyacrylamide gels at rates corresponding to molecular weights 28,000 and 38,000. After reduction, the toxin loses its effect on cells, while its ability to inhibit cell-free protein synthesis increases. Like abrin and ricin it inhibits protein synthesis by inactivating the 60S ribosomal subunits. Modeccin binds to surface receptors containing terminal galactose residues. Competition experiments with various glycoproteins indicate that the modeccin receptors are different from the abrin receptors. In addition, they were present on HeLa cells in much smaller numbers. Moreover, mutant lines resistant to abrin and ricin were not resistant to modeccin and vice-versa. The toxin resistance of various mutant cell lines could not be accounted for by a reduced number of binding sites on cells. The data are consistent with the view that the cells possesss different populations of binding sites with differences in ability to facilitate the uptake of the toxins and that in the resistant lines the most active receptors have been reduced or eliminated.

Shigella cytotoxin was purified from the culture medium of Shigella shigae 60 R and from the bacterial pellet by an extensive purification scheme involving chromatography on Cibachron Blue F3GA, chitin, and DEAE-cellulose columns, sucrose gradient centrifugation, and finally polyacrylamide gel electrophoresis under nondenaturing conditions. In sodium dodecyl sulfate polyacrylamide gels, purified toxin showed two bands, a heavy one with the molecular weight of 30,500 (A chain), and a broad band migrating near the dye front (B chain). The A chain was easily cleaved by proteases into two fragments, A1 (Mr = 27,500) and A2 (Mr = 3,000). These fragments were linked with a disulfide bridge (nicked toxin). The molecular weight of native toxin was estimated by the method of Ferguson to be 68,000 +/- 4,000. Cross-linking experiments indicated that the native toxin consists of one A chain and six to seven B chains, with each B chain having a molecular weight of congruent to 5,000. The isolated A and B chains were not toxic to cells nor did they bind to cells to a measurable extent. In a cell-free system, the A1 fragment strongly inhibited protein synthesis. The possibility that the B chains form the binding moiety of the toxin is discussed.

Patients with cholestasis-lymphedema syndrome (CLS) suffer severe neonatal cholestasis that usually lessens during early childhood and becomes episodic; they also develop chronic severe lymphedema. The genetic cause of CLS is unknown. We performed a genome screen, using DNA from eight Norwegian patients with CLS and from seven unaffected relatives, all from an extended pedigree. Regions potentially shared identical by descent in patients were further characterized in a larger set of Norwegian patients. The patients manifest extensive allele and haplotype sharing over the 6.6-cM D15S979–D15S652 region: 30 (83.3%) of 36 chromosomes of affected individuals carry a six-marker haplotype not found on any of the 32 nontransmitted parental chromosomes. All Norwegian patients with CLS are likely homozygous for the same disease mutation, inherited from a shared ancestor.

In attempts to assess how many molecules of the toxic lectins abrin, ricin and modeccin are needed in the cytosol to kill HeLa cells the effect of these toxins on protein synthesis and plating efficiency was studied. The incubation time of the cells after a 1 h exposure to the toxins influenced strongly the extent of inhibition of protein synthesis. The full toxic effect was expressed about 20 h of incubation after the exposure. On further incubation, protein synthesis again increased at a rate comparable to that in the control cells. After exposure to increasing concentrations of toxins the inhibition of cellular protein synthesis measured after 20 h showed excellent agreement with the inhibition of plating efficiency, indicating that the inhibition of protein synthesis can be used as a measure of cell killing. The inhibition of protein synthesis by toxins was found to follow first order kinetics, indicating that the cells are killed by an all- or none-effect. Autoradiographic studies indicated that after exposure to intermediate toxin concentrations protein synthesis was completely abolished in some cells, whereas it appeared to proceed at a normal rate in the remaining cells. The results provide evidence that penetration of one molecule of abrin, ricin or modeccin into cytosol is lethal to HeLa cells and that the efficiency of toxin entry into the cytoplasm is very low compared to the rate of bulk toxin uptake.

Carnitine concentration was measured in plasma, muscle, and dialysate before and after haemodialysis in patients with renal failure and in plasma and muscle of healthy controls. In eight of the nine patients carnitine concentration in muscle after haemodialysis was only 10% of the concentration in controls. Plasma-carnitine varied in patients before dialysis and in all of them was reduced by dialysis. The loss of carnitine into the dialysate (190--2100 mumol/treatment) greatly exceeded the normal loss in urine for most of patients, and was only partly compensated for. In some patients normal or high plasma-carnitine and low concentrations in muscle indicated that the carnitine-concentrating mechanisms in the muscle cell had failed. The reduction in carnitine will interfere seriously with normal cellular functions and this may help to explain the clinical syndrome of cardiomyopathy and cardiac failure which has been observed in some patients treated for a long time with intermittent haemodialysis.

The specificity and characteristics of the uptake mechanism for radiolabeled l -carnitine has been studied in cultured human heart cells (CCL 27). Iodoacetate, 2,4-dinitrophenol, arseneoxide and potassium cyanide do not reduce the uptake significantly, while sodium fluoride in high concentration (25–50 mM) inhibits the transport. Sulfhydryl blocking agents like N- ethylmaleimide , 2,4-dinitrofluorobenzene and Ellman reagent [5,5-dithiobis-(2-nitrobenzoic acid)] all reduce the uptake of radiolabeled carnitine; while phloridzin (0.1 mM), ouabain or amino acids (1–5 mM) do not. Thus it seems that the carnitine transport depends upon free sulfhydryl groups, and is neither linked to the transport of amino acids or glucose, nor to the activity of (Na + + K + )-ATPase . Variation in osmolality in the incubation medium within 225 to 450 mosM/kg water does not influence the uptake. An increase in pH from 7 to 8 reduces the transport approx. 40%. Compounds structurally related to carnitine, containing a trimethylamino group and a carboxylic group, reduce the uptake. l -Carnitine has a greater affinity for binding to the transport mechanism than d -carnitine. Acylcarnitines with varying acyl group length inhibit the transport, and the l isomers more than their d counterparts. The 50% inhibiting concentration on the uptake of 2 μM l -carnitine, was 90 μM for betaine, 11 · 10 3 μ M for choline, 2 μM for butyrobetaine, 20 μM for d -carnitine, 14 μM for 5-trimethylaminovaleric acid, 8 μM for l -acetylcarnitine and 3 μM for l -palmitoylcarnitine. Radiolabeled l -acetylcarnitine is also transported into the cells with a K m of 8 μM and a V of 10 pmol · μ g −1 DNA · h −1 . There is a competitive type of inhibition between l -carnitine and l -acetylcarnitine. Since l -acetylcarnitine constitutes 30% of total carnitine in rat plasma, both l -carnitine and l -acetylcarnitine can be physiological substrates for this active transport mechanism in vivo.

Diphtheria toxin and ricin, the toxins most frequently used in attempts to construct target-specific cytostatic agents, belong to a group of closely related toxic proteins produced by bacteria and plants. The main reason why these toxins are used for this purpose is their extreme toxicity. This is due to the fact that the active moieties of the toxins possess enzymatic activity. A single molecule may inactivate components required for protein synthesis more rapidly than the cell can produce new ones and hence kill the cell in the course of a few hours.

The cytotoxic test system for Shigella shigae toxin was improved and used to study the stability of the toxin to various pH values, temperature, and chemicals. Inhibition of protein synthesis is the first demonstrable effect in cells treated with Shigella toxin. This inhibition appears to be at the level of peptide chain elongation. An inhibition effect on cell-free protein synthesis is exhibited by toxin pretreated first with trypsin and then with dithiothreitol and 8 M urea or 1% sodium dodecyl sulfate. Ribosomes treated with toxin or its A1 fragment had lost most of their ability to polymerize [14C]phenylalanine in a poly(U)-dependent cell-free system. Salt-washed ribosomes in simple buffered solutions were inactivated at a rate of at least 40 ribosomes/(min) (A1 fragment). Addition of antitoxin immediately stopped further inactivation, but it did not reactivate the inactivated ribosomes. 60 S ribosomal subunits from toxin-treated ribosomes had a marked reduction in ability to support polyphenylanine synthesis, whereas 40 S subunits from toxin-treated ribosomes retained their activity. Toxin-treated ribosomes retained their ability to incorporate [3H]puromycin into growing peptide chains, indicating that the peptide bond formation is not the function inhibited.

The uptake of radiolabeled carnitine and butyrobetaine has been studied in human heart cells (CCL 27). The uptake of carinitine is 3-10-fold higher in heart cells than in fibroblasts (pmol - mug DNA-1). The uptake of carnitine increases with temperature coefficient KT of 1.6 in the interval 10-20 degrees C and with a negligible uptake at 4 and 10 degrees C. The uptake of carnitine follows Michaelis-Menten kinetics with a KM of 4.8 +/- 2.2 muM and V = 8.7 +/- 3.2 pmol - mug DNA-1 - H-1. Carnitine uptake is suppressed 90% by NaF (24MM). Butyrobetaine is taken up into heart cells to the same extent as carnitine with a KM of 5.7-17.3 muM and V = 8.7-9.3 pmol - mug DNA-1 - h-1. Butyrobetaine inhibits competitively the uptake of carnitine and carnitine inhibits the uptake of butyrobetaine to the same extent. No conversion of radiolabeled butyrobetaine to carnitine, or carnitine to methyl choline was observed intra- or extracellulary during incubation. These data are compatible with a selective transport mechanism for carnitine which is also responsible for the uptake of butyrobetaine.

To the Editor: Intracytoplasmic sperm injection (ICSI) has been established as an efficient treatment for male infertility and also as a supplement to in vitro fertilization (IVF) without obvious male infertility. ICSI is now regarded as a procedure that is safe overall, and no increase in developmental delay was found in a follow-up of 221 ICSI-conceived children in the 2nd year of life (Sutcliffe et al. 2001). However, the possibility of an increased risk of imprinting defects has been raised (Manning et al. 2000). Two children conceived by ICSI who had Angelman syndrome (AS [MIM 105830]) due to a presumably sporadic imprinting defect have recently been reported (Cox et al. 2002). We here report a 3.5-year-old girl with AS due to a sporadic imprinting defect, born of a pregnancy that was also the result of ICSI. The girl was the first child of a 35-year-old mother and a 36-year-old father. The father has a healthy daughter by another partner, and sperm analysis was normal on three different occasions. The mother had one spontaneous abortion and two extrauterine pregnancies before treatment with IVF. Traditional IVF did not result in fertilized eggs, and ICSI was therefore performed in spite of the normal sperm analysis of the father. The first ICSI pregnancy resulted in another spontaneous abortion, whereas the second ICSI procedure resulted in a normal pregnancy. Birth was at term, birth weight was 3,760 g, length was 54 cm, and head circumference was 36 cm (75th percentile). Development was considered normal for the first 3–4 mo, after which she started to have infections. She walked at age 2 years. She had no epilepsy but had an abnormal electroencephalogram with large-amplitude slow-spike waves. There was no language development. Chromosomes, including subtelomeres, were normal. At age 3 years, her height and weight were at the 50th percentile, whereas her head circumference was 1 cm below the 2.5th percentile. She was mentally retarded and atactic. She was dysmorphic, with a square face, deep-set eyes, and a protruding tongue. FISH analysis using the SNRPN probe (MIM 182279), as well as microsatellite studies, revealed normal chromosomes 15 of biparental origin. A common large deletion of 15q11-q13 and uniparental paternal disomy could therefore be excluded. Methylation-specific Southern blot analysis and methylation-specific PCR (Zeschnigk et al. 1997) for the SNRPN locus showed the presence of a normal unmethylated paternal band and the complete absence of a methylated maternal band, indicating that the patient had an imprinting defect. Quantitative Southern blot analysis of the critical AS imprinting center (IC) region (Buiting et al. 1999) showed a normal dosage; therefore, an IC deletion was unlikely. This result was confirmed by sequence analysis of the 880-bp AS-IC element, where the patient was heterozygous for three different SNPs. Both parents had normal chromosomes and a normal methylation pattern. These findings suggest that the patient belongs to the group of patients with a sporadic imprinting defect (Buiting et al. 1998). Both patients reported by Cox et al. (2002) had fathers with sperm abnormalities, and the possibility that the imprinting defect could be related to male infertility was discussed. The father of the child reported here had normal sperm, and a relationship to male infertility is therefore unlikely. However, similar to the mother, the maternal grandmother also had a history of reproductive difficulties. In addition to three healthy children, she had four spontaneous abortions and one daughter who was stillborn at term. Since both the maternal grandmother and the mother had reproductive difficulties, a maternal oogenesis defect cannot be excluded. A sporadic imprinting defect is a very rare cause of AS, and Cox et al. (2002) therefore considered a relationship to the ICSI procedure to be likely. The report of a third patient with this rare disorder further supports the assumption that ICSI can lead to an increased risk for imprinting defects.